Academic Appointments

Honors & Awards

  • EMBO, European Lectureship (1976)
  • Teacher-Scholar Grant, Dreyfus (1976)
  • Young Faculty Research Award, Society of the Sigma Xi (1976)
  • Award in Microbiology and Immunology, Eli Lilly and Co. (1976)
  • Chaired, Nucleic Acid Gordon Conference (1980)
  • Recipient of, United States Steel Award (1981)
  • Elected to the, National Academy of Sciences-Genetics (1983)
  • Distinguished Alumni Award, Eastern Illinois University (1984)
  • Louis S. Rosentiel Award for, Distinguished Work in Basic Medical Research (1992)
  • Biotechnology Research Award, Chiron Corporation (1998)
  • Genetics Society of America Award, Genetics Society of America (1998)
  • Senior Scholar Award in Global Infectious Disease, Ellison Medical Foundation (2002)
  • Herbert A. Sober Award, ASBMB/IUBMB (2004)
  • Lifetime Achievement Award, Genetics Society of America (2004)
  • Recipient of, Dickson Prize in Medicine, University of Pittsburgh (2005-2004)
  • Distinguished Alumni Award, California Institute of Technology (2007)
  • Inventor of the Year, SVIPLA (2011)
  • Gruber Genetics Prize, Gruber Foundation (2011)
  • Warren Alpert Foundation Prize, Warren Alpert Foundation (2013)
  • Named among the top 7 of "Today's Greatest Inventors", Atlantic Magazine (2013)

Research & Scholarship

Current Research and Scholarly Interests

We are using Saccharomyces cerevisiae and Human to conduct whole genome analysis projects. The yeast genome sequence has approximately 6,000 genes. We have made a set of haploid and diploid strains (21,000) containing a complete deletion of each gene. In order to facilitate whole genome analysis each deletion is molecularly tagged with a unique 20-mer DNA sequence. This sequence acts as a molecular bar code and makes it easy to identify the presence of each deletion. The mixture of all such tag strains then allows for the analysis of the entire genome with the manipulation of a single culture. During growth under a variety of conditions the loss of a tag indicates the loss of a deletion from the population. The concentration of each tag is determined by PCR amplification of the tags and hybridization to an Affymetrix DNA chip that contains the complement to all of the DNA sequence tags. This approach is being applied to other microorganisms.

We have identified a number of wild isolates of yeast that grow at much higher temperatures than is typical for Saccharomyces cerevisiae and are pathogenic and can kill a mouse. Microarrays have been used to map complex genetic traits such as virulence traits in pathogenic Saccharomyces cerevisiae using hybridization to detect single nucleotide polymorphisms. We have developed a new technology termed Reciprocal Hemizygosity Scanning that allows the determination of the contribution to the phenotype of all pair wise alleles for the whole genome from 2 independent strains. Using this technology we can map and quantitate all of the alleles in the genome for any complex quantitative trait in a single tube assay. These technologies will allow us to explore allelic contributions in complex mixed culture real environments and to investigate ecology at the genome level.

We are conducting a whole genome analysis (transcriptome and proteome) from blood of Human trauma patients. In this large clinical study we are establishing the standards for clinical genomics. We have developed 2 new technologies, "Molecular Inversion Probes" (MIP) for massive multiplex analysis of SNP and DNA content in Human and, "Mismatch Repair Detection" for discovery of rare Human polymorphisms. Both technologies are being applied to numerous clinical investigations.

Clinical Trials

  • Effect of Celecoxib on Perioperative Inflammatory Response in Colon Cancer Not Recruiting

    The proposed study aims to investigate how the administration of a drug known to reduce inflammation in humans, Celecoxib, will effect the peri-operative inflammatory response of a patient undergoing primary tumor resection surgery for colon cancer. The proposed project is an exploratory study, and will use data from blood samples and tumor samples to attempt to elucidate the immune and inflammatory response in colon cancer patients undergoing primary resection of their tumors.

    Stanford is currently not accepting patients for this trial. For more information, please contact Julia McNeal, (650) 723 - 9433.

    View full details


2017-18 Courses

Stanford Advisees

Graduate and Fellowship Programs


All Publications

  • Autonomous sweat extraction and analysis applied to cystic fibrosis and glucose monitoring using a fully integrated wearable platform PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Emaminejad, S., Gao, W., Wu, E., Davies, Z. A., Nyein, H. Y., Challa, S., Ryan, S. P., Fahad, H. M., Chen, K., Shahpar, Z., Talebi, S., Milla, C., Javey, A., Davis, R. W. 2017; 114 (18): 4625-4630


    Perspiration-based wearable biosensors facilitate continuous monitoring of individuals' health states with real-time and molecular-level insight. The inherent inaccessibility of sweat in sedentary individuals in large volume (≥10 µL) for on-demand and in situ analysis has limited our ability to capitalize on this noninvasive and rich source of information. A wearable and miniaturized iontophoresis interface is an excellent solution to overcome this barrier. The iontophoresis process involves delivery of stimulating agonists to the sweat glands with the aid of an electrical current. The challenge remains in devising an iontophoresis interface that can extract sufficient amount of sweat for robust sensing, without electrode corrosion and burning/causing discomfort in subjects. Here, we overcame this challenge through realizing an electrochemically enhanced iontophoresis interface, integrated in a wearable sweat analysis platform. This interface can be programmed to induce sweat with various secretion profiles for real-time analysis, a capability which can be exploited to advance our knowledge of the sweat gland physiology and the secretion process. To demonstrate the clinical value of our platform, human subject studies were performed in the context of the cystic fibrosis diagnosis and preliminary investigation of the blood/sweat glucose correlation. With our platform, we detected the elevated sweat electrolyte content of cystic fibrosis patients compared with that of healthy control subjects. Furthermore, our results indicate that oral glucose consumption in the fasting state is followed by increased glucose levels in both sweat and blood. Our solution opens the possibility for a broad range of noninvasive diagnostic and general population health monitoring applications.

    View details for DOI 10.1073/pnas.1701740114

    View details for Web of Science ID 000400358000035

    View details for PubMedID 28416667

  • Multifunctional, inexpensive, and reusable nanoparticle-printed biochip for cell manipulation and diagnosis. Proceedings of the National Academy of Sciences of the United States of America Esfandyarpour, R., DiDonato, M. J., Yang, Y., Durmus, N. G., Harris, J. S., Davis, R. W. 2017; 114 (8): E1306-E1315


    Isolation and characterization of rare cells and molecules from a heterogeneous population is of critical importance in diagnosis of common lethal diseases such as malaria, tuberculosis, HIV, and cancer. For the developing world, point-of-care (POC) diagnostics design must account for limited funds, modest public health infrastructure, and low power availability. To address these challenges, here we integrate microfluidics, electronics, and inkjet printing to build an ultra-low-cost, rapid, and miniaturized lab-on-a-chip (LOC) platform. This platform can perform label-free and rapid single-cell capture, efficient cellular manipulation, rare-cell isolation, selective analytical separation of biological species, sorting, concentration, positioning, enumeration, and characterization. The miniaturized format allows for small sample and reagent volumes. By keeping the electronics separate from microfluidic chips, the former can be reused and device lifetime is extended. Perhaps most notably, the device manufacturing is significantly less expensive, time-consuming, and complex than traditional LOC platforms, requiring only an inkjet printer rather than skilled personnel and clean-room facilities. Production only takes 20 min (vs. up to weeks) and $0.01-an unprecedented cost in clinical diagnostics. The platform works based on intrinsic physical characteristics of biomolecules (e.g., size and polarizability). We demonstrate biomedical applications and verify cell viability in our platform, whose multiplexing and integration of numerous steps and external analyses enhance its application in the clinic, including by nonspecialists. Through its massive cost reduction and usability we anticipate that our platform will enable greater access to diagnostic facilities in developed countries as well as POC diagnostics in resource-poor and developing countries.

    View details for DOI 10.1073/pnas.1621318114

    View details for PubMedID 28167769

  • A method for high-throughput production of sequence-verified DNA libraries and strain collections. Molecular systems biology Smith, J. D., Schlecht, U., Xu, W., Suresh, S., Horecka, J., Proctor, M. J., Aiyar, R. S., Bennett, R. A., Chu, A., Li, Y. F., Roy, K., Davis, R. W., Steinmetz, L. M., Hyman, R. W., Levy, S. F., St Onge, R. P. 2017; 13 (2): 913-?


    The low costs of array-synthesized oligonucleotide libraries are empowering rapid advances in quantitative and synthetic biology. However, high synthesis error rates, uneven representation, and lack of access to individual oligonucleotides limit the true potential of these libraries. We have developed a cost-effective method called Recombinase Directed Indexing (REDI), which involves integration of a complex library into yeast, site-specific recombination to index library DNA, and next-generation sequencing to identify desired clones. We used REDI to generate a library of ~3,300 DNA probes that exhibited > 96% purity and remarkable uniformity (> 95% of probes within twofold of the median abundance). Additionally, we created a collection of ~9,000 individually accessible CRISPR interference yeast strains for > 99% of genes required for either fermentative or respiratory growth, demonstrating the utility of REDI for rapid and cost-effective creation of strain collections from oligonucleotide pools. Our approach is adaptable to any complex DNA library, and fundamentally changes how these libraries can be parsed, maintained, propagated, and characterized.

    View details for DOI 10.15252/msb.20167233

    View details for PubMedID 28193641

    View details for PubMedCentralID PMC5327727

  • Next-Generation Molecular Testing of Newborn Dried Blood Spots for Cystic Fibrosis. journal of molecular diagnostics Lefterova, M. I., Shen, P., Odegaard, J. I., Fung, E., Chiang, T., Peng, G., Davis, R. W., Wang, W., Kharrazi, M., Schrijver, I., Scharfe, C. 2016; 18 (2): 267-282


    Newborn screening for cystic fibrosis enables early detection and management of this debilitating genetic disease. Implementing comprehensive CFTR analysis using Sanger sequencing as a component of confirmatory testing of all screen-positive newborns has remained impractical due to relatively lengthy turnaround times and high cost. Here, we describe CFseq, a highly sensitive, specific, rapid (<3 days), and cost-effective assay for comprehensive CFTR gene analysis from dried blood spots, the common newborn screening specimen. The unique design of CFseq integrates optimized dried blood spot sample processing, a novel multiplex amplification method from as little as 1 ng of genomic DNA, and multiplex next-generation sequencing of 96 samples in a single run to detect all relevant CFTR mutation types. Sequence data analysis utilizes publicly available software supplemented by an expert-curated compendium of >2000 CFTR variants. Validation studies across 190 dried blood spots demonstrated 100% sensitivity and a positive predictive value of 100% for single-nucleotide variants and insertions and deletions and complete concordance across the polymorphic poly-TG and consecutive poly-T tracts. Additionally, we accurately detected both a known exon 2,3 deletion and a previously undetected exon 22,23 deletion. CFseq is thus able to replace all existing CFTR molecular assays with a single robust, definitive assay at significant cost and time savings and could be adapted to high-throughput screening of other inherited conditions.

    View details for DOI 10.1016/j.jmoldx.2015.11.005

    View details for PubMedID 26847993

  • Integrating Cell Phone Imaging with Magnetic Levitation (i-LEV) for Label-Free Blood Analysis at the Point-of-Living. Small Baday, M., Calamak, S., Durmus, N. G., Davis, R. W., Steinmetz, L. M., Demirci, U. 2016; 12 (9): 1222-1229


    There is an emerging need for portable, robust, inexpensive, and easy-to-use disease diagnosis and prognosis monitoring platforms to share health information at the point-of-living, including clinical and home settings. Recent advances in digital health technologies have improved early diagnosis, drug treatment, and personalized medicine. Smartphones with high-resolution cameras and high data processing power enable intriguing biomedical applications when integrated with diagnostic devices. Further, these devices have immense potential to contribute to public health in resource-limited settings where there is a particular need for portable, rapid, label-free, easy-to-use, and affordable biomedical devices to diagnose and continuously monitor patients for precision medicine, especially those suffering from rare diseases, such as sickle cell anemia, thalassemia, and chronic fatigue syndrome. Here, a magnetic levitation-based diagnosis system is presented in which different cell types (i.e., white and red blood cells) are levitated in a magnetic gradient and separated due to their unique densities. Moreover, an easy-to-use, smartphone incorporated levitation system for cell analysis is introduced. Using our portable imaging magnetic levitation (i-LEV) system, it is shown that white and red blood cells can be identified and cell numbers can be quantified without using any labels. In addition, cells levitated in i-LEV can be distinguished at single-cell resolution, potentially enabling diagnosis and monitoring, as well as clinical and research applications.

    View details for DOI 10.1002/smll.201501845

    View details for PubMedID 26523938

    View details for PubMedCentralID PMC4775401

  • Nanoelectronic three-dimensional (3D) nanotip sensing array for real-time, sensitive, label-free sequence specific detection of nucleic acids. Biomedical microdevices Esfandyarpour, R., Yang, L., Koochak, Z., Harris, J. S., Davis, R. W. 2016; 18 (1): 7-?


    The improvements in our ability to sequence and genotype DNA have opened up numerous avenues in the understanding of human biology and medicine with various applications, especially in medical diagnostics. But the realization of a label free, real time, high-throughput and low cost biosensing platforms to detect molecular interactions with a high level of sensitivity has been yet stunted due to two factors: one, slow binding kinetics caused by the lack of probe molecules on the sensors and two, limited mass transport due to the planar structure (two-dimensional) of the current biosensors. Here we present a novel three-dimensional (3D), highly sensitive, real-time, inexpensive and label-free nanotip array as a rapid and direct platform to sequence-specific DNA screening. Our nanotip sensors are designed to have a nano sized thin film as their sensing area (~ 20 nm), sandwiched between two sensing electrodes. The tip is then conjugated to a DNA oligonucleotide complementary to the sequence of interest, which is electrochemically detected in real-time via impedance changes upon the formation of a double-stranded helix at the sensor interface. This 3D configuration is specifically designed to improve the biomolecular hit rate and the detection speed. We demonstrate that our nanotip array effectively detects oligonucleotides in a sequence-specific and highly sensitive manner, yielding concentration-dependent impedance change measurements with a target concentration as low as 10 pM and discrimination against even a single mismatch. Notably, our nanotip sensors achieve this accurate, sensitive detection without relying on signal indicators or enhancing molecules like fluorophores. It can also easily be scaled for highly multiplxed detection with up to 5000 sensors/square centimeter, and integrated into microfluidic devices. The versatile, rapid, and sensitive performance of the nanotip array makes it an excellent candidate for point-of-care diagnostics, and high-throughput DNA analysis applications.

    View details for DOI 10.1007/s10544-016-0032-8

    View details for PubMedID 26780442

  • Quantitative CRISPR interference screens in yeast identify chemical-genetic interactions and new rules for guide RNA design. Genome biology Smith, J. D., Suresh, S., Schlecht, U., Wu, M., Wagih, O., Peltz, G., Davis, R. W., Steinmetz, L. M., Parts, L., St Onge, R. P. 2016; 17 (1): 45-?


    Genome-scale CRISPR interference (CRISPRi) has been used in human cell lines; however, the features of effective guide RNAs (gRNAs) in different organisms have not been well characterized. Here, we define rules that determine gRNA effectiveness for transcriptional repression in Saccharomyces cerevisiae.We create an inducible single plasmid CRISPRi system for gene repression in yeast, and use it to analyze fitness effects of gRNAs under 18 small molecule treatments. Our approach correctly identifies previously described chemical-genetic interactions, as well as a new mechanism of suppressing fluconazole toxicity by repression of the ERG25 gene. Assessment of multiple target loci across treatments using gRNA libraries allows us to determine generalizable features associated with gRNA efficacy. Guides that target regions with low nucleosome occupancy and high chromatin accessibility are clearly more effective. We also find that the best region to target gRNAs is between the transcription start site (TSS) and 200 bp upstream of the TSS. Finally, unlike nuclease-proficient Cas9 in human cells, the specificity of truncated gRNAs (18 nt of complementarity to the target) is not clearly superior to full-length gRNAs (20 nt of complementarity), as truncated gRNAs are generally less potent against both mismatched and perfectly matched targets.Our results establish a powerful functional and chemical genomics screening method and provide guidelines for designing effective gRNAs, which consider chromatin state and position relative to the target gene TSS. These findings will enable effective library design and genome-wide programmable gene repression in many genetic backgrounds.

    View details for DOI 10.1186/s13059-016-0900-9

    View details for PubMedID 26956608

    View details for PubMedCentralID PMC4784398

  • A Rapid, High-Quality, Cost-Effective, Comprehensive and Expandable Targeted Next-Generation Sequencing Assay for Inherited Heart Diseases. Circulation research Wilson, K. D., Shen, P., Fung, E., Karakikes, I., Zhang, A., Inanloorahatloo, K., Odegaard, J., Sallam, K., Davis, R. W., Lui, G. K., Ashley, E. A., Scharfe, C., Wu, J. C. 2015; 117 (7): 603-611


    Thousands of mutations across >50 genes have been implicated in inherited cardiomyopathies. However, options for sequencing this rapidly evolving gene set are limited because many sequencing services and off-the-shelf kits suffer from slow turnaround, inefficient capture of genomic DNA, and high cost. Furthermore, customization of these assays to cover emerging targets that suit individual needs is often expensive and time consuming.We sought to develop a custom high throughput, clinical-grade next-generation sequencing assay for detecting cardiac disease gene mutations with improved accuracy, flexibility, turnaround, and cost.We used double-stranded probes (complementary long padlock probes), an inexpensive and customizable capture technology, to efficiently capture and amplify the entire coding region and flanking intronic and regulatory sequences of 88 genes and 40 microRNAs associated with inherited cardiomyopathies, congenital heart disease, and cardiac development. Multiplexing 11 samples per sequencing run resulted in a mean base pair coverage of 420, of which 97% had >20× coverage and >99% were concordant with known heterozygous single nucleotide polymorphisms. The assay correctly detected germline variants in 24 individuals and revealed several polymorphic regions in miR-499. Total run time was 3 days at an approximate cost of $100 per sample.Accurate, high-throughput detection of mutations across numerous cardiac genes is achievable with complementary long padlock probe technology. Moreover, this format allows facile insertion of additional probes as more cardiomyopathy and congenital heart disease genes are discovered, giving researchers a powerful new tool for DNA mutation detection and discovery.

    View details for DOI 10.1161/CIRCRESAHA.115.306723

    View details for PubMedID 26265630

    View details for PubMedCentralID PMC4568077

  • Magnetic levitation of single cells. Proceedings of the National Academy of Sciences of the United States of America Durmus, N. G., Tekin, H. C., Guven, S., Sridhar, K., Arslan Yildiz, A., Calibasi, G., Ghiran, I., Davis, R. W., Steinmetz, L. M., Demirci, U. 2015; 112 (28): E3661-8


    Several cellular events cause permanent or transient changes in inherent magnetic and density properties of cells. Characterizing these changes in cell populations is crucial to understand cellular heterogeneity in cancer, immune response, infectious diseases, drug resistance, and evolution. Although magnetic levitation has previously been used for macroscale objects, its use in life sciences has been hindered by the inability to levitate microscale objects and by the toxicity of metal salts previously applied for levitation. Here, we use magnetic levitation principles for biological characterization and monitoring of cells and cellular events. We demonstrate that each cell type (i.e., cancer, blood, bacteria, and yeast) has a characteristic levitation profile, which we distinguish at an unprecedented resolution of 1 × 10(-4) g⋅mL(-1). We have identified unique differences in levitation and density blueprints between breast, esophageal, colorectal, and nonsmall cell lung cancer cell lines, as well as heterogeneity within these seemingly homogenous cell populations. Furthermore, we demonstrate that changes in cellular density and levitation profiles can be monitored in real time at single-cell resolution, allowing quantification of heterogeneous temporal responses of each cell to environmental stressors. These data establish density as a powerful biomarker for investigating living systems and their responses. Thereby, our method enables rapid, density-based imaging and profiling of single cells with intriguing applications, such as label-free identification and monitoring of heterogeneous biological changes under various physiological conditions, including antibiotic or cancer treatment in personalized medicine.

    View details for DOI 10.1073/pnas.1509250112

    View details for PubMedID 26124131

    View details for PubMedCentralID PMC4507238

  • Magnetic levitation of single cells. Proceedings of the National Academy of Sciences of the United States of America Durmus, N. G., Tekin, H. C., Guven, S., Sridhar, K., Arslan Yildiz, A., Calibasi, G., Ghiran, I., Davis, R. W., Steinmetz, L. M., Demirci, U. 2015; 112 (28): E3661-8

    View details for DOI 10.1073/pnas.1509250112

    View details for PubMedID 26124131

  • Multielectrode Sensing for Extraction of Signal From Noise in Impedance Cytometry IEEE SENSORS JOURNAL Emaminejad, S., Talebi, S., Davis, R. W., Javanmard, M. 2015; 15 (5): 2715-2716
  • A Hybrid Approach of Gene Sets and Single Genes for the Prediction of Survival Risks with Gene Expression Data PLOS ONE Seok, J., Davis, R. W., Xiao, W. 2015; 10 (5)

    View details for DOI 10.1371/journal.pone.0122103

    View details for Web of Science ID 000353887100006

    View details for PubMedID 25933378

  • Robust Optimization of Biological Protocols TECHNOMETRICS Flaherty, P., Davis, R. W. 2015; 57 (2): 234-244
  • Tunable control of antibody immobilization using electric field. Proceedings of the National Academy of Sciences of the United States of America Emaminejad, S., Javanmard, M., Gupta, C., Chang, S., Davis, R. W., Howe, R. T. 2015; 112 (7): 1995-1999


    The controlled immobilization of proteins on solid-state surfaces can play an important role in enhancing the sensitivity of both affinity-based biosensors and probe-free sensing platforms. Typical methods of controlling the orientation of probe proteins on a sensor surface involve surface chemistry-based techniques. Here, we present a method of tunably controlling the immobilization of proteins on a solid-state surface using electric field. We study the ability to orient molecules by immobilizing IgG molecules in microchannels while applying lateral fields. We use atomic force microscopy to both qualitatively and quantitatively study the orientation of antibodies on glass surfaces. We apply this ability for controlled orientation to enhance the performance of affinity-based assays. As a proof of concept, we use fluorescence detection to indirectly verify the modulation of the orientation of proteins bound to the surface. We studied the interaction of fluorescently tagged anti-IgG with surface immobilized IgG controlled by electric field. Our study demonstrates that the use of electric field can result in more than 100% enhancement in signal-to-noise ratio compared with normal physical adsorption.

    View details for DOI 10.1073/pnas.1424592112

    View details for PubMedID 25650429

    View details for PubMedCentralID PMC4343132

  • A Simple Method for Encapsulating Single Cells in Alginate Microspheres Allows for Direct PCR and Whole Genome Amplification PLOS ONE Bigdeli, S., Dettloff, R. O., Frank, C. W., Davis, R. W., Crosby, L. D. 2015; 10 (2)


    Microdroplets are an effective platform for segregating individual cells and amplifying DNA. However, a key challenge is to recover the contents of individual droplets for downstream analysis. This paper offers a method for embedding cells in alginate microspheres and performing multiple serial operations on the isolated cells. Rhodobacter sphaeroides cells were diluted in alginate polymer and sprayed into microdroplets using a fingertip aerosol sprayer. The encapsulated cells were lysed and subjected either to conventional PCR, or whole genome amplification using either multiple displacement amplification (MDA) or a two-step PCR protocol. Microscopic examination after PCR showed that the lumen of the occupied microspheres contained fluorescently stained DNA product, but multiple displacement amplification with phi29 produced only a small number of polymerase colonies. The 2-step WGA protocol was successful in generating fluorescent material, and quantitative PCR from DNA extracted from aliquots of microspheres suggested that the copy number inside the microspheres was amplified up to 3 orders of magnitude. Microspheres containing fluorescent material were sorted by a dilution series and screened with a fluorescent plate reader to identify single microspheres. The DNA was extracted from individual isolates, re-amplified with full-length sequencing adapters, and then a single isolate was sequenced using the Illumina MiSeq platform. After filtering the reads, the only sequences that collectively matched a genome in the NCBI nucleotide database belonged to R. sphaeroides. This demonstrated that sequencing-ready DNA could be generated from the contents of a single microsphere without culturing. However, the 2-step WGA strategy showed limitations in terms of low genome coverage and an uneven frequency distribution of reads across the genome. This paper offers a simple method for embedding cells in alginate microspheres and performing PCR on isolated cells in common bulk reactions, although further work must be done to improve the amplification coverage of single genomes.

    View details for DOI 10.1371/journal.pone.0117738

    View details for Web of Science ID 000350322700069

    View details for PubMedID 25689864

    View details for PubMedCentralID PMC4331554

  • The conserved histone deacetylase Rpd3 and its DNA binding subunit Ume6 control dynamic transcript architecture during mitotic growth and meiotic development NUCLEIC ACIDS RESEARCH Lardenois, A., Stuparevic, I., Liu, Y., Law, M. J., Becker, E., Smagulova, F., Waern, K., Guilleux, M., Horecka, J., Chu, A., Kervarrec, C., Strich, R., Snyder, M., Davis, R. W., Steinmetz, L. M., Primig, M. 2015; 43 (1): 115-128


    It was recently reported that the sizes of many mRNAs change when budding yeast cells exit mitosis and enter the meiotic differentiation pathway. These differences were attributed to length variations of their untranslated regions. The function of UTRs in protein translation is well established. However, the mechanism controlling the expression of distinct transcript isoforms during mitotic growth and meiotic development is unknown. In this study, we order developmentally regulated transcript isoforms according to their expression at specific stages during meiosis and gametogenesis, as compared to vegetative growth and starvation. We employ regulatory motif prediction, in vivo protein-DNA binding assays, genetic analyses and monitoring of epigenetic amino acid modification patterns to identify a novel role for Rpd3 and Ume6, two components of a histone deacetylase complex already known to repress early meiosis-specific genes in dividing cells, in mitotic repression of meiosis-specific transcript isoforms. Our findings classify developmental stage-specific early, middle and late meiotic transcript isoforms, and they point to a novel HDAC-dependent control mechanism for flexible transcript architecture during cell growth and differentiation. Since Rpd3 is highly conserved and ubiquitously expressed in many tissues, our results are likely relevant for development and disease in higher eukaryotes.

    View details for DOI 10.1093/nar/gku1185

    View details for Web of Science ID 000350207100017

    View details for PubMedID 25477386

    View details for PubMedCentralID PMC4288150

  • RASA: Robust Alternative Splicing Analysis for Human Transcriptome Arrays. Scientific reports Seok, J., Xu, W., Davis, R. W., Xiao, W. 2015; 5: 11917-?


    Human transcriptome arrays (HTA) have recently been developed for high-throughput alternative splicing analysis by measuring signals not only from exons but also from exon-exon junctions. Effective use of these rich signals requires the development of computational methods for better gene and alternative splicing analyses. In this work, we introduce a computational method, Robust Alternative Splicing Analysis (RASA), for the analysis of the new transcriptome arrays by effective integration of the exon and junction signals. To increase robustness, RASA calculates the expression of each gene by selecting exons classified as not alternatively spliced. It then identifies alternatively spliced exons that are supported by both exon and junction signals to reduce the false positives. Finally, it detects additional alternative splicing candidates that are supported by only exon signals because the signals from the corresponding junctions are not well detected. RASA was demonstrated with Affymetrix HTAs and its performance was evaluated with mRNA-Seq and RT-PCR. The validation rate is 52.4%, which is a 60% increase when compared with previous methods that do not use selected exons for gene expression calculation and junction signals for splicing detection. These results suggest that RASA significantly improves alternative splicing analyses on HTA platforms.

    View details for DOI 10.1038/srep11917

    View details for PubMedID 26145443

    View details for PubMedCentralID PMC4491729

  • A hybrid approach of gene sets and single genes for the prediction of survival risks with gene expression data. PloS one Seok, J., Davis, R. W., Xiao, W. 2015; 10 (5)


    Accumulated biological knowledge is often encoded as gene sets, collections of genes associated with similar biological functions or pathways. The use of gene sets in the analyses of high-throughput gene expression data has been intensively studied and applied in clinical research. However, the main interest remains in finding modules of biological knowledge, or corresponding gene sets, significantly associated with disease conditions. Risk prediction from censored survival times using gene sets hasn't been well studied. In this work, we propose a hybrid method that uses both single gene and gene set information together to predict patient survival risks from gene expression profiles. In the proposed method, gene sets provide context-level information that is poorly reflected by single genes. Complementarily, single genes help to supplement incomplete information of gene sets due to our imperfect biomedical knowledge. Through the tests over multiple data sets of cancer and trauma injury, the proposed method showed robust and improved performance compared with the conventional approaches with only single genes or gene sets solely. Additionally, we examined the prediction result in the trauma injury data, and showed that the modules of biological knowledge used in the prediction by the proposed method were highly interpretable in biology. A wide range of survival prediction problems in clinical genomics is expected to benefit from the use of biological knowledge.

    View details for DOI 10.1371/journal.pone.0122103

    View details for PubMedID 25933378

    View details for PubMedCentralID PMC4416884

  • Surface charge sensing by altering the phase transition in VO2 JOURNAL OF APPLIED PHYSICS Kumar, S., Esfandyarpour, R., Davis, R., Nishi, Y. 2014; 116 (7)

    View details for DOI 10.1063/1.4893577

    View details for Web of Science ID 000341189400062

  • Targeted and highly multiplexed detection of microorganisms by employing an ensemble of molecular probes. Applied and environmental microbiology Xu, W., Krishnakumar, S., Miranda, M., Jensen, M. A., Fukushima, M., Palm, C., Fung, E., Davis, R. W., St Onge, R. P., Hyman, R. W. 2014; 80 (14): 4153-4161


    The vast majority of microscopic life on earth consists of microbes that do not grow in laboratory culture. To profile the microbial diversity in environmental and clinical samples, we have devised and employed molecular probe technology, which detects and identifies bacteria that do and do not grow in culture. The only requirement is a short sequence of contiguous bases (currently 60 bases) unique to the genome of the organism of interest. The procedure is relatively fast, inexpensive, customizable, robust, and culture independent and uses commercially available reagents and instruments. In this communication, we report improving the specificity of the molecular probes substantially and increasing the complexity of the molecular probe set by over an order of magnitude (>1,200 probes) and introduce a new final readout method based upon Illumina sequencing. In addition, we employed molecular probes to identify the bacteria from vaginal swabs and demonstrate how a deliberate selection of molecular probes can identify less abundant bacteria even in the presence of much more abundant species.

    View details for DOI 10.1128/AEM.00666-14

    View details for PubMedID 24795371

    View details for PubMedCentralID PMC4068679

  • Multiplexed actuation using ultra dielectrophoresis for proteomics applications: a comprehensive electrical and electrothermal design methodology. Lab on a chip Emaminejad, S., Dutton, R. W., Davis, R. W., Javanmard, M. 2014; 14 (12): 2105-2114


    In this work, we present a methodological approach to analyze an enhanced dielectrophoresis (DEP) system from both a circuit analysis and electrothermal view points. In our developed model, we have taken into account various phenomena and constraints such as voltage degradation (due to the presence of the protecting oxide layer), oxide breakdown, instrumentation limitations, and thermal effects. The results from this analysis are applicable generally to a wide variety of geometries and high voltage microsystems. Here, these design guidelines were applied to develop a robust electronic actuation system to perform a multiplexed bead-based protein assay. To carry out the multiplexed functionality, along a single microfluidic channel, an array of proteins is patterned, where each element is targeting a specific secondary protein coated on micron-sized beads in the subsequently introduced sample solution. Below each element of the array, we have a pair of addressable interdigitated electrodes. By selectively applying voltage at the terminals of each interdigitated electrode pair, the enhanced DEP, or equivalently 'ultra'-DEP (uDEP) force detaches protein-bound beads from each element of the array, one by one, without disturbing the bound beads in the neighboring regions. The detached beads can be quantified optically or electrically downstream. For proof of concept, we illustrated 16-plex actuation capability of our device to elute micron-sized beads that are bound to the surface through anti-IgG and IgG interaction which is on the same order of magnitude in strength as typical antibody-antigen interactions. In addition to its application in multiplexed protein analysis, our platform can be potentially utilized to statistically characterize the strength profile of biological bonds, since the multiplexed format allows for high throughput force spectroscopy using the array of uDEP devices, under the same buffer and assay preparation conditions.

    View details for DOI 10.1039/c4lc00036f

    View details for PubMedID 24801800

  • Multiplexed actuation using ultra dielectrophoresis for proteomics applications: a comprehensive electrical and electrothermal design methodology. Lab on a chip Emaminejad, S., Dutton, R. W., Davis, R. W., Javanmard, M. 2014; 14 (12): 2105-2114

    View details for DOI 10.1039/c4lc00036f

    View details for PubMedID 24801800

  • Single cell mutational analysis of PIK3CA in circulating tumor cells and metastases in breast cancer reveals heterogeneity, discordance, and mutation persistence in cultured disseminated tumor cells from bone marrow BMC CANCER Deng, G., Krishnakumar, S., Powell, A. A., Zhang, H., Mindrinos, M. N., Telli, M. L., Davis, R. W., Jeffrey, S. S. 2014; 14


    Therapeutic decisions in cancer are generally guided by molecular biomarkers or, for some newer therapeutics, primary tumor genotype. However, because biomarkers or genotypes may change as new metastases emerge, circulating tumor cells (CTCs) from blood are being investigated for a role in guiding real-time drug selection during disease progression, expecting that CTCs will comprehensively represent the full spectrum of genomic changes in metastases. However, information is limited regarding mutational heterogeneity among CTCs and metastases in breast cancer as discerned by single cell analysis. The presence of disseminated tumor cells (DTCs) in bone marrow also carry prognostic significance in breast cancer, but with variability between CTC and DTC detection. Here we analyze a series of single tumor cells, CTCs, and DTCs for PIK3CA mutations and report CTC and corresponding metastatic genotypes.We used the MagSweeper, an immunomagnetic separation device, to capture live single tumor cells from breast cancer patients' primary and metastatic tissues, blood, and bone marrow. Single cells were screened for known hotspot mutations in exons 9 and 20 of the PIK3CA gene. Captured DTCs grown in cell culture were also sequenced for PIK3CA mutations.Among 242 individual tumor cells isolated from 17 patients and tested for mutations, 48 mutated tumor cells were identified in three patients. Single cell analyses revealed mutational heterogeneity among CTCs and tumor cells in tissues. In a patient followed serially, there was mutational discordance between CTCs, DTCs, and metastases, and among CTCs isolated at different time points. DTCs from this patient propagated in vitro contained a PIK3CA mutation, which was maintained despite morphological changes during 21 days of cell culture.Single cell analysis of CTCs can demonstrate genotypic heterogeneity, changes over time, and discordance from DTCs and distant metastases. We present a cautionary case showing that CTCs from any single blood draw do not always reflect metastatic genotype, and that CTC and DTC analyses may provide independent clinical information. Isolated DTCs remain viable and can be propagated in culture while maintaining their original mutational status, potentially serving as a future resource for investigating new drug therapies.

    View details for DOI 10.1186/1471-2407-14-456

    View details for Web of Science ID 000338965100001

  • Nanoelectronic impedance detection of target cells. Biotechnology and bioengineering Esfandyarpour, R., Javanmard, M., Koochak, Z., Harris, J. S., Davis, R. W. 2014; 111 (6): 1161-1169


    Detection of cells is typically performed using optical fluorescence based techniques such as flow cytometry. Here we present the impedance detection of target cells using a nanoelectronic probe we have developed, which we refer to as the nanoneedle biosensor. The nanoneedle consists of a thin film conducting electrode layer at the bottom, an insulative oxide layer above, another conductive electrode layer above, and a protective oxide above. The electrical impedance is measured between the two electrode layers. Cells captured on the surface of the nanoneedle tip results in a decrease in the impedance across the sensing electrodes. The basic mechanisms behind the electrical response of cells in solution under an applied alternating electrical field stems from modulation of the relative permittivity at the interface. In this paper we discuss, the circuit model, the nanofabrication, and the testing and characterization of the sensor. We demonstrate proof of concept for detection of yeast cells with specificity. We envision the sensor presented in this paper to be combined with microfluidic pre-concentration technologies to develop low cost point-of-care diagnostic assays for the clinical setting.

    View details for DOI 10.1002/bit.25171

    View details for PubMedID 24338648

  • IpO: plasmids and methods for simplified, PCR-based DNA transplant in yeast YEAST Horecka, J., Chu, A. M., Davis, R. W. 2014; 31 (5): 185-193


    Many yeast experiments require strains modified by recombinant DNA methods. Some experiments require precise insertion of a DNA segment into the genome without a selectable marker remaining. For these applications, we developed a new PCR-based method for marker-free DNA transplant. The current PCR-based method requires the labour-intensive construction of a PCR template plasmid with repeats of the DNA segment flanking URA3. The design of a new vector, IpO, reduces the work in cloning a single copy of the DNA segment between overlapping URA3 fragments present in the vector. Two PCRs are performed that capture the DNA segment and one or the other URA3 fragment. When the PCR products are co-transformed into yeast, recombination between the overlapping URA3 fragments restores URA3 and transposes the cloned DNA segment inside out, creating a repeat-URA3-repeat cassette. Sequences designed into the PCR primers target integration of the cassette into the genome. Subsequent selection with 5-fluoro-orotic acid yields strains that have 'popped out' URA3 via recombination between the DNA repeats, with the result being the precise insertion of the DNA segment minus the selectable marker. An additional advantage of the IpO method is that it eliminates PCR artifacts that can plague the current method's repeat-containing templates.

    View details for DOI 10.1002/yea.3006

    View details for Web of Science ID 000335210800004

    View details for PubMedID 24604451

    View details for PubMedCentralID PMC4013213

  • A functional screen for copper homeostasis genes identifies a pharmacologically tractable cellular system BMC GENOMICS Schlecht, U., Suresh, S., Xu, W., Aparicio, A. M., Chu, A., Proctor, M. J., Davis, R. W., Scharfe, C., St Onge, R. P. 2014; 15


    Copper is essential for the survival of aerobic organisms. If copper is not properly regulated in the body however, it can be extremely cytotoxic and genetic mutations that compromise copper homeostasis result in severe clinical phenotypes. Understanding how cells maintain optimal copper levels is therefore highly relevant to human health.We found that addition of copper (Cu) to culture medium leads to increased respiratory growth of yeast, a phenotype which we then systematically and quantitatively measured in 5050 homozygous diploid deletion strains. Cu's positive effect on respiratory growth was quantitatively reduced in deletion strains representing 73 different genes, the function of which identify increased iron uptake as a cause of the increase in growth rate. Conversely, these effects were enhanced in strains representing 93 genes. Many of these strains exhibited respiratory defects that were specifically rescued by supplementing the growth medium with Cu. Among the genes identified are known and direct regulators of copper homeostasis, genes required to maintain low vacuolar pH, and genes where evidence supporting a functional link with Cu has been heretofore lacking. Roughly half of the genes are conserved in man, and several of these are associated with Mendelian disorders, including the Cu-imbalance syndromes Menkes and Wilson's disease. We additionally demonstrate that pharmacological agents, including the approved drug disulfiram, can rescue Cu-deficiencies of both environmental and genetic origin.A functional screen in yeast has expanded the list of genes required for Cu-dependent fitness, revealing a complex cellular system with implications for human health. Respiratory fitness defects arising from perturbations in this system can be corrected with pharmacological agents that increase intracellular copper concentrations.

    View details for DOI 10.1186/1471-2164-15-263

    View details for Web of Science ID 000334957400001

    View details for PubMedID 24708151

    View details for PubMedCentralID PMC4023593

  • Scan statistics analysis for detection of introns in time-course tiling array data STATISTICAL APPLICATIONS IN GENETICS AND MOLECULAR BIOLOGY Reiner-Benaim, A., Davis, R. W., Juneau, K. 2014; 13 (2): 173-190


    A tiling array yields a series of abundance measurements across the genome using evenly spaced probes. These data can be used for detecting sequences that exhibit a particular behavior. Scanning window statistics are often employed for testing each probe while accounting for local correlation and smoothing noisy measurements. However, window testing may yield false probe discoveries around the sequences and false non-discoveries within the sequences, resulting in biased predicted intervals. We propose to avoid this problem by stipulating that a sequence of interest can appear at most once within a defined region, such as a gene; thus, only one window statistic is considered per region. This substantially reduces the number of tests and hence, is potentially more powerful. We compare this approach to a genome-wise scan that does not require pre-defined search regions, but considers clumps of adjacent probe discoveries. Simulations show that the gene-wise search maintains the nominal FDR level, while the genome-wise scan yields FDR that exceeds the nominal level for low interval effects, and achieves slightly less power. Using arrays to map introns in yeast, we identified 71% of the previously published introns, detected nine previously undiscovered introns, and observed no false intron discoveries by either method.

    View details for DOI 10.1515/sagmb-2013-0038

    View details for Web of Science ID 000333184800004

    View details for PubMedID 24572987

  • The 50:50 method for PCR-based seamless genome editing in yeast YEAST Horecka, J., Davis, R. W. 2014; 31 (3): 103-112


    The ability to edit the yeast genome with relative ease has contributed to the organism being a model eukaryote for decades. Most methods for deleting, inserting or altering genomic sequences require transformation with DNA that carries the desired change and a selectable marker. One-step genome editing methods retain the selectable marker. Seamless genome editing methods require more steps and a marker that can be used for both positive and negative selection, such as URA3. Here we describe the PCR-based 50:50 method for seamless genome editing, which requires only two primers, one PCR with a URA3 cassette, and a single yeast transformation. Our method is based on pop-in/pop-out gene replacement and is amenable to the facile creation of genomic deletions and short insertions or substitutions. We used the 50:50 method to make two conservative loss-of-function mutations in MATα1, with results suggesting that the wild-type gene has a new function outside of that presently known.

    View details for DOI 10.1002/yea.2992

    View details for Web of Science ID 000332982800004

    View details for PubMedID 24639370

  • Depletion of cells and abundant proteins from biological samples by enhanced dielectrophoresis 17th International Conference on Solid-State Sensors, Actuators and Microsystems Javanmard, M., Emaminejad, S., Gupta, C., Provine, J., Davis, R. W., Howe, R. T. ELSEVIER SCIENCE SA. 2014: 918–924
  • Digital microfluidic assay for protein detection. Proceedings of the National Academy of Sciences of the United States of America Mok, J., Mindrinos, M. N., Davis, R. W., Javanmard, M. 2014; 111 (6): 2110-2115


    Global studies of the human proteome have revealed a plethora of putative protein biomarkers. However, their application for early disease detection remains at a standstill without suitable methods to realize their utility in the clinical setting. There thus continues to be tremendous interest in developing new technology for sensitive protein detection that is both low in cost and carries a small footprint to be able to be used at the point of care. The current gold standard method for protein biomarker detection is the ELISA, which measures protein abundance using bulky fluorescent scanners that lack portability. Here, we present a digital microfluidic platform for protein biomarker detection that is low in cost compared with standard optical detection methods, without any compromise in sensitivity. This platform furthermore makes use of simple electronics, enabling its translation into a portable handheld device, and has been developed in a manner that can easily be adapted to assay different types of proteomic biomarkers. We demonstrate its utility in quantifying not only protein abundance, but also activity. Interleukin-6 abundance could be assayed from concentrations as low as 50 pM (an order of magnitude lower than that detectable by a comparable laboratory designed ELISA) using less than 5 μL of sample, and Abelson tyrosine kinase activity was detectable in samples containing 100 pM of kinase.

    View details for DOI 10.1073/pnas.1323998111

    View details for PubMedID 24449893

  • Next generation 1536-well oligonucleotide synthesizer with on-the-fly dispense. Journal of biotechnology Jensen, M., Roberts, L., Johnson, A., Fukushima, M., Davis, R. 2014; 171: 76-81


    Here we report the development of our Next Generation Automated Multiplexed Oligonucleotide Synthesizer (NG-1536-AMOS), capable of producing 1536 samples in a single run using a multi-well filtered titer plate. With the potential to synthesize up to 3456 samples per plate, we converted the BioRAPTR Flying Reagent Dispenser into an open-well system where spent reagents are drained to waste under vacuum. During synthesis, reagents are delivered on-the-fly to each micro-titer well at volumes ≤5μl with plate speeds up to 150mm/s. Using gas-phase cleavage and deprotection, a full plate of 1536 60mers may be processed with same-day turnaround with an average yield per well at 3.5nmol. Final product at only $0.00277/base is eluted into a low-volume collection plate for immediate use in downstream application (e.g. Biomek FX for versatile sample handling). Also, crude oligonucleotide quality is comparable to that of commercial synthesis instrumentation, with an error rate on the NG-1536-AMOS platform of 1.53/717 bases. Furthermore, mass spectral analysis on strands synthesized up to 80 bases showed high purity with an average coupling efficiency of 99.5%.

    View details for DOI 10.1016/j.jbiotec.2013.11.027

    View details for PubMedID 24355807

  • Molecular indexing enables quantitative targeted RNA sequencing and reveals poor efficiencies in standard library preparations PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Fu, G. K., Xu, W., Wilhelmy, J., Mindrinos, M. N., Davis, R. W., Xiao, W., Fodor, S. P. 2014; 111 (5): 1891-1896


    We present a simple molecular indexing method for quantitative targeted RNA sequencing, in which mRNAs of interest are selectively captured from complex cDNA libraries and sequenced to determine their absolute concentrations. cDNA fragments are individually labeled so that each molecule can be tracked from the original sample through the library preparation and sequencing process. Multiple copies of cDNA fragments of identical sequence become distinct through labeling, and replicate clones created during PCR amplification steps can be identified and assigned to their distinct parent molecules. Selective capture enables efficient use of sequencing for deep sampling and for the absolute quantitation of rare or transient transcripts that would otherwise escape detection by standard sequencing methods. We have also constructed a set of synthetic barcoded RNA molecules, which can be introduced as controls into the sample preparation mix and used to monitor the efficiency of library construction. The quantitative targeted sequencing revealed extremely low efficiency in standard library preparations, which were further confirmed by using synthetic barcoded RNA molecules. This finding shows that standard library preparation methods result in the loss of rare transcripts and highlights the need for monitoring library efficiency and for developing more efficient sample preparation methods.

    View details for DOI 10.1073/pnas.1323732111

    View details for Web of Science ID 000330587600060

    View details for PubMedID 24449890

    View details for PubMedCentralID PMC3918775

  • Surface-Enhanced Raman Scattering (SERS) for Detection of Phenylketonuria for Newborn Screening Conference on Nanoscale Imaging, Sensing, and Actuation for Biomedical Applications XI Javanmard, M., Davis, R. W. SPIE-INT SOC OPTICAL ENGINEERING. 2014

    View details for DOI 10.1117/12.2040845

    View details for Web of Science ID 000337063000015

  • Label-free Electronic Detection of Target Cells Conference on Microfluidics, BioMEMS, and Medical Microsystems XII Esfandyarpour, R., Javanmard, M., Harris, J., Davis, R. W. SPIE-INT SOC OPTICAL ENGINEERING. 2014

    View details for DOI 10.1117/12.2037966

    View details for Web of Science ID 000336033500033

  • Computational identification and analysis of orphan assembly-line polyketide synthases JOURNAL OF ANTIBIOTICS O'Brien, R. V., Davis, R. W., Khosla, C., Hillenmeyer, M. E. 2014; 67 (1): 89-97


    The increasing availability of DNA sequence data offers an opportunity for identifying new assembly-line polyketide synthases (PKSs) that produce biologically active natural products. We developed an automated method to extract and consolidate all multimodular PKS sequences (including hybrid PKS/non-ribosomal peptide synthetases) in the National Center for Biotechnology Information (NCBI) database, generating a non-redundant catalog of 885 distinct assembly-line PKSs, the majority of which were orphans associated with no known polyketide product. Two in silico experiments highlight the value of this search method and resulting catalog. First, we identified an orphan that could be engineered to produce an analog of albocycline, an interesting antibiotic whose gene cluster has not yet been sequenced. Second, we identified and analyzed a hitherto overlooked family of metazoan multimodular PKSs, including one from Caenorhabditis elegans. We also developed a comparative analysis method that identified sequence relationships among known and orphan PKSs. As expected, PKS sequences clustered according to structural similarities between their polyketide products. The utility of this method was illustrated by highlighting an interesting orphan from the genus Burkholderia that has no close relatives. Our search method and catalog provide a community resource for the discovery of new families of assembly-line PKSs and their antibiotic products.

    View details for DOI 10.1038/ja.2013.125

    View details for Web of Science ID 000330222300013

    View details for PubMedID 24301183

  • Diversity of the Vaginal Microbiome Correlates With Preterm Birth REPRODUCTIVE SCIENCES Hyman, R. W., Fukushima, M., Jiang, H., Fung, E., Rand, L., Johnson, B., Vo, K. C., Caughey, A. B., Hilton, J. F., Davis, R. W., Giudice, L. C. 2014; 21 (1): 32-40


    Reproductive tract infection is a major initiator of preterm birth (PTB). The objective of this prospective cohort study of 88 participants was to determine whether PTB correlates with the vaginal microbiome during pregnancy. Total DNA was purified from posterior vaginal fornix swabs during gestation. The 16S ribosomal RNA gene was amplified using polymerase chain reaction primers, followed by chain-termination sequencing. Bacteria were identified by comparing contig consensus sequences with the Ribosomal Database Project. Dichotomous responses were summarized via proportions and continuous variables via means ± standard deviation. Mean Shannon Diversity index differed by Welch t test (P = .00016) between caucasians with PTB and term gestation. Species diversity was greatest among African Americans (P = .0045). Change in microbiome/Lactobacillus content and presence of putative novel/noxious bacteria did not correlate with PTB. We conclude that uncultured vaginal bacteria play an important role in PTB and race/ethnicity and sampling location are important determinants of the vaginal microbiome.

    View details for DOI 10.1177/1933719113488838

    View details for Web of Science ID 000327927200004

    View details for PubMedID 23715799


    View details for DOI 10.1214/13-AOAS659

    View details for Web of Science ID 000330044900023

  • Simulation and fabrication of a new novel 3D injectable biosensor for high throughput genomics and proteomics in a lab-on-a-chip device. Nanotechnology Esfandyarpour, R., Esfandyarpour, H., Harris, J. S., Davis, R. W. 2013; 24 (46): 465301-?


    Biosensors are used for the detection of biochemical molecules such as proteins and nucleic acids. Traditional techniques, such as enzyme-linked immuno-sorbent assay (ELISA), are sensitive but require several hours to yield a result and usually require the attachment of a fluorophore molecule to the target molecule. Micromachined biosensors that employ electrical detection are now being developed. Here we describe one such device, which is ultrasensitive, real-time, label free and localized. It is called the nanoneedle biosensor and shows promise to overcome some of the current limitations of biosensors. The key element of this device is a 10 nm wide annular gap at the end of the needle, which is the sensitive part of the sensor. The total diameter of the sensor is about 100 nm. Any change in the population of molecules in this gap results in a change of impedance across the gap. Single molecule detection should be possible because the sensory part of the sensor is in the range of bio-molecules of interest. To increase throughput we can flow the solution containing the target molecules over an array of such structures, each with its own integrated read-out circuitry to allow 'real-time' detection (i.e. several minutes) of label free molecules without sacrificing sensitivity. To fabricate the arrays we used electron beam lithography together with associated pattern transfer techniques. Preliminary measurements on individual needle structures in water are consistent with the design. Since the proposed sensor has a rigid nano-structure, this technology, once fully developed, could ultimately be used to directly monitor protein quantities within a single living cell, an application that would have significant utility for drug screening and studying various intracellular signaling pathways.

    View details for DOI 10.1088/0957-4484/24/46/465301

    View details for PubMedID 24149048

  • Simulation and fabrication of a new novel 3D injectable biosensor for high throughput genomics and proteomics in a lab-on-a-chip device. Nanotechnology Esfandyarpour, R., Esfandyarpour, H., Harris, J. S., Davis, R. W. 2013; 24 (46): 465301-?


    Biosensors are used for the detection of biochemical molecules such as proteins and nucleic acids. Traditional techniques, such as enzyme-linked immuno-sorbent assay (ELISA), are sensitive but require several hours to yield a result and usually require the attachment of a fluorophore molecule to the target molecule. Micromachined biosensors that employ electrical detection are now being developed. Here we describe one such device, which is ultrasensitive, real-time, label free and localized. It is called the nanoneedle biosensor and shows promise to overcome some of the current limitations of biosensors. The key element of this device is a 10 nm wide annular gap at the end of the needle, which is the sensitive part of the sensor. The total diameter of the sensor is about 100 nm. Any change in the population of molecules in this gap results in a change of impedance across the gap. Single molecule detection should be possible because the sensory part of the sensor is in the range of bio-molecules of interest. To increase throughput we can flow the solution containing the target molecules over an array of such structures, each with its own integrated read-out circuitry to allow 'real-time' detection (i.e. several minutes) of label free molecules without sacrificing sensitivity. To fabricate the arrays we used electron beam lithography together with associated pattern transfer techniques. Preliminary measurements on individual needle structures in water are consistent with the design. Since the proposed sensor has a rigid nano-structure, this technology, once fully developed, could ultimately be used to directly monitor protein quantities within a single living cell, an application that would have significant utility for drug screening and studying various intracellular signaling pathways.

    View details for DOI 10.1088/0957-4484/24/46/465301

    View details for PubMedID 24149048

  • The Sequencing Bead Array (SBA), a Next-Generation Digital Suspension Array PLOS ONE Akhras, M. S., Pettersson, E., Diamond, L., Unemo, M., Okamoto, J., Davis, R. W., Pourmand, N. 2013; 8 (10)


    Here we describe the novel Sequencing Bead Array (SBA), a complete assay for molecular diagnostics and typing applications. SBA is a digital suspension array using Next-Generation Sequencing (NGS), to replace conventional optical readout platforms. The technology allows for reducing the number of instruments required in a laboratory setting, where the same NGS instrument could be employed from whole-genome and targeted sequencing to SBA broad-range biomarker detection and genotyping. As proof-of-concept, a model assay was designed that could distinguish ten Human Papillomavirus (HPV) genotypes associated with cervical cancer progression. SBA was used to genotype 20 cervical tumor samples and, when compared with amplicon pyrosequencing, was able to detect two additional co-infections due to increased sensitivity. We also introduce in-house software Sphix, enabling easy accessibility and interpretation of results. The technology offers a multi-parallel, rapid, robust, and scalable system that is readily adaptable for a multitude of microarray diagnostic and typing applications, e.g. genetic signatures, single nucleotide polymorphisms (SNPs), structural variations, and immunoassays. SBA has the potential to dramatically change the way we perform probe-based applications, and allow for a smooth transition towards the technology offered by genomic sequencing.

    View details for DOI 10.1371/journal.pone.0076696

    View details for Web of Science ID 000325501300080

    View details for PubMedID 24116138

    View details for PubMedCentralID PMC3792038

  • Direct oligonucleotide synthesis onto super-paramagnetic beads. Journal of biotechnology Jensen, M. A., Akhras, M. S., Fukushima, M., Pourmand, N., Davis, R. W. 2013; 167 (4): 448-453


    Super-paramagnetic beads (SPMB)s used for a variety of molecular diagnostic assays are prepared by attaching pre-synthesized oligonucleotides to the surface via a cumbersome and low efficient method of carbodiimide-mediated amide bond formation. To mainstream the process, we describe a novel procedure of direct oligonucleotide synthesis onto the surface of SPMBs (e.g. MyOne Dynabeads). With the many challenges surrounding containment of paramagnetic beads (≤1μm) during automated oligonucleotide synthesis, we show that by applying a magnetic force directly to the SPMBs we prevent their loss caused by high-pressure drain steps during synthesis. To date we have synthesized 40mers using a Spacer 9 phosphoramidite (triethylene glycol) coupled to the surface of hydroxylated SPMBs. HPLC analysis shows successful product generation with an average yield of 200pmol per sample. Furthermore, because of the versatility of this powerful research tool, we envision its use in any laboratory working with conventional synthesis automation, as employed for single columns and for multi-well titer plates. In addition to direct synthesis of oligodeoxynucleotides (DNA) onto SPMBs, this platform also has the potential for RNA and peptide nucleic acid synthesis.

    View details for DOI 10.1016/j.jbiotec.2013.08.006

    View details for PubMedID 23942380

  • Label-free electronic probing of nucleic acids and proteins at the nanoscale using the nanoneedle biosensor BIOMICROFLUIDICS Esfandyarpour, R., Javanmard, M., Koochak, Z., Esfandyarpour, H., Harris, J. S., Davis, R. W. 2013; 7 (4)

    View details for DOI 10.1063/1.4817771

    View details for Web of Science ID 000323907600016

  • Determination of Burn Patient Outcome by Large-Scale Quantitative Discovery Proteomics CRITICAL CARE MEDICINE Finnerty, C. C., Jeschke, M. G., Qian, W., Kaushal, A., Xiao, W., Liu, T., Gritsenko, M. A., Moore, R. J., Camp, D. G., Moldawer, L. L., Elson, C., Schoenfeld, D., Gamelli, R., Gibran, N., Klein, M., Arnoldo, B., Remick, D., Smith, R. D., Davis, R., Tompkins, R. G., Herndon, D. N. 2013; 41 (6): 1421-1434


    OBJECTIVES:: Emerging proteomics techniques can be used to establish proteomic outcome signatures and to identify candidate biomarkers for survival following traumatic injury. We applied high-resolution liquid chromatography-mass spectrometry and multiplex cytokine analysis to profile the plasma proteome of survivors and nonsurvivors of massive burn injury to determine the proteomic survival signature following a major burn injury. DESIGN:: Proteomic discovery study. SETTING:: Five burn hospitals across the United States. PATIENTS:: Thirty-two burn patients (16 nonsurvivors and 16 survivors), 19-89 years old, were admitted within 96 hours of injury to the participating hospitals with burns covering more than 20% of the total body surface area and required at least one surgical intervention. INTERVENTIONS:: None. MEASUREMENTS AND MAIN RESULTS:: We found differences in circulating levels of 43 proteins involved in the acute-phase response, hepatic signaling, the complement cascade, inflammation, and insulin resistance. Thirty-two of the proteins identified were not previously known to play a role in the response to burn. Interleukin-4, interleukin-8, granulocyte macrophage colony-stimulating factor, monocyte chemotactic protein-1, and β2-microglobulin correlated well with survival and may serve as clinical biomarkers. CONCLUSIONS:: These results demonstrate the utility of these techniques for establishing proteomic survival signatures and for use as a discovery tool to identify candidate biomarkers for survival. This is the first clinical application of a high-throughput, large-scale liquid chromatography-mass spectrometry-based quantitative plasma proteomic approach for biomarker discovery for the prediction of patient outcome following burn, trauma, or critical illness.

    View details for DOI 10.1097/CCM.0b013e31827c072e

    View details for Web of Science ID 000319269400024

    View details for PubMedID 23507713

  • Multiplex target capture with double-stranded DNA probes GENOME MEDICINE Shen, P., Wang, W., Chi, A., Fan, Y., Davis, R. W., Scharfe, C. 2013; 5


    Target enrichment technologies utilize single-stranded oligonucleotide probes to capture candidate genomic regions from a DNA sample before sequencing. We describe target capture using double-stranded probes, which consist of single-stranded, complementary long padlock probes (cLPPs), each selectively capturing one strand of a genomic target through circularization. Using two probes per target increases sensitivity for variant detection and cLPPs are easily produced by PCR at low cost. Additionally, we introduce an approach for generating capture libraries with uniformly randomized template orientations. This facilitates bidirectional sequencing of both the sense and antisense template strands during one paired-end read, which maximizes target coverage.

    View details for DOI 10.1186/gm454

    View details for Web of Science ID 000321978500001

    View details for PubMedID 23718862

    View details for PubMedCentralID PMC3706973

  • HIGH THROUGHPUT HIGH FIDELITY HLA TYPING WITH DEEP SEQUENCING 27th Annual EFI European Immunogenetics and Histocompatibility Conference Mindros, M., Wang, C., Krishnakumar, S., Barsakis, K., Miranda, M., Babrzadeh, F., Suresh, S., Kuehn, R. T., Davis, M. M., Davis, R., Fernandez-Vina, M. A. WILEY-BLACKWELL. 2013: 344–44
  • Coded Corrugated Microfluidic Sidewalls for Code Division Multiplexing IEEE SENSORS JOURNAL Javanmard, M., Davis, R. W. 2013; 13 (5): 1399-1400
  • Rare variant detection using family-based sequencing analysis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Peng, G., Fan, Y., Palculict, T. B., Shen, P., Ruteshouser, E. C., Chi, A., Davis, R. W., Huff, V., Scharfe, C., Wang, W. 2013; 110 (10): 3985-3990


    Next-generation sequencing is revolutionizing genomic analysis, but this analysis can be compromised by high rates of missing true variants. To develop a robust statistical method capable of identifying variants that would otherwise not be called, we conducted sequence data simulations and both whole-genome and targeted sequencing data analysis of 28 families. Our method (Family-Based Sequencing Program, FamSeq) integrates Mendelian transmission information and raw sequencing reads. Sequence analysis using FamSeq reduced the number of false negative variants by 14-33% as assessed by HapMap sample genotype confirmation. In a large family affected with Wilms tumor, 84% of variants uniquely identified by FamSeq were confirmed by Sanger sequencing. In children with early-onset neurodevelopmental disorders from 26 families, de novo variant calls in disease candidate genes were corrected by FamSeq as mendelian variants, and the number of uniquely identified variants in affected individuals increased proportionally as additional family members were included in the analysis. To gain insight into maximizing variant detection, we studied factors impacting actual improvements of family-based calling, including pedigree structure, allele frequency (common vs. rare variants), prior settings of minor allele frequency, sequence signal-to-noise ratio, and coverage depth (∼20× to >200×). These data will help guide the design, analysis, and interpretation of family-based sequencing studies to improve the ability to identify new disease-associated genes.

    View details for DOI 10.1073/pnas.1222158110

    View details for Web of Science ID 000316377400068

    View details for PubMedID 23426633

    View details for PubMedCentralID PMC3593912

  • Genomic responses in mouse models poorly mimic human inflammatory diseases PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Seok, J., Warren, H. S., Cuenca, A. G., Mindrinos, M. N., Baker, H. V., Xu, W., Richards, D. R., McDonald-Smith, G. P., Gao, H., Hennessy, L., Finnerty, C. C., Lopez, C. M., Honari, S., Moore, E. E., Minei, J. P., Cuschieri, J., Bankey, P. E., Johnson, J. L., Sperry, J., Nathens, A. B., Billiar, T. R., West, M. A., Jeschke, M. G., Klein, M. B., Gamelli, R. L., Gibran, N. S., Brownstein, B. H., Miller-Graziano, C., Calvano, S. E., Mason, P. H., Cobb, J. P., Rahme, L. G., Lowry, S. F., Maier, R. V., Moldawer, L. L., Herndon, D. N., Davis, R. W., Xiao, W., Tompkins, R. G. 2013; 110 (9): 3507-3512


    A cornerstone of modern biomedical research is the use of mouse models to explore basic pathophysiological mechanisms, evaluate new therapeutic approaches, and make go or no-go decisions to carry new drug candidates forward into clinical trials. Systematic studies evaluating how well murine models mimic human inflammatory diseases are nonexistent. Here, we show that, although acute inflammatory stresses from different etiologies result in highly similar genomic responses in humans, the responses in corresponding mouse models correlate poorly with the human conditions and also, one another. Among genes changed significantly in humans, the murine orthologs are close to random in matching their human counterparts (e.g., R(2) between 0.0 and 0.1). In addition to improvements in the current animal model systems, our study supports higher priority for translational medical research to focus on the more complex human conditions rather than relying on mouse models to study human inflammatory diseases.

    View details for DOI 10.1073/pnas.1222878110

    View details for Web of Science ID 000315841900063

    View details for PubMedID 23401516

  • Microneedle biosensor: A method for direct label-free real time protein detection SENSORS AND ACTUATORS B-CHEMICAL Esfandyarpour, R., Esfandyarpour, H., Javanmard, M., Harris, J. S., Davis, R. W. 2013; 177: 848-855


    Here we present the development of an array of electrical micro-biosensors in a microfluidic channel, called microneedle biosensors. A microneedle biosensor is a real-time, label-free, direct electrical detection platform, which is capable of high sensitivity detection, measuring the change in ionic current and impedance modulation, due to the presence or reaction of biomolecules such as proteins and nucleic acids. In this study, we successfully fabricated and electrically characterized the sensors and demonstrated successful detection of target protein. In this study, we used biotinylated bovine serum albumin as the receptor and streptavidin as the target analyte.

    View details for DOI 10.1016/j.snb.2012.11.064

    View details for Web of Science ID 000315751000113

    View details for PubMedCentralID PMC3551587

  • Classification of patients from time-course gene expression BIOSTATISTICS Zhang, Y., Tibshirani, R., Davis, R. 2013; 14 (1): 87-98


    Classifying patients into different risk groups based on their genomic measurements can help clinicians design appropriate clinical treatment plans. To produce such a classification, gene expression data were collected on a cohort of burn patients, who were monitored across multiple time points. This led us to develop a new classification method using time-course gene expressions. Our results showed that making good use of time-course information of gene expression improved the performance of classification compared with using gene expression from individual time points only. Our method is implemented into an R-package: time-course prediction analysis using microarray.

    View details for DOI 10.1093/biostatistics/kxs027

    View details for Web of Science ID 000312636300007

    View details for PubMedID 22926914

  • Ultra Dielectrophoresis Using Atomic Layer Deposited Films: Electrothermal Analysis 9th International Symposium on Atomic Layer Deposition Applications held during the 224th Meeting of the Electrochemical-Society (ECS) Emaminejad, S., Javanmard, M., DUTTON, R. W., Davis, R. W. ELECTROCHEMICAL SOC INC. 2013: 67–72
  • Smart Surface for Elution of Protein-Protein Bound Particles: Nanonewton Dielectrophoretic Forces Using Atomic Layer Deposited Oxides ANALYTICAL CHEMISTRY Emaminejad, S., Javanmard, M., Dutton, R. W., Davis, R. W. 2012; 84 (24): 10793-10801


    By increasing the strength of the negative dielectrophoresis force, we demonstrated a significantly improved electrokinetic actuation and switching microsystem that can be used to elute specifically bound beads from the surface. In this work using atomic layer deposition we deposited a pinhole free nanometer-scale thin film oxide as a protective layer to prevent electrodes from corrosion, when applying high voltages (>20 V(pp)) at the electrodes. Then, by exciting the electrodes at high frequency, we capacitively coupled the electrodes to the buffer in order to avoid electric field degradation and, hence, reduction in dielectrophoresis force due to the presence of the insulating oxide layer. To illustrate the functionality of our system, we demonstrated 100% detachment of anti-IgG and IgG bound beads (which is on the same order of magnitude in strength as typical antibody-antigen interactions) from the surface, upon applying the improved negative dielectrophoresis force. The significantly enhanced switching performance presented in this work shows orders of magnitude of improvement in on-to-off ratio and switching response time, without any need for chemical eluting agents, as compared to the previous work. The promising results from this work vindicates that the functionality of this singleplexed platform can be extended to perform a multiplexed bead-based assay where in a single channel an array of proteins are patterned each targeting a different antigen or protein.

    View details for DOI 10.1021/ac302857z

    View details for Web of Science ID 000312429800043

    View details for PubMedID 23176521

  • Identification of drug targets by chemogenomic and metabolomic profiling in yeast PHARMACOGENETICS AND GENOMICS Wu, M., Zheng, M., Zhang, W., Suresh, S., Schlecht, U., Fitch, W. L., Aronova, S., Baumann, S., Davis, R., St Onge, R., Dill, D. L., Peltz, G. 2012; 22 (12): 877-886


    To advance our understanding of disease biology, the characterization of the molecular target for clinically proven or new drugs is very important. Because of its simplicity and the availability of strains with individual deletions in all of its genes, chemogenomic profiling in yeast has been used to identify drug targets. As measurement of drug-induced changes in cellular metabolites can yield considerable information about the effects of a drug, we investigated whether combining chemogenomic and metabolomic profiling in yeast could improve the characterization of drug targets.We used chemogenomic and metabolomic profiling in yeast to characterize the target for five drugs acting on two biologically important pathways. A novel computational method that uses a curated metabolic network was also developed, and it was used to identify the genes that are likely to be responsible for the metabolomic differences found.The combination of metabolomic and chemogenomic profiling, along with data analyses carried out using a novel computational method, could robustly identify the enzymes targeted by five drugs. Moreover, this novel computational method has the potential to identify genes that are causative of metabolomic differences or drug targets.

    View details for DOI 10.1097/FPC.0b013e32835aa888

    View details for Web of Science ID 000311031800006

    View details for PubMedID 23076370

  • A Peripheral Blood Diagnostic Test for Acute Rejection in Renal Transplantation AMERICAN JOURNAL OF TRANSPLANTATION Li, L., Khatri, P., Sigdel, T. K., Tran, T., Ying, L., Vitalone, M. J., Chen, A., Hsieh, S., Dai, H., Zhang, M., Naesens, M., Zarkhin, V., Sansanwal, P., Chen, R., Mindrinos, M., Xiao, W., Benfield, M., Ettenger, R. B., Dharnidharka, V., Mathias, R., Portale, A., McDonald, R., Harmon, W., Kershaw, D., Vehaskari, V. M., Kamil, E., Baluarte, H. J., Warady, B., Davis, R., Butte, A. J., Salvatierra, O., Sarwal, M. M. 2012; 12 (10): 2710-2718


    Monitoring of renal graft status through peripheral blood (PB) rather than invasive biopsy is important as it will lessen the risk of infection and other stresses, while reducing the costs of rejection diagnosis. Blood gene biomarker panels were discovered by microarrays at a single center and subsequently validated and cross-validated by QPCR in the NIH SNSO1 randomized study from 12 US pediatric transplant programs. A total of 367 unique human PB samples, each paired with a graft biopsy for centralized, blinded phenotype classification, were analyzed (115 acute rejection (AR), 180 stable and 72 other causes of graft injury). Of the differentially expressed genes by microarray, Q-PCR analysis of a five gene-set (DUSP1, PBEF1, PSEN1, MAPK9 and NKTR) classified AR with high accuracy. A logistic regression model was built on independent training-set (n = 47) and validated on independent test-set (n = 198)samples, discriminating AR from STA with 91% sensitivity and 94% specificity and AR from all other non-AR phenotypes with 91% sensitivity and 90% specificity. The 5-gene set can diagnose AR potentially avoiding the need for invasive renal biopsy. These data support the conduct of a prospective study to validate the clinical predictive utility of this diagnostic tool.

    View details for DOI 10.1111/j.1600-6143.2012.04253.x

    View details for Web of Science ID 000309180000018

    View details for PubMedID 23009139

  • Microfluidic Platform for Electrical Monitoring of Enzyme Activity IEEE SENSORS JOURNAL Javanmard, M., Davis, R. W. 2012; 12 (9): 2733-2734
  • Cationic Amphiphilic Drugs Are Potent Inhibitors of Yeast Sporulation PLOS ONE Schlecht, U., St Onge, R. P., Walther, T., Francois, J., Davis, R. W. 2012; 7 (8)


    Meiosis is a highly regulated developmental process that occurs in all eukaryotes that engage in sexual reproduction. Previous epidemiological work shows that male and female infertility is rising and environmental factors, including pollutants such as organic solvents, are thought to play a role in this phenomenon. To better understand how organic compounds interfere with meiotic development, the model organism Saccharomyces cerevisiae was exposed to 446 bioactive molecules while undergoing meiotic development, and sporulation efficiency was quantified employing two different high-throughput assays. 12 chemicals were identified that strongly inhibited spore formation but did not interfere with vegetative growth. Many of these chemicals are known to bind to monoamine-receptors in higher eukaryotes and are cationic amphiphilic drugs. A detailed analysis of one of these drugs, tripelennamine, revealed that it induces sporulation-specific cytotoxicity and a strong inhibition of meiotic M phase. The drug, however, only mildly interfered with pre-meiotic DNA synthesis and the early meiotic transcriptional program. Chemical-genomic screening identified genes involved in autophagy as hypersensitive to tripelennamine. In addition, we found that growing and sporulating yeast cells heterozygous for the aminophospholipid translocase, NEO1, are haploinsufficient in the presence of the drug.

    View details for DOI 10.1371/journal.pone.0042853

    View details for Web of Science ID 000307331100069

    View details for PubMedID 22905177

  • Improvement in cell capture throughput using parallel bioactivated microfluidic channels BIOMEDICAL MICRODEVICES Javanmard, M., Babrzadeh, F., Nyren, P., Davis, R. W. 2012; 14 (4): 625-629


    Optimization of targeted cell capture with microfluidic devices continues to be a challenge. On the one hand, microfluidics allow working with microliter volumes of liquids, whereas various applications in the real world require detection of target analyte in large volumes, such as capture of rare cell types in several ml of blood. This contrast of volumes (microliter vs. ml) has prevented the emergence of microfluidic cell capture sensors in the clinical setting. Here, we study the improvement in cell capture and throughput achieved using parallel bioactivated microfluidic channels. The device consists of channels in parallel with each other tied to a single channel. We discuss fabrication and testing of our devices, and show the ability for an improvement in throughput detection of target cells.

    View details for DOI 10.1007/s10544-012-9643-x

    View details for Web of Science ID 000305951500001

    View details for PubMedID 22367556

  • Control of DNA Capture by Nanofluidic Transistors ACS NANO Paik, K., Liu, Y., Tabard-Cossa, V., Waugh, M. J., Huber, D. E., Provine, J., Howe, R. T., Dutton, R. W., Davis, R. W. 2012; 6 (8): 6767-6775


    We report the use of an array of electrically gated ~200 nm solid-state pores as nanofluidic transistors to manipulate the capture and passage of DNA. The devices are capable of reversibly altering the rate of DNA capture by over 3 orders of magnitude using sub-1 V biasing of a gate electrode. This efficient gating originates from the counter-balance of electrophoresis and electroosmosis, as revealed by quantitative numerical simulations. Such a reversible electronically tunable biomolecular switch may be used to manipulate nucleic acid delivery in a fluidic circuit, and its development is an important first step toward active control of DNA motion through solid-state nanopores for sensing applications.

    View details for DOI 10.1021/nn3014917

    View details for Web of Science ID 000307988900029

    View details for PubMedID 22762282

    View details for PubMedCentralID PMC3429714

  • Multiplex assay for condition-dependent changes in protein-protein interactions PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Schlecht, U., Miranda, M., Suresh, S., Davis, R. W., St Onge, R. P. 2012; 109 (23): 9213-9218


    Changes in protein-protein interactions that occur in response to environmental cues are difficult to uncover and have been poorly characterized to date. Here we describe a yeast-based assay that allows many binary protein interactions to be assessed in parallel and under various conditions. This method combines molecular bar-coding and tag array technology with the murine dihydrofolate reductase-based protein-fragment complementation assay. A total of 238 protein-fragment complementation assay strains, each representing a unique binary protein complex, were tagged with molecular barcodes, pooled, and then interrogated against a panel of 80 diverse small molecules. Our method successfully identified specific disruption of the Hom3:Fpr1 interaction by the immunosuppressant FK506, illustrating the assay's capacity to identify chemical inhibitors of protein-protein interactions. Among the additional findings was specific cellular depletion of the Dst1:Rbp9 complex by the anthracycline drug doxorubicin, but not by the related drug idarubicin. The assay also revealed chemical-induced accumulation of several binary multidrug transporter complexes that largely paralleled increases in transcript levels. Further assessment of two such interactions (Tpo1:Pdr5 and Snq2:Pdr5) in the presence of 1,246 unique chemical compounds revealed a positive correlation between drug lipophilicity and the drug response in yeast.

    View details for DOI 10.1073/pnas.1204952109

    View details for Web of Science ID 000304991100085

    View details for PubMedID 22615397

  • Whole-genome sequencing of the efficient industrial fuel-ethanol fermentative Saccharomyces cerevisiae strain CAT-1 MOLECULAR GENETICS AND GENOMICS Babrzadeh, F., Jalili, R., Wang, C., Shokralla, S., Pierce, S., Robinson-Mosher, A., Nyren, P., Shafer, R. W., Basso, L. C., de Amorim, H. V., de Oliveira, A. J., Davis, R. W., Ronaghi, M., Gharizadeh, B., Stambuk, B. U. 2012; 287 (6): 485-494


    The Saccharomyces cerevisiae strains widely used for industrial fuel-ethanol production have been developed by selection, but their underlying beneficial genetic polymorphisms remain unknown. Here, we report the draft whole-genome sequence of the S. cerevisiae strain CAT-1, which is a dominant fuel-ethanol fermentative strain from the sugarcane industry in Brazil. Our results indicate that strain CAT-1 is a highly heterozygous diploid yeast strain, and the ~12-Mb genome of CAT-1, when compared with the reference S228c genome, contains ~36,000 homozygous and ~30,000 heterozygous single nucleotide polymorphisms, exhibiting an uneven distribution among chromosomes due to large genomic regions of loss of heterozygosity (LOH). In total, 58 % of the 6,652 predicted protein-coding genes of the CAT-1 genome constitute different alleles when compared with the genes present in the reference S288c genome. The CAT-1 genome contains a reduced number of transposable elements, as well as several gene deletions and duplications, especially at telomeric regions, some correlated with several of the physiological characteristics of this industrial fuel-ethanol strain. Phylogenetic analyses revealed that some genes were likely associated with traits important for bioethanol production. Identifying and characterizing the allelic variations controlling traits relevant to industrial fermentation should provide the basis for a forward genetics approach for developing better fermenting yeast strains.

    View details for DOI 10.1007/s00438-012-0695-7

    View details for Web of Science ID 000304555800004

    View details for PubMedID 22562254

  • HIGH THROUGHPUT, HIGH FIDELITY HLA GENOTYPING WITH ULTRA DEEP SEQUENCING Joint 16th International HLA and Immunogenetics Workshop/26th European Federation for Immunogenetics Conference/23rd British-Society-of-Histocompatibility-and-Immunogenetics Conference Krishnakumar, S., Wang, C., Wilhelmy, J., Babrzadeh, F., Su, L., Levinson, D., Fernandez-Vina, M., Davis, R., Davis, M., Mindrinos, M. N. WILEY-BLACKWELL. 2012: 426–26
  • Coding SNPs as intrinsic markers for sample tracking in large-scale transcriptome studies BIOTECHNIQUES Xu, W., Gao, H., Seok, J., Wilhelmy, J., Mindrinos, M. N., Davis, R. W., Xiao, W. 2012; 52 (6): 386-388


    Large-scale transcriptome profiling in clinical studies often involves assaying multiple samples of a patient to monitor disease progression, treatment effect, and host response in multiple tissues. Such profiling is prone to human error, which often results in mislabeled samples. Here, we present a method to detect mislabeled sample outliers using coding single nucleotide polymorphisms (cSNPs) specifically designed on the microarray and demonstrate that the mislabeled samples can be efficiently identified by either simple clustering of allele-specific expression scores or Mahalanobis distance-based outlier detection method. Based on our results, we recommend the incorporation of cSNPs into future transcriptome array designs as intrinsic markers for sample tracking.

    View details for DOI 10.2144/0000113879

    View details for Web of Science ID 000305300200007

    View details for PubMedID 22668418

  • High-throughput, high-fidelity HLA genotyping with deep sequencing PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wang, C., Krishnakumar, S., Wilhelmy, J., Babrzadeh, F., Stepanyan, L., Su, L. F., Levinson, D., Fernandez-Vina, M. A., Davis, R. W., Davis, M. M., Mindrinos, M. 2012; 109 (22): 8676-8681


    Human leukocyte antigen (HLA) genes are the most polymorphic in the human genome. They play a pivotal role in the immune response and have been implicated in numerous human pathologies, especially autoimmunity and infectious diseases. Despite their importance, however, they are rarely characterized comprehensively because of the prohibitive cost of standard technologies and the technical challenges of accurately discriminating between these highly related genes and their many allelles. Here we demonstrate a high-resolution, and cost-effective methodology to type HLA genes by sequencing, which combines the advantage of long-range amplification, the power of high-throughput sequencing platforms, and a unique genotyping algorithm. We calibrated our method for HLA-A, -B, -C, and -DRB1 genes with both reference cell lines and clinical samples and identified several previously undescribed alleles with mismatches, insertions, and deletions. We have further demonstrated the utility of this method in a clinical setting by typing five clinical samples in an Illumina MiSeq instrument with a 5-d turnaround. Overall, this technology has the capacity to deliver low-cost, high-throughput, and accurate HLA typing by multiplexing thousands of samples in a single sequencing run, which will enable comprehensive disease-association studies with large cohorts. Furthermore, this approach can also be extended to include other polymorphic genes.

    View details for DOI 10.1073/pnas.1206614109

    View details for Web of Science ID 000304881700065

    View details for PubMedID 22589303

  • Single Cell Profiling of Circulating Tumor Cells: Transcriptional Heterogeneity and Diversity from Breast Cancer Cell Lines PLOS ONE Powell, A. A., Talasaz, A. H., Zhang, H., Coram, M. A., Reddy, A., Deng, G., Telli, M. L., Advani, R. H., Carlson, R. W., Mollick, J. A., Sheth, S., Kurian, A. W., Ford, J. M., Stockdale, F. E., Quake, S. R., Pease, R. F., Mindrinos, M. N., Bhanot, G., Dairkee, S. H., Davis, R. W., Jeffrey, S. S. 2012; 7 (5)


    To improve cancer therapy, it is critical to target metastasizing cells. Circulating tumor cells (CTCs) are rare cells found in the blood of patients with solid tumors and may play a key role in cancer dissemination. Uncovering CTC phenotypes offers a potential avenue to inform treatment. However, CTC transcriptional profiling is limited by leukocyte contamination; an approach to surmount this problem is single cell analysis. Here we demonstrate feasibility of performing high dimensional single CTC profiling, providing early insight into CTC heterogeneity and allowing comparisons to breast cancer cell lines widely used for drug discovery.We purified CTCs using the MagSweeper, an immunomagnetic enrichment device that isolates live tumor cells from unfractionated blood. CTCs that met stringent criteria for further analysis were obtained from 70% (14/20) of primary and 70% (21/30) of metastatic breast cancer patients; none were captured from patients with non-epithelial cancer (n = 20) or healthy subjects (n = 25). Microfluidic-based single cell transcriptional profiling of 87 cancer-associated and reference genes showed heterogeneity among individual CTCs, separating them into two major subgroups, based on 31 highly expressed genes. In contrast, single cells from seven breast cancer cell lines were tightly clustered together by sample ID and ER status. CTC profiles were distinct from those of cancer cell lines, questioning the suitability of such lines for drug discovery efforts for late stage cancer therapy.For the first time, we directly measured high dimensional gene expression in individual CTCs without the common practice of pooling such cells. Elevated transcript levels of genes associated with metastasis NPTN, S100A4, S100A9, and with epithelial mesenchymal transition: VIM, TGFß1, ZEB2, FOXC1, CXCR4, were striking compared to cell lines. Our findings demonstrate that profiling CTCs on a cell-by-cell basis is possible and may facilitate the application of 'liquid biopsies' to better model drug discovery.

    View details for DOI 10.1371/journal.pone.0033788

    View details for Web of Science ID 000305335000005

    View details for PubMedID 22586443

    View details for PubMedCentralID PMC3346739

  • JETTA: junction and exon toolkits for transcriptome analysis BIOINFORMATICS Seok, J., Xu, W., Gao, H., Davis, R. W., Xiao, W. 2012; 28 (9): 1274-1275


    High-throughput genome-wide studies of alternatively spliced mRNA transcripts have become increasingly important in clinical research. Consequently, easy-to-use software tools are required to process data from these studies, for example, using exon and junction arrays. Here, we introduce JETTA, an integrated software package for the calculation of gene expression indices as well as the identification and visualization of alternative splicing events. We demonstrate the software using data of human liver and muscle samples hybridized on an exon-junction array.JETTA and its demonstrations are freely available at

    View details for DOI 10.1093/bioinformatics/bts134

    View details for Web of Science ID 000303338200013

    View details for PubMedID 22433281

  • Benchmarking Outcomes in the Critically Injured Trauma Patient and the Effect of Implementing Standard Operating Procedures ANNALS OF SURGERY Cuschieri, J., Johnson, J. L., Sperry, J., West, M. A., Moore, E. E., Minei, J. P., Bankey, P. E., Nathens, A. B., Cuenca, A. G., Efron, P. A., Hennessy, L., Xiao, W., Mindrinos, M. N., McDonald-Smith, G. P., Mason, P. H., Billiar, T. R., Schoenfeld, D. A., Warren, H. S., Cobb, J. P., Moldawer, L. L., Davis, R. W., Maier, R. V., Tompkins, R. G. 2012; 255 (5): 993-999


    To determine and compare outcomes with accepted benchmarks in trauma care at 7 academic level I trauma centers in which patients were treated on the basis of a series of standard operating procedures (SOPs).Injury remains the leading cause of death for those younger than 45 years. This study describes the baseline patient characteristics and well-defined outcomes of persons hospitalized in the United States for severe blunt trauma.We followed 1637 trauma patients from 2003 to 2009 up to 28 hospital days using SOPs developed at the onset of the study. An extensive database on patient and injury characteristics, clinical treatment, and outcomes was created. These data were compared with existing trauma benchmarks.The study patients were critically injured and were in shock. SOP compliance improved 10% to 40% during the study period. Multiple organ failure and mortality rates were 34.8% and 16.7%, respectively. Time to recovery, defined as the time until the patient was free of organ failure for at least 2 consecutive days, was developed as a new outcome measure. There was a reduction in mortality rate in the cohort during the study that cannot be explained by changes in the patient population.This study provides the current benchmark and the overall positive effect of implementing SOPs for severely injured patients. Over the course of the study, there were improvements in morbidity and mortality rates and increasing compliance with SOPs. Mortality was surprisingly low, given the degree of injury, and improved over the duration of the study, which correlated with improved SOP compliance.

    View details for DOI 10.1097/SLA.0b013e31824f1ebc

    View details for Web of Science ID 000302939500038

    View details for PubMedID 22470077

  • A Rapid, Cost-Effective Method of Assembly and Purification of Synthetic DNA Probes > 100 bp PLOS ONE Jensen, M. A., Jauregui, L., Davis, R. W. 2012; 7 (4)


    Here we introduce a rapid, cost-effective method of generating molecular DNA probes in just under 15 minutes without the need for expensive, time-consuming gel-extraction steps. As an example, we enzymatically concatenated six variable strands (50 bp) with a common strand sequence (51 bp) in a single pool using Fast-Link DNA ligase to produce 101 bp targets (10 min). Unincorporated species were then filtered out by passing the crude reaction through a size-exclusion column (<5 min). We then compared full-length product yield of crude and purified samples using HPLC analysis; the results of which clearly show our method yields three-quarters that of the crude sample (50% higher than by gel-extraction). And while we substantially reduced the amount of unligated product with our filtration process, higher purity and yield, with an increase in number of stands per reaction (>12) could be achieved with further optimization. Moreover, for large-scale assays, we envision this method to be fully automated with the use of robotics such as the Biomek FX; here, potentially thousands of samples could be pooled, ligated and purified in either a 96, 384 or 1536-well platform in just minutes.

    View details for DOI 10.1371/journal.pone.0034373

    View details for Web of Science ID 000305012700030

    View details for PubMedID 22493688

    View details for PubMedCentralID PMC3321010

  • Molecular probe technology detects bacteria without culture BMC MICROBIOLOGY Hyman, R. W., St Onge, R. P., Kim, H., Tamaresis, J. S., Miranda, M., Aparicio, A. M., Fukushima, M., Pourmand, N., Giudice, L. C., Davis, R. W. 2012; 12


    Our ultimate goal is to detect the entire human microbiome, in health and in disease, in a single reaction tube, and employing only commercially available reagents. To that end, we adapted molecular inversion probes to detect bacteria using solely a massively multiplex molecular technology. This molecular probe technology does not require growth of the bacteria in culture. Rather, the molecular probe technology requires only a sequence of forty sequential bases unique to the genome of the bacterium of interest. In this communication, we report the first results of employing our molecular probes to detect bacteria in clinical samples.While the assay on Affymetrix GenFlex Tag16K arrays allows the multiplexing of the detection of the bacteria in each clinical sample, one Affymetrix GenFlex Tag16K array must be used for each clinical sample. To multiplex the clinical samples, we introduce a second, independent assay for the molecular probes employing Sequencing by Oligonucleotide Ligation and Detection. By adding one unique oligonucleotide barcode for each clinical sample, we combine the samples after processing, but before sequencing, and sequence them together.Overall, we have employed 192 molecular probes representing 40 bacteria to detect the bacteria in twenty-one vaginal swabs as assessed by the Affymetrix GenFlex Tag16K assay and fourteen of those by the Sequencing by Oligonucleotide Ligation and Detection assay. The correlations among the assays were excellent.

    View details for DOI 10.1186/1471-2180-12-29

    View details for Web of Science ID 000302353500001

    View details for PubMedID 22404909

    View details for PubMedCentralID PMC3316761

  • Use of Negative Dielectrophoresis for Selective Elution of Protein-Bound Particles ANALYTICAL CHEMISTRY Javanmard, M., Emaminejad, S., Dutton, R. W., Davis, R. W. 2012; 84 (3): 1432-1438


    In this paper with the aid of negative dielectrophoresis force in conjunction with shear force and at an optimal sodium hydroxide concentration we demonstrated a switchlike functionality to elute specifically bound beads from the surface. At an optimal flow rate and sodium hydroxide concentration, negative dielectrophoresis turned on results in bead detachment, whereas when negative dielectrophoresis is off, the beads remain attached. This platform offers the potential for performing a bead-based multiplexed assay where in a single channel various regions are immobilized with a different antibody, each targeting a different antigen. To develop the proof of concept and to demonstrate the switchlike functionality in eluting specifically bound beads from the surface we looked at two different protein interactions. We chose interactions that were in the same order of magnitude in strength as typical antibody-antigen interactions. The first was protein G-IgG interaction, and the second was the interaction between anti-IgG and IgG.

    View details for DOI 10.1021/ac202508u

    View details for Web of Science ID 000299863200036

    View details for PubMedID 22242790

  • The dynamics of the vaginal microbiome during infertility therapy with in vitro fertilization-embryo transfer JOURNAL OF ASSISTED REPRODUCTION AND GENETICS Hyman, R. W., Herndon, C. N., Jiang, H., Palm, C., Fukushima, M., Bernstein, D., Vo, K. C., Zelenko, Z., Davis, R. W., Giudice, L. C. 2012; 29 (2): 105-115


    To determine the vaginal microbiome in women undergoing IVF-ET and investigate correlations with clinical outcomes.Thirty patients had blood drawn for estradiol (E(2)) and progesterone (P(4)) at four time points during the IVF-ET cycle and at 4-6 weeks of gestation, if pregnant. Vaginal swabs were obtained in different hormonal milieu, and the vaginal microbiome determined by deep sequencing of the 16S ribosomal RNA gene.The vaginal microbiome underwent a transition during therapy in some but not all patients. Novel bacteria were found in 33% of women tested during the treatment cycle, but not at 6-8 weeks of gestation. Diversity of species varied across different hormonal milieu, and on the day of embryo transfer correlated with outcome (live birth/no live birth). The species diversity index distinguished women who had a live birth from those who did not.This metagenomics approach has enabled discovery of novel, previously unidentified bacterial species in the human vagina in different hormonal milieu and supports a shift in the vaginal microbiome during IVF-ET therapy using standard protocols. Furthermore, the data suggest that the vaginal microbiome on the day of embryo transfer affects pregnancy outcome.

    View details for DOI 10.1007/s10815-011-9694-6

    View details for Web of Science ID 000300063400002

    View details for PubMedID 22222853

  • Knowledge-Based Reconstruction of mRNA Transcripts with Short Sequencing Reads for Transcriptome Research PLOS ONE Seok, J., Xu, W., Jiang, H., Davis, R. W., Xiao, W. 2012; 7 (2)


    While most transcriptome analyses in high-throughput clinical studies focus on gene level expression, the existence of alternative isoforms of gene transcripts is a major source of the diversity in the biological functionalities of the human genome. It is, therefore, essential to annotate isoforms of gene transcripts for genome-wide transcriptome studies. Recently developed mRNA sequencing technology presents an unprecedented opportunity to discover new forms of transcripts, and at the same time brings bioinformatic challenges due to its short read length and incomplete coverage for the transcripts. In this work, we proposed a computational approach to reconstruct new mRNA transcripts from short sequencing reads with reference information of known transcripts in existing databases. The prior knowledge helped to define exon boundaries and fill in the transcript regions not covered by sequencing data. This approach was demonstrated using a deep sequencing data set of human muscle tissue with transcript annotations in RefSeq as prior knowledge. We identified 2,973 junctions, 7,471 exons, and 7,571 transcripts not previously annotated in RefSeq. 73% of these new transcripts found supports from UCSC Known Genes, Ensembl or EST transcript annotations. In addition, the reconstructed transcripts were much longer than those from de novo approaches that assume no prior knowledge. These previously un-annotated transcripts can be integrated with known transcript annotations to improve both the design of microarrays and the follow-up analyses of isoform expression. The overall results demonstrated that incorporating transcript annotations from genomic databases significantly helps the reconstruction of novel transcripts from short sequencing reads for transcriptome research.

    View details for DOI 10.1371/journal.pone.0031440

    View details for Web of Science ID 000301977500037

    View details for PubMedID 22312447

  • Microfluidic diagnostic tool for the developing world: contactless impedance flow cytometry LAB ON A CHIP Emaminejad, S., Javanmard, M., Dutton, R. W., Davis, R. W. 2012; 12 (21): 4499-4507


    In this work, we demonstrate a novel and cost-effective approach to implement a disposable microfluidic contactless impedance cytometer. Conventional methods for single cell impedance cytometry use microfabricated electrodes in direct contact with the buffer to measure changes of its electrical impedance when cells pass through the applied electric field. However, this approach requires expensive microfabrication of electrodes, and also, the fabricated electrodes cannot be reused without thorough and time-consuming cleaning process. Here, we introduce a novel approach to allow for single cell impedance cytometry using electrodes that can be reused, without the need for microfabrication of the electrodes. This disposable device can be potentially inserted onto a printed circuit board (PCB) which has a non-disposable, yet inexpensive, electronic reading apparatus. This significantly reduces the manufacturing costs, making it suitable for low resource settings, such as point-of-care testing in the developing countries.

    View details for DOI 10.1039/c2lc40759k

    View details for Web of Science ID 000310916100038

    View details for PubMedID 22971813

  • Electrochemical quantum tunneling for electronic detection and characterization of biological toxins Conference on Micro- and Nanotechnology Sensors, Systems, and Applications IV Gupta, C., Walker, R. M., Gharpuray, R., Shulaker, M. M., Zhang, Z., Javanmard, M., Davis, R. W., Murmann, B., Howe, R. T. SPIE-INT SOC OPTICAL ENGINEERING. 2012

    View details for DOI 10.1117/12.920692

    View details for Web of Science ID 000306560400002

  • The Human OligoGenome Resource: a database of oligonucleotide capture probes for resequencing target regions across the human genome NUCLEIC ACIDS RESEARCH Newburger, D. E., Natsoulis, G., Grimes, S., Bell, J. M., Davis, R. W., Batzoglou, S., Ji, H. P. 2012; 40 (D1): D1137-D1143

    View details for DOI 10.1093/nar/gkr973

    View details for Web of Science ID 000298601300170

  • The Human OligoGenome Resource: a database of oligonucleotide capture probes for resequencing target regions across the human genome. Nucleic acids research Newburger, D. E., Natsoulis, G., Grimes, S., Bell, J. M., Davis, R. W., Batzoglou, S., Ji, H. P. 2012; 40 (Database issue): D1137-43


    Recent exponential growth in the throughput of next-generation DNA sequencing platforms has dramatically spurred the use of accessible and scalable targeted resequencing approaches. This includes candidate region diagnostic resequencing and novel variant validation from whole genome or exome sequencing analysis. We have previously demonstrated that selective genomic circularization is a robust in-solution approach for capturing and resequencing thousands of target human genome loci such as exons and regulatory sequences. To facilitate the design and production of customized capture assays for any given region in the human genome, we developed the Human OligoGenome Resource ( This online database contains over 21 million capture oligonucleotide sequences. It enables one to create customized and highly multiplexed resequencing assays of target regions across the human genome and is not restricted to coding regions. In total, this resource provides 92.1% in silico coverage of the human genome. The online server allows researchers to download a complete repository of oligonucleotide probes and design customized capture assays to target multiple regions throughout the human genome. The website has query tools for selecting and evaluating capture oligonucleotides from specified genomic regions.

    View details for DOI 10.1093/nar/gkr973

    View details for PubMedID 22102592

  • High-throughput VDJ sequencing for quantification of minimal residual disease in chronic lymphocytic leukemia and immune reconstitution assessment PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Logan, A. C., Gao, H., Wang, C., Sahaf, B., Jones, C. D., Marshall, E. L., Buno, I., Armstrong, R., Fire, A. Z., Weinberg, K. I., Mindrinos, M., Zehnder, J. L., Boyd, S. D., Xiao, W., Davis, R. W., Miklos, D. B. 2011; 108 (52): 21194-21199


    The primary cause of poor outcome following allogeneic hematopoietic cell transplantation (HCT) for chronic lymphocytic leukemia (CLL) is disease recurrence. Detection of increasing minimal residual disease (MRD) following HCT may permit early intervention to prevent clinical relapse; however, MRD quantification remains an uncommon diagnostic test because of logistical and financial barriers to widespread use. Here we describe a method for quantifying CLL MRD using widely available consensus primers for amplification of all Ig heavy chain (IGH) genes in a mixture of peripheral blood mononuclear cells, followed by high-throughput sequencing (HTS) for disease-specific IGH sequence quantification. To achieve accurate MRD quantification, we developed a systematic bioinformatic methodology to aggregate cancer clone sequence variants arising from systematic and random artifacts occurring during IGH-HTS. We then compared the sensitivity of IGH-HTS, flow cytometry, and allele-specific oligonucleotide PCR for MRD quantification in 28 samples collected from 6 CLL patients following allogeneic HCT. Using amplimer libraries generated with consensus primers from patient blood samples, we demonstrate the sensitivity of IGH-HTS with 454 pyrosequencing to be 10(-5), with a high correlation between quantification by allele-specific oligonucleotide PCR and IGH-HTS (r = 0.85). From the same dataset used to quantify MRD, IGH-HTS also allowed us to profile IGH repertoire reconstitution after HCT-information not provided by the other MRD methods. IGH-HTS using consensus primers will broaden the availability of MRD quantification in CLL and other B cell malignancies, and this approach has potential for quantitative evaluation of immune diversification following transplant and nontransplant therapies.

    View details for DOI 10.1073/pnas.1118357109

    View details for Web of Science ID 000298479900065

    View details for PubMedID 22160699

  • A genomic storm in critically injured humans JOURNAL OF EXPERIMENTAL MEDICINE Xiao, W., Mindrinos, M. N., Seok, J., Cuschieri, J., Cuenca, A. G., Gao, H., Hayden, D. L., Hennessy, L., Moore, E. E., Minei, J. P., Bankey, P. E., Johnson, J. L., Sperry, J., Nathens, A. B., Billiar, T. R., West, M. A., Brownstein, B. H., Mason, P. H., Baker, H. V., Finnerty, C. C., Jeschke, M. G., Lopez, M. C., Klein, M. B., Gamelli, R. L., Gibran, N. S., Arnoldo, B., Xu, W., Zhang, Y., Calvano, S. E., McDonald-Smith, G. P., Schoenfeld, D. A., Storey, J. D., Cobb, J. P., Warren, H. S., Moldawer, L. L., Herndon, D. N., Lowry, S. F., Maier, R. V., Davis, R. W., Tompkins, R. G. 2011; 208 (13): 2581-2590


    Human survival from injury requires an appropriate inflammatory and immune response. We describe the circulating leukocyte transcriptome after severe trauma and burn injury, as well as in healthy subjects receiving low-dose bacterial endotoxin, and show that these severe stresses produce a global reprioritization affecting >80% of the cellular functions and pathways, a truly unexpected "genomic storm." In severe blunt trauma, the early leukocyte genomic response is consistent with simultaneously increased expression of genes involved in the systemic inflammatory, innate immune, and compensatory antiinflammatory responses, as well as in the suppression of genes involved in adaptive immunity. Furthermore, complications like nosocomial infections and organ failure are not associated with any genomic evidence of a second hit and differ only in the magnitude and duration of this genomic reprioritization. The similarities in gene expression patterns between different injuries reveal an apparently fundamental human response to severe inflammatory stress, with genomic signatures that are surprisingly far more common than different. Based on these transcriptional data, we propose a new paradigm for the human immunological response to severe injury.

    View details for DOI 10.1084/jem.20111354

    View details for Web of Science ID 000299045000005

    View details for PubMedID 22110166

    View details for PubMedCentralID PMC3244029

  • Evidence-Based Annotation of Gene Function in Shewanella oneidensis MR-1 Using Genome-Wide Fitness Profiling across 121 Conditions PLOS GENETICS Deutschbauer, A., Price, M. N., Wetmore, K. M., Shao, W., Baumohl, J. K., Xu, Z., Michelle Nguyen, M., Tamse, R., Davis, R. W., Arkin, A. P. 2011; 7 (11)


    Most genes in bacteria are experimentally uncharacterized and cannot be annotated with a specific function. Given the great diversity of bacteria and the ease of genome sequencing, high-throughput approaches to identify gene function experimentally are needed. Here, we use pools of tagged transposon mutants in the metal-reducing bacterium Shewanella oneidensis MR-1 to probe the mutant fitness of 3,355 genes in 121 diverse conditions including different growth substrates, alternative electron acceptors, stresses, and motility. We find that 2,350 genes have a pattern of fitness that is significantly different from random and 1,230 of these genes (37% of our total assayed genes) have enough signal to show strong biological correlations. We find that genes in all functional categories have phenotypes, including hundreds of hypotheticals, and that potentially redundant genes (over 50% amino acid identity to another gene in the genome) are also likely to have distinct phenotypes. Using fitness patterns, we were able to propose specific molecular functions for 40 genes or operons that lacked specific annotations or had incomplete annotations. In one example, we demonstrate that the previously hypothetical gene SO_3749 encodes a functional acetylornithine deacetylase, thus filling a missing step in S. oneidensis metabolism. Additionally, we demonstrate that the orphan histidine kinase SO_2742 and orphan response regulator SO_2648 form a signal transduction pathway that activates expression of acetyl-CoA synthase and is required for S. oneidensis to grow on acetate as a carbon source. Lastly, we demonstrate that gene expression and mutant fitness are poorly correlated and that mutant fitness generates more confident predictions of gene function than does gene expression. The approach described here can be applied generally to create large-scale gene-phenotype maps for evidence-based annotation of gene function in prokaryotes.

    View details for DOI 10.1371/journal.pgen.1002385

    View details for Web of Science ID 000297264500035

    View details for PubMedID 22125499

    View details for PubMedCentralID PMC3219624

  • A microfluidic platform for electrical detection of DNA hybridization 15th Transducers Conference Javanmard, M., Davis, R. W. ELSEVIER SCIENCE SA. 2011: 22–27
  • Distinctive Responsiveness to Stromal Signaling Accompanies Histologic Grade Programming of Cancer Cells PLOS ONE Luciani, M. g., Seok, J., Sayeed, A., Champion, S., Goodson, W. H., Jeffrey, S. S., Xiao, W., Mindrinos, M., Davis, R. W., Dairkee, S. H. 2011; 6 (5)


    Whether stromal components facilitate growth, invasion, and dissemination of cancer cells or suppress neoplastic lesions from further malignant progression is a continuing conundrum in tumor biology. Conceptualizing a dynamic picture of tumorigenesis is complicated by inter-individual heterogeneity. In the post genomic era, unraveling such complexity remains a challenge for the cancer biologist. Towards establishing a functional association between cellular crosstalk and differential cancer aggressiveness, we identified a signature of malignant breast epithelial response to stromal signaling. Proximity to fibroblasts resulted in gene transcript alterations of >2-fold for 107 probes, collectively designated as Fibroblast Triggered Gene Expression in Tumor (FTExT). The hazard ratio predicted by the FTExT classifier for distant relapse in patients with intermediate and high grade breast tumors was significant compared to routine clinical variables (dataset 1, n = 258, HR--2.11, 95% CI 1.17-3.80, p-value 0.01; dataset 2, n = 171, HR--3.07, 95% CI 1.21-7.83, p-value 0.01). Biofunctions represented by FTExT included inflammatory signaling, free radical scavenging, cell death, and cell proliferation. Unlike genes of the 'proliferation cluster', which are overexpressed in aggressive primary tumors, FTExT genes were uniquely repressed in such cases. As proof of concept for our correlative findings, which link stromal-epithelial crosstalk and tumor behavior, we show a distinctive differential in stromal impact on prognosis-defining functional endpoints of cell cycle progression, and resistance to therapy-induced growth arrest and apoptosis in low vs. high grade cancer cells. Our experimental data thus reveal aspects of 'paracrine cooperativity' that are exclusively contingent upon the histopathologically defined grade of interacting tumor epithelium, and demonstrate that epithelial responsiveness to the tumor microenvironment is a deterministic factor underlying clinical outcome. In this light, early attenuation of epithelial-stromal crosstalk could improve the management of cases prone to be clinically challenging.

    View details for DOI 10.1371/journal.pone.0020016

    View details for Web of Science ID 000290771200040

    View details for PubMedID 21625507

    View details for PubMedCentralID PMC3098270

  • High-quality DNA sequence capture of 524 disease candidate genes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Shen, P., Wang, W., Krishnakumar, S., Palm, C., Chi, A., Enns, G. M., Davis, R. W., Speed, T. P., Mindrinos, M. N., Scharfe, C. 2011; 108 (16): 6549-6554


    The accurate and complete selection of candidate genomic regions from a DNA sample before sequencing is critical in molecular diagnostics. Several recently developed technologies await substantial improvements in performance, cost, and multiplex sample processing. Here we present the utility of long padlock probes (LPPs) for targeted exon capture followed by array-based sequencing. We found that on average 92% of 5,471 exons from 524 nuclear-encoded mitochondrial genes were successfully amplified from genomic DNA from 63 individuals. Only 144 exons did not amplify in any sample due to high GC content. One LPP was sufficient to capture sequences from <100-500 bp in length and only a single-tube capture reaction and one microarray was required per sample. Our approach was highly reproducible and quick (<8 h) and detected DNA variants at high accuracy (false discovery rate 1%, false negative rate 3%) on the basis of known sample SNPs and Sanger sequence verification. In a patient with clinical and biochemical presentation of ornithine transcarbamylase (OTC) deficiency, we identified copy-number differences in the OTC gene at exon-level resolution. This shows the ability of LPPs to accurately preserve a sample's genome information and provides a cost-effective strategy to identify both single nucleotide changes and structural variants in targeted resequencing.

    View details for DOI 10.1073/pnas.1018981108

    View details for Web of Science ID 000289680400046

    View details for PubMedID 21467225

    View details for PubMedCentralID PMC3080966

  • Human transcriptome array for high-throughput clinical studies PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Xu, W., Seok, J., Mindrinos, M. N., Schweitzer, A. C., Jiang, H., Wilhelmy, J., Clark, T. A., Kapur, K., Xing, Y., Faham, M., Storey, J. D., Moldawer, L. L., Maier, R. V., Tompkins, R. G., Wong, W. H., Davis, R. W., Xiao, W. 2011; 108 (9): 3707-3712


    A 6.9 million-feature oligonucleotide array of the human transcriptome [Glue Grant human transcriptome (GG-H array)] has been developed for high-throughput and cost-effective analyses in clinical studies. This array allows comprehensive examination of gene expression and genome-wide identification of alternative splicing as well as detection of coding SNPs and noncoding transcripts. The performance of the array was examined and compared with mRNA sequencing (RNA-Seq) results over multiple independent replicates of liver and muscle samples. Compared with RNA-Seq of 46 million uniquely mappable reads per replicate, the GG-H array is highly reproducible in estimating gene and exon abundance. Although both platforms detect similar expression changes at the gene level, the GG-H array is more sensitive at the exon level. Deeper sequencing is required to adequately cover low-abundance transcripts. The array has been implemented in a multicenter clinical program and has generated high-quality, reproducible data. Considering the clinical trial requirements of cost, sample availability, and throughput, the GG-H array has a wide range of applications. An emerging approach for large-scale clinical genomic studies is to first use RNA-Seq to the sufficient depth for the discovery of transcriptome elements relevant to the disease process followed by high-throughput and reliable screening of these elements on thousands of patient samples using custom-designed arrays.

    View details for DOI 10.1073/pnas.1019753108

    View details for Web of Science ID 000287844400051

    View details for PubMedID 21317363

  • Execution of the meiotic noncoding RNA expression program and the onset of gametogenesis in yeast require the conserved exosome subunit Rrp6 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lardenois, A., Liu, Y., Walther, T., Chalmel, F., Evrard, B., Granovskaia, M., Chu, A., Davis, R. W., Steinmetz, L. M., Primig, M. 2011; 108 (3): 1058-1063


    Budding yeast noncoding RNAs (ncRNAs) are pervasively transcribed during mitosis, and some regulate mitotic protein-coding genes. However, little is known about ncRNA expression during meiotic development. Using high-resolution profiling we identified an extensive meiotic ncRNA expression program interlaced with the protein-coding transcriptome via sense/antisense transcript pairs, bidirectional promoters, and ncRNAs that overlap the regulatory regions of genes. Meiotic unannotated transcripts (MUTs) are mitotic targets of the conserved exosome component Rrp6, which itself is degraded after the onset of meiosis when MUTs and other ncRNAs accumulate in successive waves. Diploid cells lacking Rrp6 fail to initiate premeiotic DNA replication normally and cannot undergo efficient meiotic development. The present study demonstrates a unique function for budding yeast Rrp6 in degrading distinct classes of meiotically induced ncRNAs during vegetative growth and the onset of meiosis and thus points to a critical role of differential ncRNA expression in the execution of a conserved developmental program.

    View details for DOI 10.1073/pnas.1016459108

    View details for Web of Science ID 000286310300034

    View details for PubMedID 21149693

    View details for PubMedCentralID PMC3024698

  • The automated cell: compound and environment screening system (ACCESS) for chemogenomic screening. Methods in molecular biology (Clifton, N.J.) Proctor, M., Urbanus, M. L., Fung, E. L., Jaramillo, D. F., Davis, R. W., Nislow, C., Giaever, G. 2011; 759: 239-269


    The automated cell, compound and environment screening system (ACCESS) was developed as an automated platform for chemogenomic research. In the yeast Saccharomyces cerevisiae, a number of genomic screens rely on the modulation of gene dose to determine the mode of action of bioactive compounds or the effects of environmental/compound perturbations. These and other phenotypic experiments have been shown to benefit from high-resolution growth curves and a highly automated controlled environment system that enables a wide range of multi-well assays that can be run over many days without any manual intervention. Furthermore, precise control of drug dosing, timing of drug exposure, and precise timing of cell harvesting at specific generation times are important for optimal results. Some of these benefits include the ability to derive fine distinctions between growth rates of mutant strains (1) and the discovery of novel compounds and drug targets (2). The automation has also enabled large-scale screening projects with over 100,000 unique compounds screened to date including a thousand genome-wide screens (3). The ACCESS system also has a diverse set of software tools to enable users to set up, run, annotate, and evaluate complex screens with minimal training.

    View details for DOI 10.1007/978-1-61779-173-4_15

    View details for PubMedID 21863492

  • Experimental Demonstration and Analysis of DNA Passage in Nanopore-based Nanofluidic Transistors IEEE International Electron Devices Meeting (IEDM) Paik, K., Liu, Y., Tabard-Cossa, V., Huber, D. E., Provine, J., Howe, R. T., Davis, R. W., Dutton, R. W. IEEE. 2011
  • Identification of rare DNA variants in mitochondrial disorders with improved array-based sequencing NUCLEIC ACIDS RESEARCH Wang, W., Shen, P., Thiyagarajan, S., Lin, S., Palm, C., Horvath, R., Klopstock, T., Cutler, D., Pique, L., Schrijver, I., Davis, R. W., Mindrinos, M., Speed, T. P., Scharfe, C. 2011; 39 (1): 44-58


    A common goal in the discovery of rare functional DNA variants via medical resequencing is to incur a relatively lower proportion of false positive base-calls. We developed a novel statistical method for resequencing arrays (SRMA, sequence robust multi-array analysis) to increase the accuracy of detecting rare variants and reduce the costs in subsequent sequence verifications required in medical applications. SRMA includes single and multi-array analysis and accounts for technical variables as well as the possibility of both low- and high-frequency genomic variation. The confidence of each base-call was ranked using two quality measures. In comparison to Sanger capillary sequencing, we achieved a false discovery rate of 2% (false positive rate 1.2 × 10⁻⁵, false negative rate 5%), which is similar to automated second-generation sequencing technologies. Applied to the analysis of 39 nuclear candidate genes in disorders of mitochondrial DNA (mtDNA) maintenance, we confirmed mutations in the DNA polymerase gamma POLG in positive control cases, and identified novel rare variants in previously undiagnosed cases in the mitochondrial topoisomerase TOP1MT, the mismatch repair enzyme MUTYH, and the apurinic-apyrimidinic endonuclease APEX2. Some patients carried rare heterozygous variants in several functionally interacting genes, which could indicate synergistic genetic effects in these clinically similar disorders.

    View details for DOI 10.1093/nar/gkq750

    View details for Web of Science ID 000286008500009

    View details for PubMedID 20843780

    View details for PubMedCentralID PMC3017602

  • A molecular inversion probe assay for detecting alternative splicing BMC GENOMICS Lin, S., Wang, W., Palm, C., Davis, R. W., Juneau, K. 2010; 11


    A sensitive, high-throughput method for monitoring pre-mRNA splicing on a genomic scale is needed to understand the spectrum of alternatively spliced mRNA in human cells.We adapted Molecular Inversion Probes (MIPs), a padlock-probe based technology, for the multiplexed capture and quantitation of individual splice events in human tissues. Individual MIP capture probes can be quantified using either DNA microarrays or high-throughput sequencing, which permits independent assessment of each spliced junction. Using our methodology we successfully identified 100% of our positive controls and showed that there is a strong correlation between the data from our alternative splicing MIP (asMIP) assay and quantitative PCR.The asMIP assay provides a sensitive, accurate and multiplexed means for measuring pre-mRNA splicing. Fully optimized, we estimate that the assay could accommodate a throughput of greater than 20,000 splice junctions in a single reaction. This would represent a significant improvement over existing technologies.

    View details for DOI 10.1186/1471-2164-11-712

    View details for Web of Science ID 000286242300001

    View details for PubMedID 21167051

    View details for PubMedCentralID PMC3022918

  • Gas-phase cleavage and dephosphorylation of universal linker-bound oligodeoxynucleotides. Nucleosides, nucleotides & nucleic acids Jensen, M. A., Anderson, K. M., Davis, R. W. 2010; 29 (11): 867-878


    While base-specific support is commonly used for single-column oligodeoxynucleotide synthesis, the universal linker is critical for high-throughput synthesis of potentially thousands of samples in a single run. Here, we report conditions for cleavage and complete dephosphorylation of two commercial universal linkers, UnySupport and UnyLinker, processed in the gas phase (NH(3)) using our custom device. First, we compared the average yield of T10mers over time (15, 30, 60, 120, and 240 minutes, 40 psi, 80°C and 90°C). For samples processed with water added prior to incubation, we discovered a substantial increase in yield compared to those left dry (up to 55%). This was also the case for samples subjected to increases in chamber pressure (10, 20, 30 and 40 psi, 120 minutes, 80°C and 90°C). Next, we compared the effects of increased temperature, pressure and incubation times on the rates of dephosphorylation. We found the optimum conditions to be either 10 psi, 120 minutes at 80°C or 60 minutes at 90°C; in both cases, water added to columns prior to incubation had a substantial effect on rate of reaction as well as overall yield compared with those left dry. Finally, performance between the two linkers was similar enough to conclude each fulfills the desired requirements for mainstream, high-throughput oligodeoxynucleotide cleavage/deprotection and dephosphorylation in the gas phase.

    View details for DOI 10.1080/15257770.2010.534757

    View details for PubMedID 21128173

  • Microfluidic force spectroscopy for characterization of biomolecular interactions with piconewton resolution APPLIED PHYSICS LETTERS Javanmard, M., Babrzadeh, F., Davis, R. W. 2010; 97 (17)

    View details for DOI 10.1063/1.3491547

    View details for Web of Science ID 000284233600072

  • Gene Annotation and Drug Target Discovery in Candida albicans with a Tagged Transposon Mutant Collection PLOS PATHOGENS Oh, J., Fung, E., Schlecht, U., Davis, R. W., Giaever, G., St Onge, R. P., Deutschbauer, A., Nislow, C. 2010; 6 (10)


    Candida albicans is the most common human fungal pathogen, causing infections that can be lethal in immunocompromised patients. Although Saccharomyces cerevisiae has been used as a model for C. albicans, it lacks C. albicans' diverse morphogenic forms and is primarily non-pathogenic. Comprehensive genetic analyses that have been instrumental for determining gene function in S. cerevisiae are hampered in C. albicans, due in part to limited resources to systematically assay phenotypes of loss-of-function alleles. Here, we constructed and screened a library of 3633 tagged heterozygous transposon disruption mutants, using them in a competitive growth assay to examine nutrient- and drug-dependent haploinsufficiency. We identified 269 genes that were haploinsufficient in four growth conditions, the majority of which were condition-specific. These screens identified two new genes necessary for filamentous growth as well as ten genes that function in essential processes. We also screened 57 chemically diverse compounds that more potently inhibited growth of C. albicans versus S. cerevisiae. For four of these compounds, we examined the genetic basis of this differential inhibition. Notably, Sec7p was identified as the target of brefeldin A in C. albicans screens, while S. cerevisiae screens with this compound failed to identify this target. We also uncovered a new C. albicans-specific target, Tfp1p, for the synthetic compound 0136-0228. These results highlight the value of haploinsufficiency screens directly in this pathogen for gene annotation and drug target identification.

    View details for DOI 10.1371/journal.ppat.1001140

    View details for Web of Science ID 000283652200016

    View details for PubMedID 20949076

  • Plasma Proteome Response to Severe Burn Injury Revealed by O-18-Labeled "Universal" Reference-Based Quantitative Proteomics JOURNAL OF PROTEOME RESEARCH Qian, W., Petritis, B. O., Kaushal, A., Finnerty, C. C., Jeschke, M. G., Monroe, M. E., Moore, R. J., Schepmoes, A. A., Xiao, W., Moldawer, L. L., Davis, R. W., Tompkins, R. G., Herndon, D. N., Camp, D. G., Smith, R. D. 2010; 9 (9): 4779-4789


    A burn injury represents one of the most severe forms of human trauma and is responsible for significant mortality worldwide. Here, we present the first quantitative proteomics investigation of the blood plasma proteome response to severe burn injury by comparing the plasma protein concentrations of 10 healthy control subjects with those of 15 severe burn patients at two time-points following the injury. The overall analytical strategy for this work integrated immunoaffinity depletion of the 12 most abundant plasma proteins with cysteinyl-peptide enrichment-based fractionation prior to LC-MS analyses of individual patient samples. Incorporation of an 18O-labeled "universal" reference among the sample sets enabled precise relative quantification across samples. In total, 313 plasma proteins confidently identified with two or more unique peptides were quantified. Following statistical analysis, 110 proteins exhibited significant abundance changes in response to the burn injury. The observed changes in protein concentrations suggest significant inflammatory and hypermetabolic response to the injury, which is supported by the fact that many of the identified proteins are associated with acute phase response signaling, the complement system, and coagulation system pathways. The regulation of approximately 35 proteins observed in this study is in agreement with previous results reported for inflammatory or burn response, but approximately 50 potentially novel proteins previously not known to be associated with burn response or inflammation are also found. Elucidating proteins involved in the response to severe burn injury may reveal novel targets for therapeutic interventions as well as potential predictive biomarkers for patient outcomes such as multiple organ failure.

    View details for DOI 10.1021/pr1005026

    View details for Web of Science ID 000281443700041

    View details for PubMedID 20698492

  • Clinical microfluidics for neutrophil genomics and proteomics NATURE MEDICINE Kotz, K. T., Xiao, W., Miller-Graziano, C., Qian, W., Russom, A., Warner, E. A., Moldawer, L. L., De, A., Bankey, P. E., Petritis, B. O., Camp, D. G., Rosenbach, A. E., Goverman, J., Fagan, S. P., Brownstein, B. H., Irimia, D., Xu, W., Wilhelmy, J., Mindrinos, M. N., Smith, R. D., Davis, R. W., Tompkins, R. G., Toner, M. 2010; 16 (9): 1042-U142


    Neutrophils have key roles in modulating the immune response. We present a robust methodology for rapidly isolating neutrophils directly from whole blood with 'on-chip' processing for mRNA and protein isolation for genomics and proteomics. We validate this device with an ex vivo stimulation experiment and by comparison with standard bulk isolation methodologies. Last, we implement this tool as part of a near-patient blood processing system within a multi-center clinical study of the immune response to severe trauma and burn injury. The preliminary results from a small cohort of subjects in our study and healthy controls show a unique time-dependent gene expression pattern clearly demonstrating the ability of this tool to discriminate temporal transcriptional events of neutrophils within a clinical setting.

    View details for DOI 10.1038/nm.2205

    View details for Web of Science ID 000281622900035

    View details for PubMedID 20802500

    View details for PubMedCentralID PMC3136804

  • A universal TagModule collection for parallel genetic analysis of microorganisms NUCLEIC ACIDS RESEARCH Oh, J., Fung, E., Price, M. N., Dehal, P. S., Davis, R. W., Giaever, G., Nislow, C., Arkin, A. P., Deutschbauer, A. 2010; 38 (14)


    Systems-level analyses of non-model microorganisms are limited by the existence of numerous uncharacterized genes and a corresponding over-reliance on automated computational annotations. One solution to this challenge is to disrupt gene function using DNA tag technology, which has been highly successful in parallelizing reverse genetics in Saccharomyces cerevisiae and has led to discoveries in gene function, genetic interactions and drug mechanism of action. To extend the yeast DNA tag methodology to a wide variety of microorganisms and applications, we have created a universal, sequence-verified TagModule collection. A hallmark of the 4280 TagModules is that they are cloned into a Gateway entry vector, thus facilitating rapid transfer to any compatible genetic system. Here, we describe the application of the TagModules to rapidly generate tagged mutants by transposon mutagenesis in the metal-reducing bacterium Shewanella oneidensis MR-1 and the pathogenic yeast Candida albicans. Our results demonstrate the optimal hybridization properties of the TagModule collection, the flexibility in applying the strategy to diverse microorganisms and the biological insights that can be gained from fitness profiling tagged mutant collections. The publicly available TagModule collection is a platform-independent resource for the functional genomics of a wide range of microbial systems in the post-genome era.

    View details for DOI 10.1093/nar/gkq419

    View details for Web of Science ID 000280922400003

    View details for PubMedID 20494978

  • DMSO and Betaine Greatly Improve Amplification of GC-Rich Constructs in De Novo Synthesis PLOS ONE Jensen, M. A., Fukushima, M., Davis, R. W. 2010; 5 (6)


    In Synthetic Biology, de novo synthesis of GC-rich constructs poses a major challenge because of secondary structure formation and mispriming. While there are many web-based tools for codon optimizing difficult regions, no method currently exists that allows for potentially phenotypically important sequence conservation. Therefore, to overcome these limitations in researching GC-rich genes and their non-coding elements, we explored the use of DMSO and betaine in two conventional methods of assembly and amplification. For this study, we compared the polymerase (PCA) and ligase-based (LCR) methods for construction of two GC-rich gene fragments implicated in tumorigenesis, IGF2R and BRAF. Though we found no benefit in employing either DMSO or betaine during the assembly steps, both additives greatly improved target product specificity and yield during PCR amplification. Of the methods tested, LCR assembly proved far superior to PCA, generating a much more stable template to amplify from. We further report that DMSO and betaine are highly compatible with all other reaction components of gene synthesis and do not require any additional protocol modifications. Furthermore, we believe either additive will allow for the production of a wide variety of GC-rich gene constructs without the need for expensive and time-consuming sample extraction and purification prior to downstream application.

    View details for DOI 10.1371/journal.pone.0011024

    View details for Web of Science ID 000278662900007

    View details for PubMedID 20552011

    View details for PubMedCentralID PMC2883997

  • Analysis of factorial time-course microarrays with application to a clinical study of burn injury. Proceedings of the National Academy of Sciences of the United States of America Zhou, B., Xu, W., Herndon, D., Tompkins, R., Davis, R., Xiao, W., Wong, W. H., Toner, M., Warren, H. S., Schoenfeld, D. A., Rahme, L., McDonald-Smith, G. P., Hayden, D., Mason, P., Fagan, S., Yu, Y., Cobb, J. P., Remick, D. G., Mannick, J. A., Lederer, J. A., Gamelli, R. L., Silver, G. M., West, M. A., Shapiro, M. B., Smith, R., Camp, D. G., Qian, W., Storey, J., Mindrinos, M., Tibshirani, R., Lowry, S., Calvano, S., Chaudry, I., West, M. A., Cohen, M., Moore, E. E., Johnson, J., Moldawer, L. L., Baker, H. V., Efron, P. A., Balis, U. G., Billiar, T. R., Ochoa, J. B., Sperry, J. L., Miller-Graziano, C. L., De, A. K., Bankey, P. E., Finnerty, C. C., Jeschke, M. G., Minei, J. P., Arnoldo, B. D., Hunt, J. L., Horton, J., Cobb, J. P., Brownstein, B., Freeman, B., Maier, R. V., Nathens, A. B., Cuschieri, J., Gibran, N., Klein, M., O'Keefe, G. 2010; 107 (22): 9923-9928


    Time-course microarray experiments are capable of capturing dynamic gene expression profiles. It is important to study how these dynamic profiles depend on the multiple factors that characterize the experimental condition under which the time course is observed. Analytic methods are needed to simultaneously handle the time course and factorial structure in the data. We developed a method to evaluate factor effects by pooling information across the time course while accounting for multiple testing and nonnormality of the microarray data. The method effectively extracts gene-specific response features and models their dependency on the experimental factors. Both longitudinal and cross-sectional time-course data can be handled by our approach. The method was used to analyze the impact of age on the temporal gene response to burn injury in a large-scale clinical study. Our analysis reveals that 21% of the genes responsive to burn are age-specific, among which expressions of mitochondria and immunoglobulin genes are differentially perturbed in pediatric and adult patients by burn injury. These new findings in the body's response to burn injury between children and adults support further investigations of therapeutic options targeting specific age groups. The methodology proposed here has been implemented in R package "TANOVA" and submitted to the Comprehensive R Archive Network at It is also available for download at

    View details for DOI 10.1073/pnas.1002757107

    View details for PubMedID 20479259

    View details for PubMedCentralID PMC2890487

  • Analysis of factorial time-course microarrays with application to a clinical study of burn injury PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zhou, B., Xu, W., Herndon, D., Tompkins, R., Davis, R., Xiao, W., Wong, W. H. 2010; 107 (22): 9923-9928
  • Multiplex Identification of Microbes APPLIED AND ENVIRONMENTAL MICROBIOLOGY Hyman, R. W., St Onge, R. P., Allen, E. A., Miranda, M., Aparicio, A. M., Fukushima, M., Davis, R. W. 2010; 76 (12): 3904-3910


    We have adapted molecular inversion probe technology to identify microbes in a highly multiplexed procedure. This procedure does not require growth of the microbes. Rather, the technology employs DNA homology twice: once for the molecular probe to hybridize to its homologous DNA and again for the 20-mer oligonucleotide barcode on the molecular probe to hybridize to a commercially available molecular barcode array. As proof of concept, we have designed, tested, and employed 192 molecular probes for 40 microbes. While these particular molecular probes are aimed at our interest in the microbes in the human vagina, this molecular probe method could be employed to identify the microbes in any ecological niche.

    View details for DOI 10.1128/AEM.02785-09

    View details for Web of Science ID 000278415300016

    View details for PubMedID 20418427

  • Sensitive giant magnetoresistive-based immunoassay for multiplex mycotoxin detection BIOSENSORS & BIOELECTRONICS Mak, A. C., Osterfeld, S. J., Yu, H., Wang, S. X., Davis, R. W., Jejelowo, O. A., Pourmand, N. 2010; 25 (7): 1635-1639


    Rapid and multiplexed measurement is vital in the detection of food-borne pathogens. While highly specific and sensitive, traditional immunochemical assays such as enzyme-linked immunosorbent assays (ELISAs) often require expensive read-out equipment (e.g. fluorescent labels) and lack the capability of multiplex detection. By combining the superior specificity of immunoassays with the sensitivity and simplicity of magnetic detection, we have developed a novel multiplex magnetic nanotag-based detection platform for mycotoxins that functions on a sub-picomolar concentration level. Unlike fluorescent labels, magnetic nanotags (MNTs) can be detected with inexpensive giant magnetoresistive (GMR) sensors such as spin-valve sensors. In the system presented here, each spin-valve sensor has an active area of 90 microm x 90 microm, arranged in an 8 x 8 array. Sample is added to the antibody-immobilized sensor array prior to the addition of the biotinylated detection antibody. The sensor response is recorded in real time upon the addition of streptavidin-linked MNTs on the chip. Here we demonstrate the simultaneous detection of multiple mycotoxins (aflatoxins B(1), zearalenone and HT-2) and show that a detection limit of 50 pg/mL can be achieved.

    View details for DOI 10.1016/j.bios.2009.11.028

    View details for Web of Science ID 000275978700013

    View details for PubMedID 20047828

  • High throughput sequencing reveals a complex pattern of dynamic interrelationships among human T cell subsets PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wang, C., Sanders, C. M., Yang, Q., Schroeder, H. W., Wang, E., Babrzadeh, F., Gharizadeh, B., Myers, R. M., Hudson, J. R., Davis, R. W., Han, J. 2010; 107 (4): 1518-1523


    Developing T cells face a series of cell fate choices in the thymus and in the periphery. The role of the individual T cell receptor (TCR) in determining decisions of cell fate remains unresolved. The stochastic/selection model postulates that the initial fate of the cell is independent of TCR specificity, with survival dependent on additional TCR/coreceptor "rescue" signals. The "instructive" model holds that cell fate is initiated by the interaction of the TCR with a cognate peptide-MHC complex. T cells are then segregated on the basis of TCR specificity with the aid of critical coreceptors and signal modulators [Chan S, Correia-Neves M, Benoist C, Mathis (1998) Immunol Rev 165: 195-207]. The former would predict a random representation of individual TCR across divergent T cell lineages whereas the latter would predict minimal overlap between divergent T cell subsets. To address this issue, we have used high-throughput sequencing to evaluate the TCR distribution among key T cell developmental and effector subsets from a single donor. We found numerous examples of individual subsets sharing identical TCR sequence, supporting a model of a stochastic process of cell fate determination coupled with dynamic patterns of clonal expansion of T cells bearing the same TCR sequence among both CD4(+) and CD8+ populations.

    View details for DOI 10.1073/pnas.0913939107

    View details for Web of Science ID 000273974600053

    View details for PubMedID 20080641

  • Predicting Patient Survival from Longitudinal Gene Expression STATISTICAL APPLICATIONS IN GENETICS AND MOLECULAR BIOLOGY Zhang, Y., Tibshirani, R. J., Davis, R. W. 2010; 9 (1)


    Characterizing dynamic gene expression pattern and predicting patient outcome is now significant and will be of more interest in the future with large scale clinical investigation of microarrays. However, there is currently no method that has been developed for prediction of patient outcome using longitudinal gene expression, where gene expression of patients is being monitored across time. Here, we propose a novel prediction approach for patient survival time that makes use of time course structure of gene expression. This method is applied to a burn study. The genes involved in the final predictors are enriched in the inflammatory response and immune system related pathways. Moreover, our method is consistently better than prediction methods using individual time point gene expression or simply pooling gene expression from each time point.

    View details for DOI 10.2202/1544-6115.1617

    View details for Web of Science ID 000284905500002

    View details for PubMedID 21126232

    View details for PubMedCentralID PMC3004784

  • Method Systematic analysis of genome-wide fitness data in yeast reveals novel gene function and drug action GENOME BIOLOGY Hillenmeyer, M. E., Ericson, E., Davis, R. W., Nislow, C., Koller, D., Giaever, G. 2010; 11 (3)


    We systematically analyzed the relationships between gene fitness profiles (co-fitness) and drug inhibition profiles (co-inhibition) from several hundred chemogenomic screens in yeast. Co-fitness predicted gene functions distinct from those derived from other assays and identified conditionally dependent protein complexes. Co-inhibitory compounds were weakly correlated by structure and therapeutic class. We developed an algorithm predicting protein targets of chemical compounds and verified its accuracy with experimental testing. Fitness data provide a novel, systems-level perspective on the cell.

    View details for DOI 10.1186/gb-2010-11-3-r30

    View details for Web of Science ID 000277309100015

    View details for PubMedID 20226027

  • Knowledge-based analysis of microarrays for the discovery of transcriptional regulation relationships 8th Asia Pacific Bioinformatics Conference Seok, J., Kaushal, A., Davis, R. W., Xiao, W. BIOMED CENTRAL LTD. 2010


    The large amount of high-throughput genomic data has facilitated the discovery of the regulatory relationships between transcription factors and their target genes. While early methods for discovery of transcriptional regulation relationships from microarray data often focused on the high-throughput experimental data alone, more recent approaches have explored the integration of external knowledge bases of gene interactions.In this work, we develop an algorithm that provides improved performance in the prediction of transcriptional regulatory relationships by supplementing the analysis of microarray data with a new method of integrating information from an existing knowledge base. Using a well-known dataset of yeast microarrays and the Yeast Proteome Database, a comprehensive collection of known information of yeast genes, we show that knowledge-based predictions demonstrate better sensitivity and specificity in inferring new transcriptional interactions than predictions from microarray data alone. We also show that comprehensive, direct and high-quality knowledge bases provide better prediction performance. Comparison of our results with ChIP-chip data and growth fitness data suggests that our predicted genome-wide regulatory pairs in yeast are reasonable candidates for follow-up biological verification.High quality, comprehensive, and direct knowledge bases, when combined with appropriate bioinformatic algorithms, can significantly improve the discovery of gene regulatory relationships from high throughput gene expression data.

    View details for DOI 10.1186/1471-2105-11-S1-S8

    View details for Web of Science ID 000273969500009

    View details for PubMedID 20122245

  • A direct comparison of the KB™ Basecaller and phred for identifying the bases from DNA sequencing using chain termination chemistry. BMC research notes Hyman, R. W., Jiang, H., Fukushima, M., Davis, R. W. 2010; 3: 257-?


    Relatively recently, the software KB™ Basecaller has replaced phred for identifying the bases from raw sequence data in DNA sequencing employing dideoxy chemistry. We have measured quantitatively the consequences of that change.The high quality sequence segment of reads derived from the KB™ Basecaller were, on average, 30-to-50 bases longer than reads derived from phred. However, microbe identification appeared to have been unaffected by the change in software.We have demonstrated a modest, but statistically significant, superiority in high quality read length of the KB™ Basecaller compared to phred. We found no statistically significant difference between the numbers of microbial species identified from the sequence data.

    View details for DOI 10.1186/1756-0500-3-257

    View details for PubMedID 20932319

  • Electrical detection of proteins and DNA using bioactivated microfluidic channels: Theoretical and experimental considerations JOURNAL OF VACUUM SCIENCE & TECHNOLOGY B Javanmard, M., Esfandyarpour, H., Pease, F., Davis, R. W. 2009; 27 (6): 3099-3103


    In order to detect diseases like cancer at an early stage while it still may be curable, it's necessary to develop a diagnostic technique which can rapidly and inexpensively detect protein and nucleic acid biomarkers, without making any sacrifice in the sensitivity. We have developed a technique, based on the use of bioactivated microfluidic channels integrated with electrodes for electrical sensing, which can be used to detect protein biomarkers, target cells, and DNA hybridization. In this paper, we discuss the theoretical detection limits of this kind of sensor, and also discuss various experimental considerations in the electrical characterization of our device. In particular, we discuss the temperature dependence, the impedance drift, the noise sources, and various methods for optimizing the signal to noise ratio.

    View details for DOI 10.1116/1.3264675

    View details for Web of Science ID 000272803400156

  • Sequential Use of Transcriptional Profiling, Expression Quantitative Trait Mapping, and Gene Association Implicates MMP20 in Human Kidney Aging PLOS GENETICS Wheeler, H. E., Metter, E. J., Tanaka, T., Absher, D., Higgins, J., Zahn, J. M., Wilhelmy, J., Davis, R. W., Singleton, A., Myers, R. M., Ferrucci, L., Kim, S. K. 2009; 5 (10)


    Kidneys age at different rates, such that some people show little or no effects of aging whereas others show rapid functional decline. We sequentially used transcriptional profiling and expression quantitative trait loci (eQTL) mapping to narrow down which genes to test for association with kidney aging. We first performed whole-genome transcriptional profiling to find 630 genes that change expression with age in the kidney. Using two methods to detect eQTLs, we found 101 of these age-regulated genes contain expression-associated SNPs. We tested the eQTLs for association with kidney aging, measured by glomerular filtration rate (GFR) using combined data from the Baltimore Longitudinal Study of Aging (BLSA) and the InCHIANTI study. We found a SNP association (rs1711437 in MMP20) with kidney aging (uncorrected p = 3.6 x 10(-5), empirical p = 0.01) that explains 1%-2% of the variance in GFR among individuals. The results of this sequential analysis may provide the first evidence for a gene association with kidney aging in humans.

    View details for DOI 10.1371/journal.pgen.1000685

    View details for Web of Science ID 000272032100023

    View details for PubMedID 19834535

  • Alternative Splicing of PTC7 in Saccharomyces cerevisiae Determines Protein Localization GENETICS Juneau, K., Nislow, C., Davis, R. W. 2009; 183 (1): 185-194


    It is well established that higher eukaryotes use alternative splicing to increase proteome complexity. In contrast, Saccharomyces cerevisiae, a single-cell eukaryote, conducts predominantly regulated splicing through retention of nonfunctional introns. In this article we describe our discovery of a functional intron in the PTC7 (YHR076W) gene that can be alternatively spliced to create two mRNAs that code for distinct proteins. These two proteins localize to different cellular compartments and have distinct cellular roles. The protein translated from the spliced mRNA localizes to the mitochondria and its expression is carbon-source dependent. In comparison, the protein translated from the unspliced mRNA contains a transmembrane domain, localizes to the nuclear envelope, and mediates the toxic effects of Latrunculin A exposure. In conclusion, we identified a definitive example of functional alternative splicing in S. cerevisiae that confers a measurable fitness benefit.

    View details for DOI 10.1534/genetics.109.105155

    View details for Web of Science ID 000272067000018

    View details for PubMedID 19564484

  • A Microfluidic Platform for Characterization of Protein-Protein Interactions IEEE SENSORS JOURNAL Javanmard, M., Talasaz, A. H., Nemat-Gorgani, M., Huber, D. E., Pease, F., Ronaghi, M., Davis, R. W. 2009; 9 (8): 883-891


    Traditionally, expensive and time consuming techniques such as mass spectrometry and Western Blotting have been used for characterization of protein-protein interactions. In this paper, we describe the design, fabrication, and testing of a rapid and inexpensive sensor, involving the use of microelectrodes in a microchannel, which can be used for real-time electrical detection of specific interactions between proteins. We have successfully demonstrated detection of target glycoprotein-glycoprotein interactions, antigen-antibody interactions, and glycoprotein-antigen interactions. We have also demonstrated the ability of this technique to distinguish between strong and weak interactions. Using this approach, it may be possible to multiplex an array of these sensors onto a chip and probe a complex mixture for various types of interactions involving protein molecules.

    View details for DOI 10.1109/JSEN.2009.2022558

    View details for Web of Science ID 000268020400003

  • Molecular inversion probes reveal patterns of 9p21 deletion and copy number aberrations in childhood leukemia CANCER GENETICS AND CYTOGENETICS Schiffman, J. D., Wang, Y., McPherson, L. A., Welch, K., Zhang, N., Davis, R., Lacayo, N. J., Dahl, G. V., Faham, M., Ford, J. M., Ji, H. P. 2009; 193 (1): 9-18


    Childhood leukemia, which accounts for >30% of newly diagnosed childhood malignancies, is one of the leading causes of death for children with cancer. Genome-wide studies using microarray chips to identify copy number changes in human cancer are becoming more common. In this pilot study, 45 pediatric leukemia samples were analyzed for gene copy aberrations using novel molecular inversion probe (MIP) technology. Acute leukemia subtypes included precursor B-cell acute lymphoblastic leukemia (ALL) (n=23), precursor T-cell ALL (n=6), and acute myeloid leukemia (n=14). The MIP analysis identified 69 regions of recurring copy number changes, of which 41 have not been identified with other DNA microarray platforms. Copy number gains and losses were validated in 98% of clinical karyotypes and 100% of fluorescence in situ hybridization studies available. We report unique patterns of copy number loss in samples with 9p21.3 (CDKN2A) deletion in the precursor B-cell ALL patients, compared with the precursor T-cell ALL patients. MIPs represent an attractive technology for identifying novel copy number aberrations, validating previously reported copy number changes, and translating molecular findings into clinically relevant targets for further investigation.

    View details for DOI 10.1016/j.cancergencyto.2009.03.005

    View details for Web of Science ID 000268922900002

    View details for PubMedID 19602459

    View details for PubMedCentralID PMC2776674

  • A dynamic network of transcription in LPS-treated human subjects BMC SYSTEMS BIOLOGY Seok, J., Xiao, W., Moldawer, L. L., Davis, R. W., Covert, M. W. 2009; 3


    Understanding the transcriptional regulatory networks that map out the coordinated dynamic responses of signaling proteins, transcription factors and target genes over time would represent a significant advance in the application of genome wide expression analysis. The primary challenge is monitoring transcription factor activities over time, which is not yet available at the large scale. Instead, there have been several developments to estimate activities computationally. For example, Network Component Analysis (NCA) is an approach that can predict transcription factor activities over time as well as the relative regulatory influence of factors on each target gene.In this study, we analyzed a gene expression data set in blood leukocytes from human subjects administered with lipopolysaccharide (LPS), a prototypical inflammatory challenge, in the context of a reconstructed regulatory network including 10 transcription factors, 99 target genes and 149 regulatory interactions. We found that the computationally estimated activities were well correlated to their coordinated action. Furthermore, we found that clustering the genes in the context of regulatory influences greatly facilitated interpretation of the expression data, as clusters of gene expression corresponded to the activity of specific factors or more interestingly, factor combinations which suggest coordinated regulation of gene expression. The resulting clusters were therefore more biologically meaningful, and also led to identification of additional genes under the same regulation.Using NCA, we were able to build a network that accounted for between 8-11% genes in the known transcriptional response to LPS in humans. The dynamic network illustrated changes of transcription factor activities and gene expressions as well as interactions of signaling proteins, transcription factors and target genes.

    View details for DOI 10.1186/1752-0509-3-78

    View details for Web of Science ID 000269747200001

    View details for PubMedID 19638230

  • A Genome-Wide Screen for Regulators of TORC1 in Response to Amino Acid Starvation Reveals a Conserved Npr2/3 Complex PLOS GENETICS Neklesa, T. K., Davis, R. W. 2009; 5 (6)


    TORC1 is a central regulator of cell growth in response to amino acid availability, yet little is known about how it is regulated. Here, we performed a reverse genetic screen in yeast for genes necessary to inactivate TORC1. The screen consisted of monitoring the expression of a TORC1 sensitive GFP-based transcriptional reporter in all yeast deletion strains using flow cytometry. We find that in response to amino acid starvation, but not to carbon starvation or rapamycin treatment, cells lacking NPR2 and NPR3 fail to fully (1) activate transcription factors Gln3/Gat1, (2) dephosphorylate TORC1 effector Npr1, and (3) repress ribosomal protein gene expression. Both mutants show proliferation defects only in media containing a low quality nitrogen source, such as proline or ammonia, whereas no defects are evident when cells are grown in the presence of glutamine or peptone mixture. Proliferation defects in npr2Delta and npr3Delta cells can be completely rescued by artificially inhibiting TORC1 by rapamycin, demonstrating that overactive TORC1 in both strains prevents their ability to adapt to an environment containing a low quality nitrogen source. A biochemical purification of each demonstrates that Npr2 and Npr3 form a heterodimer, and this interaction is evolutionarily conserved since the human homologs of NPR2 and NPR3 (NPRL2 and NPRL3, respectively) also co-immunoprecipitate. We conclude that, in yeast, the Npr2/3 complex mediates an amino acid starvation signal to TORC1.

    View details for DOI 10.1371/journal.pgen.1000515

    View details for Web of Science ID 000268444600031

    View details for PubMedID 19521502

  • Electrical detection of protein biomarkers using bioactivated microfluidic channels LAB ON A CHIP Javanmard, M., Talasaz, A. H., Nemat-Gorgani, M., Pease, F., Ronaghi, M., Davis, R. W. 2009; 9 (10): 1429-1434


    Current methods used for analyzing biomarkers involve expensive and time consuming techniques like the Sandwich ELISA which require lengthy incubation times, high reagent costs, and bulky optical equipment. We have developed a technique involving the use of a micro-channel with integrated electrodes, functionalized with receptors specific to target biomarkers. We have applied our biochip to the rapid electrical detection and quantification of target protein biomarkers using protein functionalized micro-channels. We successfully demonstrate detection of anti-hCG antibody, at a concentration of 1 ng ml(-1) and a dynamic range of three orders of magnitude, in less than one hour. We envision the use of this technique in a handheld device for multiplex high throughput analysis using an array of micro-channels for probing various protein biomarkers in clinically relevant samples such as human serum for cancer detection.

    View details for DOI 10.1039/b818872f

    View details for Web of Science ID 000268227400016

    View details for PubMedID 19417910

  • Meiotic recombination generates rich diversity in NK cell receptor genes, alleles, and haplotypes GENOME RESEARCH Norman, P. J., Abi-Rached, L., Gendzekhadze, K., Hammond, J. A., Moesta, A. K., Sharma, D., Graef, T., McQueen, K. L., Guethlein, L. A., Carrington, C. V., Chandanayingyong, D., Chang, Y., Crespi, C., Saruhan-Direskeneli, G., Hameed, K., Kamkamidze, G., Koram, K. A., Layrisse, Z., Matamoros, N., Mila, J., Park, M. H., Pitchappan, R. M., Ramdath, D. D., Shiau, M., Stephens, H. A., Struik, S., Tyan, D., Verity, D. H., Vaughan, R. W., Davis, R. W., Fraser, P. A., Riley, E. M., Ronaghi, M., Parham, P. 2009; 19 (5): 757-769


    Natural killer (NK) cells contribute to the essential functions of innate immunity and reproduction. Various genes encode NK cell receptors that recognize the major histocompatibility complex (MHC) Class I molecules expressed by other cells. For primate NK cells, the killer-cell immunoglobulin-like receptors (KIR) are a variable and rapidly evolving family of MHC Class I receptors. Studied here is KIR3DL1/S1, which encodes receptors for highly polymorphic human HLA-A and -B and comprises three ancient allelic lineages that have been preserved by balancing selection throughout human evolution. While the 3DS1 lineage of activating receptors has been conserved, the two 3DL1 lineages of inhibitory receptors were diversified through inter-lineage recombination with each other and with 3DS1. Prominent targets for recombination were D0-domain polymorphisms, which modulate enhancer function, and dimorphism at position 283 in the D2 domain, which influences inhibitory function. In African populations, unequal crossing over between the 3DL1 and 3DL2 genes produced a deleted KIR haplotype in which the telomeric "half" was reduced to a single fusion gene with functional properties distinct from its 3DL1 and 3DL2 parents. Conversely, in Eurasian populations, duplication of the KIR3DL1/S1 locus by unequal crossing over has enabled individuals to carry and express alleles of all three KIR3DL1/S1 lineages. These results demonstrate how meiotic recombination combines with an ancient, preserved diversity to create new KIR phenotypes upon which natural selection acts. A consequence of such recombination is to blur the distinction between alleles and loci in the rapidly evolving human KIR gene family.

    View details for DOI 10.1101/gr.085738.108

    View details for Web of Science ID 000265668800009

    View details for PubMedID 19411600

  • High-throughput, high-accuracy array-based resequencing PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zheng, J., Moorhead, M., Weng, L., Siddiqui, F., Carlton, V. E., Ireland, J. S., Lee, L., Peterson, J., Wilkins, J., Lin, S., Kan, Z., Seshagiri, S., Davis, R. W., Faham, M. 2009; 106 (16): 6712-6717


    Although genomewide association studies have successfully identified associations of many common single-nucleotide polymorphisms (SNPs) with common diseases, the SNPs implicated so far account for only a small proportion of the genetic variability of tested diseases. It has been suggested that common diseases may often be caused by rare alleles missed by genomewide association studies. To identify these rare alleles we need high-throughput, high-accuracy resequencing technologies. Although array-based genotyping has allowed genomewide association studies of common SNPs in tens of thousands of samples, array-based resequencing has been limited for 2 main reasons: the lack of a fully multiplexed pipeline for high-throughput sample processing, and failure to achieve sufficient performance. We have recently solved both of these problems and created a fully multiplexed high-throughput pipeline that results in high-quality data. The pipeline consists of target amplification from genomic DNA, followed by allele enrichment to generate pools of purified variant (or nonvariant) DNA and ends with interrogation of purified DNA on resequencing arrays. We have used this pipeline to resequence approximately 5 Mb of DNA (on 3 arrays) corresponding to the exons of 1,500 genes in >473 samples; in total >2,350 Mb were sequenced. In the context of this large-scale study we obtained a false positive rate of approximately 1 in 500,000 bp and a false negative rate of approximately 10%.

    View details for DOI 10.1073/pnas.0901902106

    View details for Web of Science ID 000265506800053

    View details for PubMedID 19342489

    View details for PubMedCentralID PMC2672536

  • Mapping Gene Associations in Human Mitochondria using Clinical Disease Phenotypes PLOS COMPUTATIONAL BIOLOGY Scharfe, C., Lu, H. H., Neuenburg, J. K., Allen, E. A., Li, G., Klopstock, T., Cowan, T. M., Enns, G. M., Davis, R. W. 2009; 5 (4)


    Nuclear genes encode most mitochondrial proteins, and their mutations cause diverse and debilitating clinical disorders. To date, 1,200 of these mitochondrial genes have been recorded, while no standardized catalog exists of the associated clinical phenotypes. Such a catalog would be useful to develop methods to analyze human phenotypic data, to determine genotype-phenotype relations among many genes and diseases, and to support the clinical diagnosis of mitochondrial disorders. Here we establish a clinical phenotype catalog of 174 mitochondrial disease genes and study associations of diseases and genes. Phenotypic features such as clinical signs and symptoms were manually annotated from full-text medical articles and classified based on the hierarchical MeSH ontology. This classification of phenotypic features of each gene allowed for the comparison of diseases between different genes. In turn, we were then able to measure the phenotypic associations of disease genes for which we calculated a quantitative value that is based on their shared phenotypic features. The results showed that genes sharing more similar phenotypes have a stronger tendency for functional interactions, proving the usefulness of phenotype similarity values in disease gene network analysis. We then constructed a functional network of mitochondrial genes and discovered a higher connectivity for non-disease than for disease genes, and a tendency of disease genes to interact with each other. Utilizing these differences, we propose 168 candidate genes that resemble the characteristic interaction patterns of mitochondrial disease genes. Through their network associations, the candidates are further prioritized for the study of specific disorders such as optic neuropathies and Parkinson disease. Most mitochondrial disease phenotypes involve several clinical categories including neurologic, metabolic, and gastrointestinal disorders, which might indicate the effects of gene defects within the mitochondrial system. The accompanying knowledgebase ( supports the study of clinical diseases and associated genes.

    View details for DOI 10.1371/journal.pcbi.1000374

    View details for Web of Science ID 000266214200033

    View details for PubMedID 19390613

  • Label-free biosensing with functionalized nanopipette probes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Umehara, S., Karhanek, M., Davis, R. W., Pourmand, N. 2009; 106 (12): 4611-4616


    Nanopipette technology can uniquely identify biomolecules such as proteins based on differences in size, shape, and electrical charge. These differences are determined by the detection of changes in ionic current as the proteins interact with the nanopipette tip coated with probe molecules. Here we show that electrostatic, biotin-streptavidin, and antibody-antigen interactions on the nanopipette tip surface affect ionic current flowing through a 50-nm pore. Highly charged polymers interacting with the glass surface modulated the rectification property of the nanopipette electrode. Affinity-based binding between the probes tethered to the surface and their target proteins caused a change in the ionic current due to a partial blockade or an altered surface charge. These findings suggest that nanopipettes functionalized with appropriate molecular recognition elements can be used as nanosensors in biomedical and biological research.

    View details for DOI 10.1073/pnas.0900306106

    View details for Web of Science ID 000264522600010

    View details for PubMedID 19264962

  • Isolating highly enriched populations of circulating epithelial cells and other rare cells from blood using a magnetic sweeper device PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Talasaz, A. H., Powell, A. A., Huber, D. E., Berbee, J. G., Roh, K., Yu, W., Xiao, W., Davis, M. M., Pease, R. F., Mindrinos, M. N., Jeffrey, S. S., Davis, R. W. 2009; 106 (10): 3970-3975


    The enumeration of rare circulating epithelial cells (CEpCs) in the peripheral blood of metastatic cancer patients has shown promise for improved cancer prognosis. Moving beyond enumeration, molecular analysis of CEpCs may provide candidate surrogate endpoints to diagnose, treat, and monitor malignancy directly from the blood samples. Thorough molecular analysis of CEpCs requires the development of new sample preparation methods that yield easily accessible and purified CEpCs for downstream biochemical assays. Here, we describe a new immunomagnetic cell separator, the MagSweeper, which gently enriches target cells and eliminates cells that are not bound to magnetic particles. The isolated cells are easily accessible and can be extracted individually based on their physical characteristics to deplete any cells nonspecifically bound to beads. We have shown that our device can process 9 mL of blood per hour and captures >50% of CEpCs as measured in spiking experiments. We have shown that the separation process does not perturb the gene expression of rare cells. To determine the efficiency of our platform in isolating CEpCs from patients, we have isolated CEpCs from all 47 tubes of 9-mL blood samples collected from 17 women with metastatic breast cancer. In contrast, we could not find any circulating epithelial cells in samples from 5 healthy donors. The isolated CEpCs are all stored individually for further molecular analysis.

    View details for DOI 10.1073/pnas.0813188106

    View details for Web of Science ID 000264036900059

    View details for PubMedID 19234122

    View details for PubMedCentralID PMC2645911

  • Whole Genome Survey of Coding SNPs Reveals a Reproducible Pathway Determinant of Parkinson Disease HUMAN MUTATION Srinivasan, B. S., Doostzadeh, J., Absalan, F., Mohandessi, S., Jalili, R., Bigdeli, S., Wang, J., Mahadevan, J., Lee, C. L., Davis, R. W., Langston, J. W., Ronaghi, M. 2009; 30 (2): 228-238


    It is quickly becoming apparent that situating human variation in a pathway context is crucial to understanding its phenotypic significance. Toward this end, we have developed a general method for finding pathways associated with traits that control for pathway size. We have applied this method to a new whole genome survey of coding SNP variation in 187 patients afflicted with Parkinson disease (PD) and 187 controls. We show that our dataset provides an independent replication of the axon guidance association recently reported by Lesnick et al. [PLoS Genet 2007;3:e98], and also indicates that variation in the ubiquitin-mediated proteolysis and T-cell receptor signaling pathways may predict PD susceptibility. Given this result, it is reasonable to hypothesize that pathway associations are more replicable than individual SNP associations in whole genome association studies. However, this hypothesis is complicated by a detailed comparison of our dataset to the second recent PD association study by Fung et al. [Lancet Neurol 2006;5:911-916]. Surprisingly, we find that the axon guidance pathway does not rank at the very top of the Fung dataset after controlling for pathway size. More generally, in comparing the studies, we find that SNP frequencies replicate well despite technologically different assays, but that both SNP and pathway associations are globally uncorrelated across studies. We thus have a situation in which an association between axon guidance pathway variation and PD has been found in 2 out of 3 studies. We conclude by relating this seeming inconsistency to the molecular heterogeneity of PD, and suggest future analyses that may resolve such discrepancies.

    View details for DOI 10.1002/humu.20840

    View details for Web of Science ID 000263254200013

    View details for PubMedID 18853455

    View details for PubMedCentralID PMC2793088

  • Large-Scale Multiplexed Quantitative Discovery Proteomics Enabled by the Use of an O-18-Labeled "Universal" Reference Sample JOURNAL OF PROTEOME RESEARCH Qian, W., Liu, T., Petyuk, V. A., Gritsenko, M. A., Petritis, B. O., Polpitiya, A. D., Kaushal, A., Xiao, W., Finnerty, C. C., Jeschke, M. G., Jaitly, N., Monroe, M. E., Moore, R. J., Moldawer, L. L., Davis, R. W., Tompkins, R. G., Herndon, D. N., Camp, D. G., Smith, R. D. 2009; 8 (1): 290-299


    The quantitative comparison of protein abundances across a large number of biological or patient samples represents an important proteomics challenge that needs to be addressed for proteomics discovery applications. Herein, we describe a strategy that incorporates a stable isotope (18)O-labeled "universal" reference sample as a comprehensive set of internal standards for analyzing large sample sets quantitatively. As a pooled sample, the (18)O-labeled "universal" reference sample is spiked into each individually processed unlabeled biological sample and the peptide/protein abundances are quantified based on (16)O/(18)O isotopic peptide pair abundance ratios that compare each unlabeled sample to the identical reference sample. This approach also allows for the direct application of label-free quantitation across the sample set simultaneously along with the labeling-approach (i.e., dual-quantitation) since each biological sample is unlabeled except for the labeled reference sample that is used as internal standards. The effectiveness of this approach for large-scale quantitative proteomics is demonstrated by its application to a set of 18 plasma samples from severe burn patients. When immunoaffinity depletion and cysteinyl-peptide enrichment-based fractionation with high resolution LC-MS measurements were combined, a total of 312 plasma proteins were confidently identified and quantified with a minimum of two unique peptides per protein. The isotope labeling data was directly compared with the label-free (16)O-MS intensity data extracted from the same data sets. The results showed that the (18)O reference-based labeling approach had significantly better quantitative precision compared to the label-free approach. The relative abundance differences determined by the two approaches also displayed strong correlation, illustrating the complementary nature of the two quantitative methods. The simplicity of including the (18)O-reference for accurate quantitation makes this strategy especially attractive when a large number of biological samples are involved in a study where label-free quantitation may be problematic, for example, due to issues associated with instrument platform robustness. The approach will also be useful for more effectively discovering subtle abundance changes in broad systems biology studies.

    View details for DOI 10.1021/pr800467r

    View details for Web of Science ID 000262171100030

    View details for PubMedID 19053531

  • Hyperspectral Image Correlation for Monitoring Membrane Protein Dynamics in Living Cells Conference on Three-Dimensional and Multidimensional Microscopy - Image Acquisition and Processing XVI Davis, R. W., Carson, B., Jones, H. D., Sinclair, M. B. SPIE-INT SOC OPTICAL ENGINEERING. 2009

    View details for DOI 10.1117/12.807743

    View details for Web of Science ID 000284868100008

  • Genome-wide transcriptome analysis of 150 cell samples INTEGRATIVE BIOLOGY Irimia, D., Mindrinos, M., Russom, A., Xiao, W., Wilhelmy, J., Wang, S., Heath, J. D., Kurn, N., Tompkins, R. G., Davis, R. W., Toner, M. 2009; 1 (1): 99-107


    A major challenge in molecular biology is interrogating the human transcriptome on a genome wide scale when only a limited amount of biological sample is available for analysis. Current methodologies using microarray technologies for simultaneously monitoring mRNA transcription levels require nanogram amounts of total RNA. To overcome the sample size limitation of current technologies, we have developed a method to probe the global gene expression in biological samples as small as 150 cells, or the equivalent of approximately 300 pg total RNA. The new method employs microfluidic devices for the purification of total RNA from mammalian cells and ultra-sensitive whole transcriptome amplification techniques. We verified that the RNA integrity is preserved through the isolation process, accomplished highly reproducible whole transcriptome analysis, and established high correlation between repeated isolations of 150 cells and the same cell culture sample. We validated the technology by demonstrating that the combined microfluidic and amplification protocol is capable of identifying biological pathways perturbed by stimulation, which are consistent with the information recognized in bulk-isolated samples.

    View details for DOI 10.1039/b814329c

    View details for Web of Science ID 000266978200011

    View details for PubMedID 20023796

  • Chemogenomic approaches to elucidation of gene function and genetic pathways. Methods in molecular biology (Clifton, N.J.) Pierce, S. E., Davis, R. W., Nislow, C., Giaever, G. 2009; 548: 115-143


    The approximately 6,000 strains in the yeast deletion collection can be studied in a single culture by using a microarray to detect the 20 bp DNA "barcodes" or "tags" contained in each strain. Barcode intensities measured by microarray are compared across time-points or across conditions to analyze the relative fitness of each strain. The development of this pooled fitness assay has greatly facilitated the functional annotation of the yeast genome by making genome-wide gene-deletion studies faster and easier, and has led to the development of high throughput methods for studying drug action in yeast. Pooled screens can be used for identifying gene functions, measuring the functional relatedness of gene pairs to group genes into pathways, identifying drug targets, and determining a drug's mechanism of action. This process involves five main steps: preparing aliquots of pooled cells, pooled growth, isolation of genomic DNA and PCR amplification of the barcodes, array hybridization, and data analysis. In addition to yeast fitness applications, the general method of studying pooled samples with barcode arrays can also be adapted for use with other types of samples, such as mutant collections in other organisms, siRNA vectors, and molecular inversion probes.

    View details for DOI 10.1007/978-1-59745-540-4_7

    View details for PubMedID 19521822

  • THE DIVERSITY OF NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY NATO Advanced Study Institute on Biophysics and the Challenges of Emerging Threats Liu, C. W., Alekseyev, V. Y., Allwardt, J. R., Bankovich, A. J., Cade-Menun, B. J., Davis, R. W., Du, L., Garcia, K. C., Herschlag, D., Khosla, C., Kraut, D. A., Li, Q., Null, B., Puglisi, J. D., Sigala, P. A., Stebbins, J. F., Varani, L. SPRINGER. 2009: 65–81
  • HIGH THROUGHPUT DNA SEQUENCING, APPLICATIONS AND DISEASE INVESTIGATION 6th International Forum on Post-Genome Technologies Gharizadeh, B., Wang Chunlin, C. L., Babrzadeh, F., Wang Elijah, E. J., Han Jian, J., Davis, R. W. SOUTHEAST UNIV PRESS. 2009: 27–27
  • Multiplex protein assays based on real-time magnetic nanotag sensing PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Osterfeld, S. J., Yu, H., Gaster, R. S., Caramuta, S., Xu, L., Han, S., Hall, D. A., Wilson, R. J., Sun, S., White, R. L., Davis, R. W., Pourmand, N., Wang, S. X. 2008; 105 (52): 20637-20640


    Magnetic nanotags (MNTs) are a promising alternative to fluorescent labels in biomolecular detection assays, because minute quantities of MNTs can be detected with inexpensive giant magnetoresistive (GMR) sensors, such as spin valve (SV) sensors. However, translating this promise into easy to use and multilplexed protein assays, which are highly sought after in molecular diagnostics such as cancer diagnosis and treatment monitoring, has been challenging. Here, we demonstrate multiplex protein detection of potential cancer markers at subpicomolar concentration levels and with a dynamic range of more than four decades. With the addition of nanotag amplification, the analytic sensitivity extends into the low fM concentration range. The multianalyte ability, sensitivity, scalability, and ease of use of the MNT-based protein assay technology make it a strong contender for versatile and portable molecular diagnostics in both research and clinical settings.

    View details for DOI 10.1073/pnas.0810822105

    View details for Web of Science ID 000262092800015

    View details for PubMedID 19074273

    View details for PubMedCentralID PMC2602607

  • Superoxide anions regulate TORC1 and its ability to bind Fpr1:rapamycin complex PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ronald, T. K., Davis, W. 2008; 105 (39): 15166-15171


    The small natural product rapamycin, when bound to FKBP12, is a potent inhibitor of an evolutionarily conserved Target of Rapamycin Complex 1 (TORC1), which plays a central role in mediating cellular response to nutrient availability. Given the prominent role of TORC1 in cell growth and proliferation, clinical trials have explored the possibility of using rapamycin as an anticancer agent. Unfortunately, the percentage of patients responding favorably has been low, intensifying the need to find biomarkers able to predict rapamycin sensitivity or resistance. In this study, we elucidate the molecular mechanism underlying partial rapamycin resistance in yeast. Using the yeast deletion collection, we identified 15 deletion strains leading to partial rapamycin resistance. Among these were Cu/Zn-superoxide dismutase Sod1, copper transporter Ctr1, and copper chaperone Lys7, suggesting a role for oxidative stress in rapamycin resistance. Further analysis revealed that all 15 strains exhibit elevated levels of superoxide anions, and we show that elevated levels of reactive oxygen species specifically modify TORC1 such that it is no longer able to fully bind FKBP12:rapamycin. Therefore, elevated oxidative stress modifies TORC1 and prevents its binding to the FKBP12:rapamycin complex, ultimately leading to rapamycin resistance. These results warrant an examination into whether similar reasons explain rapamycin resistance observed in various clinical samples.

    View details for DOI 10.1073/pnas.0807712105

    View details for Web of Science ID 000261914300060

    View details for PubMedID 18812505

  • An integrated platform of genomic assays reveals small-molecule bioactivities NATURE CHEMICAL BIOLOGY Hoon, S., Smith, A. M., Wallace, I. M., Suresh, S., Miranda, M., Fung, E., Proctor, M., Shokat, K. M., Zhang, C., Davis, R. W., Giaever, G., StOnge, R. P., Nislow, C. 2008; 4 (8): 498-506


    Bioactive compounds are widely used to modulate protein function and can serve as important leads for drug development. Identifying the in vivo targets of these compounds remains a challenge. Using yeast, we integrated three genome-wide gene-dosage assays to measure the effect of small molecules in vivo. A single TAG microarray was used to resolve the fitness of strains derived from pools of (i) homozygous deletion mutants, (ii) heterozygous deletion mutants and (iii) genomic library transformants. We demonstrated, with eight diverse reference compounds, that integration of these three chemogenomic profiles improves the sensitivity and specificity of small-molecule target identification. We further dissected the mechanism of action of two protein phosphatase inhibitors and in the process developed a framework for the rational design of multidrug combinations to sensitize cells with specific genotypes more effectively. Finally, we applied this platform to 188 novel synthetic chemical compounds and identified both potential targets and structure-activity relationships.

    View details for DOI 10.1038/nchembio.100

    View details for Web of Science ID 000257852500016

    View details for PubMedID 18622389

  • High-Resolution, In Vivo Magnetic Resonance Imaging of Drosophila at 18.8 Tesla PLOS ONE Null, B., Liu, C. W., Hedehus, M., Conolly, S., Davis, R. W. 2008; 3 (7)


    High resolution MRI of live Drosophila was performed at 18.8 Tesla, with a field of view less than 5 mm, and administration of manganese or gadolinium-based contrast agents. This study demonstrates the feasibility of MR methods for imaging the fruit fly Drosophila with an NMR spectrometer, at a resolution relevant for undertaking future studies of the Drosophila brain and other organs. The fruit fly has long been a principal model organism for elucidating biology and disease, but without capabilities like those of MRI. This feasibility marks progress toward the development of new in vivo research approaches in Drosophila without the requirement for light transparency or destructive assays.

    View details for DOI 10.1371/journal.pone.0002817

    View details for Web of Science ID 000264304300036

    View details for PubMedID 18665264

    View details for PubMedCentralID PMC2474967

  • High Throughput Automated Allele Frequency Estimation by Pyrosequencing PLOS ONE Doostzadeh, J., Shokralla, S., Absalan, F., Jalili, R., Mohandessi, S., Langston, J. W., Davis, R. W., Ronaghi, M., Gharizadeh, B. 2008; 3 (7)


    Pyrosequencing is a DNA sequencing method based on the principle of sequencing-by-synthesis and pyrophosphate detection through a series of enzymatic reactions. This bioluminometric, real-time DNA sequencing technique offers unique applications that are cost-effective and user-friendly. In this study, we have combined a number of methods to develop an accurate, robust and cost efficient method to determine allele frequencies in large populations for association studies. The assay offers the advantage of minimal systemic sampling errors, uses a general biotin amplification approach, and replaces dTTP for dATP-apha-thio to avoid non-uniform higher peaks in order to increase accuracy. We demonstrate that this newly developed assay is a robust, cost-effective, accurate and reproducible approach for large-scale genotyping of DNA pools. We also discuss potential improvements of the software for more accurate allele frequency analysis.

    View details for DOI 10.1371/journal.pone.0002693

    View details for Web of Science ID 000264057200036

    View details for PubMedID 18628978

  • A comprehensive assay for targeted multiplex amplification of human DNA sequences PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Krishnakumar, S., Zheng, J., Wilhelmy, J., Faham, M., Mindrinos, M., Davis, R. 2008; 105 (27): 9296-9301


    We developed a robust and reproducible methodology to amplify human sequences in parallel for use in downstream multiplexed sequence analyses. We call the methodology SMART (Spacer Multiplex Amplification Reaction), and it is based, in part, on padlock probe technology. As a proof of principle, we used SMART technology to simultaneously amplify 485 human exons ranging from 100 to 500 bp from human genomic DNA. In multiple repetitions, >90% of the targets were successfully amplified with a high degree of uniformity, with 70% of targets falling within a 10-fold range and all products falling within a 100-fold range of each other in abundance. We used long padlock probes (LPPs) >300 bases in length for the assay, and the increased length of these probes allowed for the capture of human sequences up to 500 bp in length, which is optimal for capturing most human exons. To engineer the LPPs, we developed a method that generates ssDNA molecules with precise ends, using an appropriately designed dsDNA template. The template has appropriate restriction sites engineered into it that can be digested to generate nucleotide overhangs that are suitable for lambda exonuclease digestion, producing a single-stranded probe from dsDNA. The SMART technology is flexible and can be easily adapted to multiplex tens of thousands of target sequences in a single reaction.

    View details for DOI 10.1073/pnas.0803240105

    View details for Web of Science ID 000257645400031

    View details for PubMedID 18599465

  • Pervasive and Persistent Redundancy among Duplicated Genes in Yeast PLOS GENETICS Dean, E. J., Davis, J. C., Davis, R. W., Petrov, D. A. 2008; 4 (7)


    The loss of functional redundancy is the key process in the evolution of duplicated genes. Here we systematically assess the extent of functional redundancy among a large set of duplicated genes in Saccharomyces cerevisiae. We quantify growth rate in rich medium for a large number of S. cerevisiae strains that carry single and double deletions of duplicated and singleton genes. We demonstrate that duplicated genes can maintain substantial redundancy for extensive periods of time following duplication ( approximately 100 million years). We find high levels of redundancy among genes duplicated both via the whole genome duplication and via smaller scale duplications. Further, we see no evidence that two duplicated genes together contribute to fitness in rich medium substantially beyond that of their ancestral progenitor gene. We argue that duplicate genes do not often evolve to behave like singleton genes even after very long periods of time.

    View details for DOI 10.1371/journal.pgen.1000113

    View details for Web of Science ID 000260410600025

    View details for PubMedID 18604285

  • Conformational flexibility of a model protein upon immobilization on self-assembled monolayers BIOTECHNOLOGY AND BIOENGINEERING Bigdeli, S., Talasaz, A. H., Stahl, P., Persson, H. H., Ronaghi, M., Davis, R. W., Nemat-Gorgani, M. 2008; 100 (1): 19-27


    The present study reports on the retention of conformational flexibility of a model allosteric protein upon immobilization on self-assembled monolayers (SAMs) on gold. Organothiolated SAMs of different compositions were utilized for adsorptive and covalent attachment of bovine liver glutamate dehydrogenase (GDH), a well-characterized allosteric enzyme. Sensitive fluorimetric assays were developed to determine immobilization capacity, specific activity, and allosteric properties of the immobilized preparations as well as the potential for repeated use and continuous catalytic transformations. The allosteric response of the free and immobilized forms towards ADP, L-leucine and high concentrations of NAD(+), some of the well-known activators for this enzyme, were determined and compared. The enzyme immobilized by adsorption or chemical binding responded similarly to the activators with a greater degree of activation, as compared to the free form. Also loss of activity involving the two immobilization procedures were similar, suggesting that residues essential for catalytic activity or allosteric properties of GDH remained unchanged in the course of chemical modification. A recently established method was used to predict GDH orientation upon immobilization, which was found to explain some of the experimental results presented. The general significance of these observations in connection with retention of native properties of protein structures upon immobilization on SAMs is discussed.

    View details for DOI 10.1002/bit.21724

    View details for Web of Science ID 000254786200002

    View details for PubMedID 18078298

  • Interference of globin genes with rejection biomarker discovery 8th American Transplant Congress Li, L., Ying, L., Naesens, M., Xiao, W., Sigdel, T., Hsieh, S., Martin, J., Chen, R., Liu, K., Mindrinos, M., Davis, R., Sarwal, M. WILEY-BLACKWELL. 2008: 636–636
  • Microfluidic leukocyte isolation for gene expression analysis in critically ill hospitalized patients CLINICAL CHEMISTRY Russom, A., Sethu, P., Irimia, D., Mindrinos, M. N., Calvan, S. E., Garcia, I., Finnerty, C., Tannahill, C., Abouhamze, A., Wilhelmy, J., Lopez, M. C., Baker, H. V., Herndon, D. N., Lowry, S. F., Maier, R. V., Davis, R. W., Moldawer, L. L., Tompkins, R. G., Toner, M. 2008; 54 (5): 891-900


    Microarray technology is becoming a powerful tool for diagnostic, therapeutic, and prognostic applications. There is at present no consensus regarding the optimal technique to isolate nucleic acids from blood leukocyte populations for subsequent expression analyses. Current collection and processing techniques pose significant challenges in the clinical setting. Here, we report the clinical validation of a novel microfluidic leukocyte nucleic acid isolation technique for gene expression analysis from critically ill, hospitalized patients that can be readily used on small volumes of blood.We processed whole blood from hospitalized patients after burn injury and severe blunt trauma according to the microfluidic and standard macroscale leukocyte isolation protocol. Side-by-side comparison of RNA quantity, quality, and genome-wide expression patterns was used to clinically validate the microfluidic technique.When the microfluidic protocol was used for processing, sufficient amounts of total RNA were obtained for genome-wide expression analysis from 0.5 mL whole blood. We found that the leukocyte expression patterns from samples processed using the 2 protocols were concordant, and there was less variability introduced as a result of harvesting method than there existed between individuals.The novel microfluidic approach achieves leukocyte isolation in <25 min, and the quality of nucleic acids and genome expression analysis is equivalent to or surpasses that obtained from macroscale approaches. Microfluidics can significantly improve the isolation of blood leukocytes for genomic analyses in the clinical setting.

    View details for DOI 10.1373/clinchem.2007.099150

    View details for Web of Science ID 000255344600016

    View details for PubMedID 18375483

    View details for PubMedCentralID PMC4011019

  • The chemical genomic portrait of yeast: Uncovering a phenotype for all genes SCIENCE Hillenmeyer, M. E., Fung, E., Wildenhain, J., Pierce, S. E., Hoon, S., Lee, W., Proctor, M., St Onge, R. P., Tyers, M., Koller, D., Altman, R. B., Davis, R. W., Nislow, C., Giaever, G. 2008; 320 (5874): 362-365


    Genetics aims to understand the relation between genotype and phenotype. However, because complete deletion of most yeast genes ( approximately 80%) has no obvious phenotypic consequence in rich medium, it is difficult to study their functions. To uncover phenotypes for this nonessential fraction of the genome, we performed 1144 chemical genomic assays on the yeast whole-genome heterozygous and homozygous deletion collections and quantified the growth fitness of each deletion strain in the presence of chemical or environmental stress conditions. We found that 97% of gene deletions exhibited a measurable growth phenotype, suggesting that nearly all genes are essential for optimal growth in at least one condition.

    View details for DOI 10.1126/science.1150021

    View details for Web of Science ID 000255026100040

    View details for PubMedID 18420932

    View details for PubMedCentralID PMC2794835

  • Structural optimization for heat detection of DNA thermosequencing platform using finite element analysis BIOMICROFLUIDICS Esfandyarpour, H., Zheng, B., Fabian, R., Pease, W., Davis, R. W. 2008; 2 (2)


    For the past three decades, Sanger's method has been the primary DNA sequencing technology; however, inherent limitations in cost and complexity have limited its usage in personalized medicine and ecological studies. A new technology called "thermosequencing" can potentially reduce both the cost and complexity of DNA sequencing by using a microfluidic platform [Esfandyarpour, Pease, and Davis, J. Vac. Sci. Technol. B26, 661 (2008)]. To optimize the efficiency of the technology, finite element analysis was used to model the thermosequencing system by simulating the DNA incorporation reaction series and the resulting product concentration and heat production. Different models of the thermosequencing platform were created to simulate the effects of the materials surrounding the system, to optimize the geometry of the system, and to concentrate reaction heat into specific regions for detection in the real system. The resulting concentrations of reaction products were used to calibrate the reaction speed and to design the heat sensors in the thermosequencing technology. We recommend a modified gated structure for the microfluidic detection platform by using control valves and show how this new platform could dramatically improve the detection efficiency.

    View details for DOI 10.1063/1.2901138

    View details for Web of Science ID 000257284300002

    View details for PubMedID 19693405

    View details for PubMedCentralID PMC2719263

  • Bisphenol A induces a profile of tumor aggressiveness in high-risk cells from breast cancer patients CANCER RESEARCH Dairkee, S. H., Seok, J., Champion, S., Sayeed, A., Mindrinos, M., Xiao, W., Davis, R. W., Goodson, W. H. 2008; 68 (7): 2076-2080


    Breast cancer outcome is highly variable. Whether inadvertent exposure to environmental xenobiotics evokes a biological response promoting cancer aggressiveness and a higher probability of tumor recurrence remains unknown. To determine specific molecular alterations which arise in high-risk breast tissue in the presence of the ubiquitous xenoestrogen, bisphenol A (BPA), we used nonmalignant random periareolar fine-needle aspirates in a novel functional assay. Early events induced by BPA in epithelial-stromal cocultures derived from the contralateral tissue of patients with breast cancer included gene expression patterns which facilitate apoptosis evasion, endurance of microenvironmental stress, and cell cycle deregulation without a detectable increase in cell numbers. This BPA response profile was significantly associated with breast tumors characterized by high histologic grade (P < 0.001) and large tumor size (P = 0.002), resulting in decreased recurrence-free patient survival (P < 0.001). Our assays show a biological "fingerprint" of probable prior exposure to endocrine-disrupting agents, and suggest a scenario in which their presence in the microenvironmental milieu of high-risk breast tissue could play a deterministic role in establishing and maintaining tumor aggressiveness and poor patient outcome.

    View details for DOI 10.1158/0008-5472.CAN-07-6526

    View details for Web of Science ID 000254738500006

    View details for PubMedID 18381411

  • Multiplexed proximity ligation assays to profile putative plasma biomarkers relevant to pancreatic and ovarian cancer CLINICAL CHEMISTRY Fredriksson, S., Horecka, J., Brustugun, O. T., Schlingemann, J., Koong, A. C., Tibshirani, R., Davis, R. W. 2008; 54 (3): 582-589


    Sensitive methods are needed for biomarker discovery and validation. We tested one promising technology, multiplex proximity ligation assay (PLA), in a pilot study profiling plasma biomarkers in pancreatic and ovarian cancer.We used 4 panels of 6- and 7-plex PLAs to detect biomarkers, with each assay consuming 1 microL plasma and using either matched monoclonal antibody pairs or single batches of polyclonal antibody. Protein analytes were converted to unique DNA amplicons by proximity ligation and subsequently detected by quantitative PCR. We profiled 18 pancreatic cancer cases and 19 controls and 19 ovarian cancer cases and 20 controls for the following proteins: a disintegrin and metalloprotease 8, CA-125, CA 19-9, carboxypeptidase A1, carcinoembryonic antigen, connective tissue growth factor, epidermal growth factor receptor, epithelial cell adhesion molecule, Her2, galectin-1, insulin-like growth factor 2, interleukin-1alpha, interleukin-7, mesothelin, macrophage migration inhibitory factor, osteopontin, secretory leukocyte peptidase inhibitor, tumor necrosis factor alpha, vascular endothelial growth factor, and chitinase 3-like 1. Probes for CA-125 were present in 3 of the multiplex panels. We measured plasma concentrations of the CA-125-mesothelin complex by use of a triple-specific PLA with 2 ligation events among 3 probes.The assays displayed consistent measurements of CA-125 independent of which other markers were simultaneously detected and showed good correlation with Luminex data. In comparison to literature reports, we achieved expected results for other putative markers.Multiplex PLA using either matched monoclonal antibodies or single batches of polyclonal antibody should prove useful for identifying and validating sets of putative disease biomarkers and finding multimarker panels.

    View details for DOI 10.1373/clinchem.2007.093195

    View details for Web of Science ID 000253570400019

    View details for PubMedID 18171715

  • Picocalorimetric method for DNA sequencing JOURNAL OF VACUUM SCIENCE & TECHNOLOGY B Esfandyarpour, H., Fabian, R., Pease, W., Davis, R. W. 2008; 26 (2): 661-665

    View details for DOI 10.1116/1.2897322

    View details for Web of Science ID 000254600600034

  • A system for multiplexed direct electrical detection of DNA synthesis SENSORS AND ACTUATORS B-CHEMICAL Anderson, E. P., Daniels, J. S., Yu, H., Karhanek, M., Lee, T. H., Davis, R. W., Pourmand, N. 2008; 129 (1): 79-86


    An electronic system for the multiplexed detection of DNA polymerization is designed and characterized. DNA polymerization is detected by the measurement of small transient currents arising from ion diffusion during polymerization. A transimpedance amplifier is used to detect these small currents; we implemented a twenty-four channel recording system on a single printed circuit board. Various contributions to the input-referred current noise are analyzed and characterized, as it limits the minimum detectable current and thus the biological limit of detection. We obtained 8.5 pA RMS mean noise current (averaged over all 24 channels) over the recording bandwidth (DC to 2 kHz). With digital filtering, the input-referred current noise of the acquisition system is reduced to 2.4 pA, which is much lower than the biological noise. Electrical crosstalk between channels is measured, and a model for the crosstalk is presented. Minimizing the crosstalk is critical because it can lead to erroneous microarray data. With proper precautions, crosstalk is reduced to a negligible value (less than 1.4%). Using a micro-fabricated array of 24 gold electrodes, we demonstrated system functionality by detecting the presence of a target DNA oligonucleotide which hybridized onto its corresponding target.

    View details for DOI 10.1016/j.snb.2007.07.105

    View details for Web of Science ID 000253172500013

    View details for PubMedCentralID PMC2344141

  • Interference of globin genes with biomarker discovery for allograft rejection in peripheral blood samples PHYSIOLOGICAL GENOMICS Li, L., Ying, L., Naesens, M., Xiao, W., Sigdel, T., Hsieh, S., Martin, J., Chen, R., Liu, K., Mindrinos, M., Davis, R., Sarwal, M. 2008; 32 (2): 190-197


    Microarray technology is a powerful tool in the discovery of new biomarkers for disease. After solid organ transplantation, where the detection of rejection is usually made on invasive biopsies, it could be hypothesized that noninvasive transcriptional profiling of peripheral blood will reveal rejection-specific expression patterns from circulating immune cells. However, in kidney transplant rejection, the analysis of gene expression data in whole blood has proven difficult for detecting significant genes specific for acute graft rejection. Previous studies have demonstrated that the abundance of globin genes in whole blood may mask the underlying biological differences between whole blood samples. In the present study, we compared the gene expression profiles of peripheral blood of nine stable renal allograft recipients with seven matched patients having an ongoing acute renal transplant rejection, using four different protocols of preparation, amplification, and synthesis of cRNA or cDNA and hybridization on the Affymetrix platform. We demonstrated that the globin reduction method is not sufficient to unmask clinically relevant rejection-specific transcriptome profiles in whole blood. Applying an additional mathematical depletion of the globin genes improves the efficacy of globin reduction but cannot remove the confounding influence of globin gene hybridization. Sampling of peripheral blood leukocytes alone, without the confounding influence of globin mRNA, provides sensitive and specific peripheral signatures for graft rejection, with many of these signals overlapping with rejection-driven tissue (kidney)-specific signatures from matched biopsies. Similar applications may exist for array-based biomarker discovery for other diseases associated with changes in leukocyte trafficking, activation, or function.

    View details for DOI 10.1152/physiolgenomics.00216.2007

    View details for Web of Science ID 000256815600004

    View details for PubMedID 17971501

  • High-throughput creation of a whole-genome collection of yeast knockout strains. Methods in molecular biology (Clifton, N.J.) Chu, A. M., Davis, R. W. 2008; 416: 205-220


    Gene disruption methods have proved to be a valuable tool for studying gene function in yeast. Gene replacement with a drug-resistant cassette renders the disruption strain selectable and is stable against reversion. Polymerase chain reaction-generated deletion cassettes are designed with homology sequences that flank the target gene. These deletion cassettes also contain unique "molecular bar code" sequence tags. Methods to generate these mutant strains are scalable and facile, allowing for the production of a collection of systematic disruptions across the Saccharomyces cerevisiae genome. The deletion strains can be studied individually or pooled together and assayed in parallel utilizing the sequence tags with microarray-based methods.

    View details for DOI 10.1007/978-1-59745-321-9_14

    View details for PubMedID 18392970

  • Real time simulation of heat detection in DNA Thermosequencing 3rd IEEE Sensors Applications Symposium Esfandyarpour, H., Zheng, B., Pease, R. F., Davis, R. W. IEEE. 2008: 89–94
  • STRUCTURAL OPTIMIZATION OF MICROFLUIDIC GATE CONTROLLED MAGNETIC BEAD SYSTEM FOR DNA SEQUENCING-BY-SYNTHESIS 6th International Conference on Nanochannels, Microchannels and Minichannels Esfandyarpour, H., Davis, R. W. AMER SOC MECHANICAL ENGINEERS. 2008: 1629–1634
  • Accurate measurement of cellular autofluorescence is critical for Imaging of host-pathogen interactions Conference on Imaging, Manipulation and Analysis of Biomolecules, Cells, and Tissues VI Timlin, J. A., Noek, R. M., Kaiser, J. N., Sinclair, M. B., Jones, H. D., Davis, R. W., Lane, T. W. SPIE-INT SOC OPTICAL ENGINEERING. 2008

    View details for DOI 10.1117/12.763915

    View details for Web of Science ID 000255316300008

  • Label-free electrical detection of antigens based on micro-channel gating 3rd IEEE Sensors Applications Symposium Javanmard, M., Nemat-Gorgani, M., Talasaz, A. H., Pease, F., Ronaghi, M., Davis, R. W. IEEE. 2008: 8–12
  • Direct electrical detection of target cells on a microfluidic biochip Conference on Microfluidics, BioMEMS, and Medical Microsystems VI Javanmard, M., Talasaz, A. H., Nemat-Gorgani, M., Pease, F., Ronaghi, M., Davis, R. W. SPIE-INT SOC OPTICAL ENGINEERING. 2008

    View details for DOI 10.1117/12.765611

    View details for Web of Science ID 000255942400010

  • Involvement of Skeletal Muscle Gene Regulatory Network in Susceptibility to Wound Infection Following Trauma PLOS ONE Apidianakis, Y., Mindrinos, M. N., Xiao, W., Tegos, G. P., Papisov, M. I., Hamblin, M. R., Davis, R. W., Tompkins, R. G., Rahme, L. G. 2007; 2 (12)


    Despite recent advances in our understanding the pathophysiology of trauma, the basis of the predisposition of trauma patients to infection remains unclear. A Drosophila melanogaster/Pseudomonas aeruginosa injury and infection model was used to identify host genetic components that contribute to the hyper-susceptibility to infection that follows severe trauma. We show that P. aeruginosa compromises skeletal muscle gene (SMG) expression at the injury site to promote infection. We demonstrate that activation of SMG structural components is under the control of cJun-N-terminal Kinase (JNK) Kinase, Hemipterous (Hep), and activation of this pathway promotes local resistance to P. aeruginosa in flies and mice. Our study links SMG expression and function to increased susceptibility to infection, and suggests that P. aeruginosa affects SMG homeostasis locally by restricting SMG expression in injured skeletal muscle tissue. Local potentiation of these host responses, and/or inhibition of their suppression by virulent P. aeruginosa cells, could lead to novel therapies that prevent or treat deleterious and potentially fatal infections in severely injured individuals.

    View details for DOI 10.1371/journal.pone.0001356

    View details for Web of Science ID 000207459700013

    View details for PubMedID 18159239

    View details for PubMedCentralID PMC2131783

  • A novel catechol-based universal support for oligonucleotide synthesis JOURNAL OF ORGANIC CHEMISTRY Anderson, K. M., Jaquinod, L., Jensen, M. A., Ngo, N., Davis, R. W. 2007; 72 (26): 9875-9880


    A novel universal support for deoxyribo- and ribonucleic acid synthesis has been developed. The support, constructed from 1,4-dimethoxycatechol, represents an improvement over existing universal supports because of its ability to cleave and deprotect under mild conditions in standard reagents. Because no nonvolatile additives are required for cleavage and deprotection, the synthesized oligonucleotides do not require purification prior to use in biochemical assays. Using reverse phase HPLC and electrospray mass spectroscopy, it was determined that oligonucleotides synthesized on the universal support (UL1) 3'-dephosphorylate quickly (9 h in 28-30% ammonium hydroxide (NH4OH) at 55 degrees C, 2 h in 28-30% NH4OH at 80 degrees C, or <1 h in ammonium hydroxide/methylamine (1:1) (AMA) at 80 degrees C). Oligonucleotides used as primers for the polymerase chain reaction (PCR) assay were found to perform identically to control primers, demonstrating full biological compatibility. In addition, a method was developed for sintering the universal support directly into a filter plug which can be pressure fit into the synthesis column of a commercial synthesizer. The universal support plugs allow the synthesis of high-quality oligonucleotides at least 120 nucleotides in length, with purity comparable to non-universal commercial supports and approximately 50% lower reagent consumption. The universal support plugs are routinely used to synthesize deoxyribo-, ribo-, 3'-modified, 5'-modified, and thioated oligonucleotides. The flexibility of the universal support and the efficiency of 3'-dephosphorylation are expected to increase the use of universal supports in oligonucleotide synthesis.

    View details for DOI 10.1021/jo071087a

    View details for Web of Science ID 000251653000003

    View details for PubMedID 18044913

  • MagSweeper: an automated system for high efficiency and specificity capture of live circulating tumor cells 30th Annual San Antonio Breast Cancer Symposium Powell, A. A., Talasaz, A. A., Mindrinos, M., Carlson, R., Pease, F. W., Davis, R. W., Jeffrey, S. S. SPRINGER. 2007: S24–S24
  • Gene-specific delineation of copy number aberrations in follicular lymphoma with molecular inversion probes 49th Annual Meeting of the American-Society-of-Hematology Ji, H. P., Welch, K. M., Wang, Y., Faham, M., Akasaka, T., Czerwinski, D., Davis, R. W., Levy, R. AMER SOC HEMATOLOGY. 2007: 766A–767A
  • Diverse cellular functions of the Hsp90 molecular chaperone uncovered using systems approaches CELL McClellan, A. J., Xia, Y., Deutschbauer, A. M., Davis, R. W., Gerstein, M., Frydman, J. 2007; 131 (1): 121-135


    A comprehensive understanding of the cellular functions of the Hsp90 molecular chaperone has remained elusive. Although Hsp90 is essential, highly abundant under normal conditions, and further induced by environmental stress, only a limited number of Hsp90 "clients" have been identified. To define Hsp90 function, a panel of genome-wide chemical-genetic screens in Saccharomyces cerevisiae were combined with bioinformatic analyses. This approach identified several unanticipated functions of Hsp90 under normal conditions and in response to stress. Under normal growth conditions, Hsp90 plays a major role in various aspects of the secretory pathway and cellular transport; during environmental stress, Hsp90 is required for the cell cycle, meiosis, and cytokinesis. Importantly, biochemical and cell biological analyses validated several of these Hsp90-dependent functions, highlighting the potential of our integrated global approach to uncover chaperone functions in the cell.

    View details for DOI 10.1016/j.cell.2007.07.036

    View details for Web of Science ID 000249934700016

    View details for PubMedID 17923092

  • Targeted cell detection based on microchannel gating BIOMICROFLUIDICS Javanmard, M., Talasaz, A. H., Nemat-Gorgani, M., Pease, F., Ronaghi, M., Davis, R. W. 2007; 1 (4)


    Currently, microbiological techniques such as culture enrichment and various plating techniques are used for detection of pathogens. These expensive and time consuming methods can take several days. Described below is the design, fabrication, and testing of a rapid and inexpensive sensor, involving the use of microelectrodes in a microchannel, which can be used to detect single bacterial cells electrically (label-free format) in real time. As a proof of principle, we have successfully demonstrated real-time detection of target yeast cells by measuring instantaneous changes in ionic impedance. We have also demonstrated the selectivity of our sensors in responding to target cells while remaining irresponsive to nontarget cells. Using this technique, it can be possible to multiplex an array of these sensors onto a chip and probe a complex mixture for various types of bacterial cells.

    View details for DOI 10.1063/1.2815760

    View details for Web of Science ID 000254901700003

    View details for PubMedID 19693402

  • A high- resolution atlas of nucleosome occupancy in yeast NATURE GENETICS Lee, W., Tillo, D., Bray, N., Morse, R. H., Davis, R. W., Hughes, T. R., Nislow, C. 2007; 39 (10): 1235-1244


    We present the first complete high-resolution map of nucleosome occupancy across the whole Saccharomyces cerevisiae genome, identifying over 70,000 positioned nucleosomes occupying 81% of the genome. On a genome-wide scale, the persistent nucleosome-depleted region identified previously in a subset of genes demarcates the transcription start site. Both nucleosome occupancy signatures and overall occupancy correlate with transcript abundance and transcription rate. In addition, functionally related genes can be clustered on the basis of the nucleosome occupancy patterns observed at their promoters. A quantitative model of nucleosome occupancy indicates that DNA structural features may account for much of the global nucleosome occupancy.

    View details for DOI 10.1038/ng2117

    View details for Web of Science ID 000249737400019

    View details for PubMedID 17873876

  • Connector Inversion Probe Technology: A Powerful One-Primer Multiplex DNA Amplification System for Numerous Scientific Applications PLOS ONE Akhras, M. S., Unemo, M., Thiyagarajan, S., Nyren, P., Davis, R. W., Fire, A. Z., Pourmand, N. 2007; 2 (9)


    We combined components of a previous assay referred to as Molecular Inversion Probe (MIP) with a complete gap filling strategy, creating a versatile powerful one-primer multiplex amplification system. As a proof-of-concept, this novel method, which employs a Connector Inversion Probe (CIPer), was tested as a genetic tool for pathogen diagnosis, typing, and antibiotic resistance screening with two distinct systems: i) a conserved sequence primer system for genotyping Human Papillomavirus (HPV), a cancer-associated viral agent and ii) screening for antibiotic resistance mutations in the bacterial pathogen Neisseria gonorrhoeae. We also discuss future applications and advances of the CIPer technology such as integration with digital amplification and next-generation sequencing methods. Furthermore, we introduce the concept of two-dimension informational barcodes, i.e. "multiplex multiplexing padlocks" (MMPs). For the readers' convenience, we also provide an on-line tutorial with user-interface software application CIP creator 1.0.1, for custom probe generation from virtually any new or established primer-pairs.

    View details for DOI 10.1371/journal.pone.0000915

    View details for Web of Science ID 000207455700023

    View details for PubMedID 17878950

  • Unusual selection on the KIR3DL1/S1 natural killer cell receptor in Africans NATURE GENETICS Norman, P. J., Abi-Rached, L., Gendzekhadze, K., Korbel, D., Gleimer, M., Rowley, D., Bruno, D., Carrington, C. V., Chandanayingyong, D., Chang, Y., Crespi, C., Saruhan-Direskeneli, G., Fraser, P. A., Hameed, K., Kamkamidze, G., Koram, K. A., Layrisse, Z., Matamoros, N., Mila, J., Park, M. H., Pitchappan, R. M., Ramdath, D. D., Shiau, M., Stephens, H. A., Struik, S., Verity, D. H., Vaughan, R. W., Tyan, D., Davis, R. W., Riley, E. M., Ronaghi, M., Parham, P. 2007; 39 (9): 1092-1099


    Interactions of killer cell immunoglobulin-like receptors (KIRs) with major histocompatibility complex (MHC) class I ligands diversify natural killer cell responses to infection. By analyzing sequence variation in diverse human populations, we show that the KIR3DL1/S1 locus encodes two lineages of polymorphic inhibitory KIR3DL1 allotypes that recognize Bw4 epitopes of protein">HLA-A and HLA-B and one lineage of conserved activating KIR3DS1 allotypes, also implicated in Bw4 recognition. Balancing selection has maintained these three lineages for over 3 million years. Variation was selected at D1 and D2 domain residues that contact HLA class I and at two sites on D0, the domain that enhances the binding of KIR3D to HLA class I. HLA-B variants that gained Bw4 through interallelic microconversion are also products of selection. A worldwide comparison uncovers unusual KIR3DL1/S1 evolution in modern sub-Saharan Africans. Balancing selection is weak and confined to D0, KIR3DS1 is rare and KIR3DL1 allotypes with similar binding sites predominate. Natural killer cells express the dominant KIR3DL1 at a high frequency and with high surface density, providing strong responses to cells perturbed in Bw4 expression.

    View details for DOI 10.1038/ng2111

    View details for Web of Science ID 000249122400018

    View details for PubMedID 17694054

  • Multigene amplification and massively parallel sequencing for cancer mutation discovery PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Dahl, F., Stenberg, J., Fredriksson, S., Welch, K., Zhang, M., Nilsson, M., Bicknell, D., Bodmer, W. F., Davis, R. W., Ji, H. 2007; 104 (22): 9387-9392


    We have developed a procedure for massively parallel resequencing of multiple human genes by combining a highly multiplexed and target-specific amplification process with a high-throughput parallel sequencing technology. The amplification process is based on oligonucleotide constructs, called selectors, that guide the circularization of specific DNA target regions. Subsequently, the circularized target sequences are amplified in multiplex and analyzed by using a highly parallel sequencing-by-synthesis technology. As a proof-of-concept study, we demonstrate parallel resequencing of 10 cancer genes covering 177 exons with average sequence coverage per sample of 93%. Seven cancer cell lines and one normal genomic DNA sample were studied with multiple mutations and polymorphisms identified among the 10 genes. Mutations and polymorphisms in the TP53 gene were confirmed by traditional sequencing.

    View details for DOI 10.1073/pnas.0702165104

    View details for Web of Science ID 000246935700055

    View details for PubMedID 17517648

  • Branch migration displacement assay with automated heuristic analysis for discrete DNA length measurement using DNA microarrays PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Pourmand, N., Caramuta, S., Villablanca, A., Mori, S., Karhanek, M., Wang, S. X., Davis, R. W. 2007; 104 (15): 6146-6151


    The analysis of short tandem repeats (STRs) plays an important role in forensic science, human identification, genetic mapping, and disease diagnostics. Traditional STR analysis utilizes gel- or column-based approaches to analyze DNA repeats. Individual STR alleles are separated and distinguished according to fragment length; thus the assay is generally hampered by its low multiplex capacity. However, use of DNA microarray would employ a simple hybridization and detection for field forensics and biology. Here we demonstrate a rapid, highly sensitive method for STR analysis that utilizes DNA microarray technology. We describe two adaptations to accomplish this: the use of competitive hybridization to remove unpaired ssDNA from an array and the use of neural network classification to automate the analysis. The competitive displacement technique mimics the branch migration process that occurs during DNA recombination. Our technique will facilitate the rapid deduction of identity, length, and number of repeats for the multiple STRs in an unknown DNA sample.

    View details for DOI 10.1073/pnas.0700921104

    View details for Web of Science ID 000245737500012

    View details for PubMedID 17389407

  • Multiplex amplification of all coding sequences within 10 cancer genes by Gene-Collector NUCLEIC ACIDS RESEARCH Fredriksson, S., Baner, J., Dahl, F., Chu, A., Ji, H., Welch, K., Davis, R. W. 2007; 35 (7)


    Herein we present Gene-Collector, a method for multiplex amplification of nucleic acids. The procedure has been employed to successfully amplify the coding sequence of 10 human cancer genes in one assay with uniform abundance of the final products. Amplification is initiated by a multiplex PCR in this case with 170 primer pairs. Each PCR product is then specifically circularized by ligation on a Collector probe capable of juxtapositioning only the perfectly matched cognate primer pairs. Any amplification artifacts typically associated with multiplex PCR derived from the use of many primer pairs such as false amplicons, primer-dimers etc. are not circularized and degraded by exonuclease treatment. Circular DNA molecules are then further enriched by randomly primed rolling circle replication. Amplification was successful for 90% of the targeted amplicons as seen by hybridization to a custom resequencing DNA micro-array. Real-time quantitative PCR revealed that 96% of the amplification products were all within 4-fold of the average abundance. Gene-Collector has utility for numerous applications such as high throughput resequencing, SNP analyses, and pathogen detection.

    View details for DOI 10.1093/nar/gkm078

    View details for Web of Science ID 000246294700001

    View details for PubMedID 17317684

  • Multiplexed protein detection by proximity ligation for cancer biomarker validation NATURE METHODS Fredriksson, S., Dixon, W., Ji, H., Koong, A. C., Mindrinos, M., Davis, R. W. 2007; 4 (4): 327-329


    We present a proximity ligation-based multiplexed protein detection procedure in which several selected proteins can be detected via unique nucleic-acid identifiers and subsequently quantified by real-time PCR. The assay requires a 1-microl sample, has low-femtomolar sensitivity as well as five-log linear range and allows for modular multiplexing without cross-reactivity. The procedure can use a single polyclonal antibody batch for each target protein, simplifying affinity-reagent creation for new biomarker candidates.

    View details for DOI 10.1038/NMETH1020

    View details for Web of Science ID 000245584900013

    View details for PubMedID 17369836

  • Accelerating the discovery of biologically active small molecules using a high-throughput yeast halo assay JOURNAL OF NATURAL PRODUCTS Gassner, N. C., Tamble, C. M., Bock, J. E., Cotton, N., White, K. N., Tenney, K., St Onge, R. P., Proctor, M. J., Giaever, G., Nislow, C., Davis, R. W., Crews, P., Holman, T. R., Lokey, R. S. 2007; 70 (3): 383-390


    The budding yeast Saccharomyces cerevisiae, a powerful model system for the study of basic eukaryotic cell biology, has been used increasingly as a screening tool for the identification of bioactive small molecules. We have developed a novel yeast toxicity screen that is easily automated and compatible with high-throughput screening robotics. The new screen is quantitative and allows inhibitory potencies to be determined, since the diffusion of the sample provides a concentration gradient and a corresponding toxicity halo. The efficacy of this new screen was illustrated by testing materials including 3104 compounds from the NCI libraries, 167 marine sponge crude extracts, and 149 crude marine-derived fungal extracts. There were 46 active compounds among the NCI set. One very active extract was selected for bioactivity-guided fractionation, resulting in the identification of crambescidin 800 as a potent antifungal agent.

    View details for DOI 10.1021/np060555t

    View details for Web of Science ID 000245118800011

    View details for PubMedID 17291044

    View details for PubMedCentralID PMC2533267

  • PathogenMip Assay: A Multiplex Pathogen Detection Assay PLOS ONE Akhras, M. S., Thiyagarajan, S., Villablanca, A. C., Davis, R. W., Nyren, P., Pourmand, N. 2007; 2 (2)


    The Molecular Inversion Probe (MIP) assay has been previously applied to a large-scale human SNP detection. Here we describe the PathogenMip Assay, a complete protocol for probe production and applied approaches to pathogen detection. We have demonstrated the utility of this assay with an initial set of 24 probes targeting the most clinically relevant HPV genotypes associated with cervical cancer progression. Probe construction was based on a novel, cost-effective, ligase-based protocol. The assay was validated by performing pyrosequencing and Microarray chip detection in parallel experiments. HPV plasmids were used to validate sensitivity and selectivity of the assay. In addition, 20 genomic DNA extracts from primary tumors were genotyped with the PathogenMip Assay results and were in 100% agreement with conventional sequencing using an L1-based HPV genotyping protocol. The PathogenMip Assay is a widely accessible protocol for producing and using highly discriminating probes, with experimentally validated results in pathogen genotyping, which could potentially be applied to the detection and characterization of any microbe.

    View details for DOI 10.1371/journal.pone.0000223

    View details for Web of Science ID 000207444500004

    View details for PubMedID 17311101

  • Systematic pathway analysis using high-resolution fitness profiling of combinatorial gene deletions NATURE GENETICS St Onge, R. P., Mani, R., Oh, J., Proctor, M., Fung, E., Davis, R. W., Nislow, C., Roth, F. P., Giaever, G. 2007; 39 (2): 199-206


    Systematic genetic interaction studies have illuminated many cellular processes. Here we quantitatively examine genetic interactions among 26 Saccharomyces cerevisiae genes conferring resistance to the DNA-damaging agent methyl methanesulfonate (MMS), as determined by chemogenomic fitness profiling of pooled deletion strains. We constructed 650 double-deletion strains, corresponding to all pairings of these 26 deletions. The fitness of single- and double-deletion strains were measured in the presence and absence of MMS. Genetic interactions were defined by combining principles from both statistical and classical genetics. The resulting network predicts that the Mph1 helicase has a role in resolving homologous recombination-derived DNA intermediates that is similar to (but distinct from) that of the Sgs1 helicase. Our results emphasize the utility of small molecules and multifactorial deletion mutants in uncovering functional relationships and pathway order.

    View details for DOI 10.1038/ng1948

    View details for Web of Science ID 000244063900017

    View details for PubMedID 17206143

  • High-density yeast-tiling array reveals previously undiscovered introns and extensive regulation of rneiotic splicing PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Juneau, K., Palm, C., Miranda, M., Davis, R. W. 2007; 104 (5): 1522-1527


    Knowing gene structure is vital to understanding gene function, and accurate genome annotation is essential for understanding cellular function. To this end, we have developed a genome-wide assay for mapping introns in Saccharomyces cerevisiae. Using high-density tiling arrays, we compared wild-type yeast to a mutant deficient for intron degradation. Our method identified 76% of the known introns, confirmed 18 previously predicted introns, and revealed 9 formerly undiscovered introns. Furthermore, we discovered that all 13 meiosis-specific intronic yeast genes undergo regulated splicing, which provides posttranscriptional regulation of the genes involved in yeast cell differentiation. Moreover, we found that approximately 16% of intronic genes in yeast are incompletely spliced during exponential growth in rich medium, which suggests that meiosis is not the only biological process regulated by splicing. Our tiling-array assay provides a snapshot of the spliced transcriptome in yeast. This robust methodology can be used to explore environmentally distinct splicing responses and should be readily adaptable to the study of other organisms, including humans.

    View details for DOI 10.1073/pnas.0610354104

    View details for Web of Science ID 000244081000017

    View details for PubMedID 17244705

  • Genome-wide analysis of barcoded Saccharomyces cerevisiae gene-deletion mutants in pooled cultures NATURE PROTOCOLS Pierce, S. E., Davis, R. W., Nislow, C., Giaever, G. 2007; 2 (11): 2958-2974


    The availability of a near-complete (96%) collection of gene-deletion mutants in Saccharomyces cerevisiae greatly facilitates the systematic analyses of gene function in yeast. The unique 20 bp DNA 'barcodes' or 'tags' in each deletion strain enable the individual fitness of thousands of deletion mutants to be resolved from a single pooled culture. Here, we present protocols for the study of pooled cultures of tagged yeast deletion mutants with a tag microarray. This process involves five main steps: pooled growth, isolation of genomic DNA, PCR amplification of the barcodes, array hybridization and data analysis. Pooled deletion screening can be used to study gene function, uncover a compound's mode of action and identify drug targets. In addition to these applications, the general method of studying pooled samples with barcode arrays can also be adapted for use with other types of samples, such as mutant collections in other organisms, short interfering RNA vectors and molecular inversion probes.

    View details for DOI 10.1038/nprot.2007.427

    View details for Web of Science ID 000253140000035

    View details for PubMedID 18007632

  • Gate-controlled microfluidic chamber with magnetic bead for DNA sequencing-by-synthesis technology 5th International Conference on Nanochannels, Microchannels and Minichannels Esfandyarpour, H., Davis, R. W. AMER SOC MECHANICAL ENGINEERS. 2007: 163–167
  • Array-based pyrosequencing 5th International Forum on Post-Genome Technologies Gharizadeh, B., Goel, S., Eltoukhy, H., Agah, M. N., Babrzadeh, F., Jalili, R., Talasaz, A. H., Davis, R. W., Ronaghi, M. PHOENIX PUBL & MEDIA NETWORK. 2007: 8–9
  • Electronic detection of bio-molecules with synthetic and organic Nanopores. 51st Annual Meeting of the Biophysical-Society Tropini, C., Talasaz, A., Marziali, A., Ronaghi, M., Davis, R. W. CELL PRESS. 2007: 649A–649A
  • Chemical genomic profiling for identifying intracellular targets of toxicants producing Parkinson's disease TOXICOLOGICAL SCIENCES Doostzadeh, J., Davis, R. W., Giaever, G. N., Nislow, C., Langston, J. W. 2007; 95 (1): 182-187


    The yeast deletion collection includes approximately 4700 strains deleted for both copies of every nonessential gene. This collection is a powerful resource for identifying the cellular pathways that functionally interact with drugs. In the present study, the complete pool of approximately 4700 barcoded homozygous deletion strains of Saccharomyces cerevisiae were surveyed to identify genes/pathways interacting with 1-methyl-4-phenylpyridinium (MPP(+)) and N,N-dimethyl-4-4-bipiridinium (paraquat), neurotoxicants that can produce Parkinson's disease. Each yeast mutant is molecularly "barcoded" the collections can be grown competitively and ranked for sensitivity by microarray hybridization. Analysis data from these screens allowed us to determine that the multivesicular body pathway is an important element of toxicity induced by both MPP(+) and paraquat. When yeast genes that when deleted showed sensitivity to MPP(+) and paraquat toxicity were analyzed for their homology to human genes, 80% were found to have highly conserved human homologs (with e < 10(-8)). Future work will address if these human genes may also functionally interact with MPP(+) and paraquat toxicity.

    View details for DOI 10.1093/toxsci/kfl131

    View details for Web of Science ID 000243072300018

    View details for PubMedID 17043098

  • Modeling and test of traveling-wave electrode Mach-Zehnder InP/InGaAsP quantum well modulators 20th Annual Meeting of the IEEE-Lasers-and-Electro-Optics-Society Kunkee, E. T., Shah, A. R., Shih, S., Smith, A. D., Davis, R. IEEE. 2007: 266–267
  • Rapid and Highly Informative Diagnostic Assay for H5N1 Influenza Viruses PLOS ONE Pourmand, N., Diamond, L., Garten, R., Erickson, J. P., Kumm, J., Donis, R. O., Davis, R. W. 2006; 1 (1)


    A highly discriminative and information-rich diagnostic assay for H5N1 avian influenza would meet immediate patient care needs and provide valuable information for public health interventions, e.g., tracking of new and more dangerous variants by geographic area as well as avian-to-human or human-to-human transmission. In the present study, we have designed a rapid assay based on multilocus nucleic acid sequencing that focuses on the biologically significant regions of the H5N1 hemagglutinin gene. This allows the prediction of viral strain, clade, receptor binding properties, low- or high-pathogenicity cleavage site and glycosylation status. H5 HA genes were selected from nine known high-pathogenicity avian influenza subtype H5N1 viruses, based on their diversity in biologically significant regions of hemagglutinin and/or their ability to cause infection in humans. We devised a consensus pre-programmed pyrosequencing strategy, which may be used as a faster, more accurate alternative to de novo sequencing. The available data suggest that the assay described here is a reliable, rapid, information-rich and cost-effective approach for definitive diagnosis of H5N1 avian influenza. Knowledge of the predicted functional sequences of the HA will enhance H5N1 avian influenza surveillance efforts.

    View details for DOI 10.1371/journal.pone.0000095

    View details for Web of Science ID 000207443600094

    View details for PubMedID 17183727

  • PathogenMIPer: a tool for the design of molecular inversion probes to detect multiple pathogens BMC BIOINFORMATICS Thiyagarajan, S., Karhanek, M., Akhras, M., Davis, R. W., Pourmand, N. 2006; 7


    Here we describe PathogenMIPer, a software program for designing molecular inversion probe (MIP) oligonucleotides for use in pathogen identification and detection. The software designs unique and specific oligonucleotide probes targeting microbial or other genomes. The tool tailors all probe sequence components (including target-specific sequences, barcode sequences, universal primers and restriction sites) and combines these components into ready-to-order probes for use in a MIP assay. The system can harness the genetic variability available in an entire genome in designing specific probes for the detection of multiple co-infections in a single tube using a MIP assay.PathogenMIPer can accept sequence data in FASTA file format, and other parameter inputs from the user through a graphical user interface. It can design MIPs not only for pathogens, but for any genome for use in parallel genomic analyses. The software was validated experimentally by applying it to the detection of human papilloma virus (HPV) as a model system, which is associated with various human malignancies including cervical and skin cancers. Initial tests of laboratory samples using the MIPs developed by the PathogenMIPer to recognize 24 different types of HPVs gave very promising results, detecting even a small viral load of single as well as multiple infections (Akhras et al, personal communication).PathogenMIPer is a software for designing molecular inversion probes for detection of multiple target DNAs in a sample using MIP assays. It enables broader use of MIP technology in the detection through genotyping of pathogens that are complex, difficult-to-amplify, or present in multiple subtypes in a sample.

    View details for DOI 10.1186/1471-2105-7-500

    View details for Web of Science ID 000242188500002

    View details for PubMedID 17105657

  • Current rectification with poly-L-lysine-coated quartz nanopipettes NANO LETTERS Umehara, S., Pourmand, N., Webb, C. D., Davis, R. W., Yasuda, K., Karhanek, M. 2006; 6 (11): 2486-2492


    Ion current rectification with quartz nanopipette electrodes was investigated through the control of the surface charge. The presence and absence of a positively charged poly-l-lysine (PLL) coating resulted in the rectified current with opposite polarity. The results agreed with the theories developed for current-rectifying conical nanopores, suggesting the similar underlying mechanism among asymmetric nanostructure in general. This surface condition dependence can be used as the fundamental principle of multi-purpose real-time in vivo biosensors.

    View details for DOI 10.1021/nl061681k

    View details for Web of Science ID 000241856700017

    View details for PubMedID 17090078

  • Cell-specific expression and pathway analyses reveal alterations in trauma-related human T cell and monocyte pathways PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Laudanski, K., Miller-Graziano, C., Xiao, W., Mindrinos, M. N., Richards, D. R., De, A., Moldawer, L. L., Maier, R. V., Bankey, P., Baker, H. V., Brownstein, B. H., Cobb, J. P., Calvano, S. E., Davis, R. W., Tompkins, R. G. 2006; 103 (42): 15564-15569


    Monitoring genome-wide, cell-specific responses to human disease, although challenging, holds great promise for the future of medicine. Patients with injuries severe enough to develop multiple organ dysfunction syndrome have multiple immune derangements, including T cell apoptosis and anergy combined with depressed monocyte antigen presentation. Genome-wide expression analysis of highly enriched circulating leukocyte subpopulations, combined with cell-specific pathway analyses, offers an opportunity to discover leukocyte regulatory networks in critically injured patients. Severe injury induced significant changes in T cell (5,693 genes), monocyte (2,801 genes), and total leukocyte (3,437 genes) transcriptomes, with only 911 of these genes common to all three cell populations (12%). T cell-specific pathway analyses identified increased gene expression of several inhibitory receptors (PD-1, CD152, NRP-1, and Lag3) and concomitant decreases in stimulatory receptors (CD28, CD4, and IL-2Ralpha). Functional analysis of T cells and monocytes confirmed reduced T cell proliferation and increased cell surface expression of negative signaling receptors paired with decreased monocyte costimulation ligands. Thus, genome-wide expression from highly enriched cell populations combined with knowledge-based pathway analyses leads to the identification of regulatory networks differentially expressed in injured patients. Importantly, application of cell separation, genome-wide expression, and cell-specific pathway analyses can be used to discover pathway alterations in human disease.

    View details for DOI 10.1073/pnas.0607028103

    View details for Web of Science ID 000241476200053

    View details for PubMedID 17032758

  • Prediction of protein orientation upon immobilization on biological and nonbiological surfaces PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Talasaz, A. H., Nemat-Gorgani, M., Liu, Y., Stahl, P., Dutton, R. W., Ronaghi, M., Davis, R. W. 2006; 103 (40): 14773-14778


    We report on a rapid simulation method for predicting protein orientation on a surface based on electrostatic interactions. New methods for predicting protein immobilization are needed because of the increasing use of biosensors and protein microarrays, two technologies that use protein immobilization onto a solid support, and because the orientation of an immobilized protein is important for its function. The proposed simulation model is based on the premise that the protein interacts with the electric field generated by the surface, and this interaction defines the orientation of attachment. Results of this model are in agreement with experimental observations of immobilization of mitochondrial creatine kinase and type I hexokinase on biological membranes. The advantages of our method are that it can be applied to any protein with a known structure; it does not require modeling of the surface at atomic resolution and can be run relatively quickly on readily available computing resources. Finally, we also propose an orientation of membrane-bound cytochrome c, a protein for which the membrane orientation has not been unequivocally determined.

    View details for DOI 10.1073/pnas.0605841103

    View details for Web of Science ID 000241069300025

    View details for PubMedID 17001006

  • Analysis of hybridization on the molecular barcode GeneChip microarray BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Walton, S. P., Mindrinos, M. N., Davis, R. W. 2006; 348 (2): 689-696


    Microarrays have been developed for analysis of transcriptional profiles in many organisms. For experimental simplicity and systems for which microarrays do not exist, it would be desirable to use a standard microarray platform for the analysis of multiple systems. The molecular barcode (MB) Affymetrix GeneChip could serve as such a platform. The reproducibility and quantitative capacity of the MB GeneChip were examined. Using mixed PCR templates of defined template quantity, the individual concentration responses of 384 array features were measured and shown to be highly reproducible. Moreover, the binding behaviors of the mismatched array features mirror those of the matched features, at reduced intensity. Additional analysis defined the importance of particular sequence motifs in the prediction of high-affinity and low-affinity target hybridization. The data support the future application of MB GeneChips for quantitative applications. It is proposed that at least seven orders-of-magnitude in accurate concentration sensitivity could be achieved.

    View details for DOI 10.1016/j.bbrc.2006.07.108

    View details for Web of Science ID 000240275000050

    View details for PubMedID 16893517

  • Data quality in genomics and microarrays NATURE BIOTECHNOLOGY Ji, H., Davis, R. W. 2006; 24 (9): 1112-1113

    View details for DOI 10.1038/nbt0906-1108

    View details for Web of Science ID 000240495200031

    View details for PubMedID 16964224

  • Introns regulate RNA and protein abundance in yeast GENETICS Juneau, K., Miranda, M., Hillenmeyer, M. E., Nislow, C., Davis, R. W. 2006; 174 (1): 511-518


    The purpose of introns in the architecturally simple genome of Saccharomyces cerevisiae is not well understood. To assay the functional relevance of introns, a series of computational analyses and several detailed deletion studies were completed on the intronic genes of S. cerevisiae. Mining existing data from genomewide studies on yeast revealed that intron-containing genes produce more RNA and more protein and are more likely to be haplo-insufficient than nonintronic genes. These observations for all intronic genes held true for distinct subsets of genes including ribosomal, nonribosomal, duplicated, and nonduplicated. Corroborating the result of computational analyses, deletion of introns from three essential genes decreased cellular RNA levels and caused measurable growth defects. These data provide evidence that introns improve transcriptional and translational yield and are required for competitive growth of yeast.

    View details for DOI 10.1534/genetics.106.058560

    View details for Web of Science ID 000241134400044

    View details for PubMedID 16816425

  • Molecular inversion probe analysis of gene copy alterations reveals distinct categories of colorectal carcinoma CANCER RESEARCH Ji, H., Kumm, J., Zhang, M., Farnam, K., Salari, K., Faham, M., Ford, J. M., Davis, R. W. 2006; 66 (16): 7910-7919


    Genomic instability is a major feature of neoplastic development in colorectal carcinoma and other cancers. Specific genomic instability events, such as deletions in chromosomes and other alterations in gene copy number, have potential utility as biologically relevant prognostic biomarkers. For example, genomic deletions on chromosome arm 18q are an indicator of colorectal carcinoma behavior and potentially useful as a prognostic indicator. Adapting a novel genomic technology called molecular inversion probes which can determine gene copy alterations, such as genomic deletions, we designed a set of probes to interrogate several hundred individual exons of >200 cancer genes with an overall distribution covering all chromosome arms. In addition, >100 probes were designed in close proximity of microsatellite markers on chromosome arm 18q. We analyzed a set of colorectal carcinoma cell lines and primary colorectal tumor samples for gene copy alterations and deletion mutations in exons. Based on clustering analysis, we distinguished the different categories of genomic instability among the colorectal cancer cell lines. Our analysis of primary tumors uncovered several distinct categories of colorectal carcinoma, each with specific patterns of 18q deletions and deletion mutations in specific genes. This finding has potential clinical ramifications given the application of 18q loss of heterozygosity events as a potential indicator for adjuvant treatment in stage II colorectal carcinoma.

    View details for DOI 10.1158/0008-5472.CAN-06-0595

    View details for Web of Science ID 000239828200013

    View details for PubMedID 16912164

  • A unique and universal molecular barcode array NATURE METHODS Pierce, S. E., Fung, E. L., Jaramillo, D. F., Chu, A. M., Davis, R. W., Nislow, C., Giaever, G. 2006; 3 (8): 601-603


    Molecular barcode arrays allow the analysis of thousands of biological samples in parallel through the use of unique 20-base-pair (bp) DNA tags. Here we present a new barcode array, which is unique among microarrays in that it includes at least five replicates of every tag feature. The use of smaller dispersed replicate features dramatically improves performance versus a single larger feature and allows the correction of previously undetectable hybridization defects.

    View details for DOI 10.1038/NMETH905

    View details for Web of Science ID 000239598600009

    View details for PubMedID 16862133

  • Transcriptional profiling of aging in human muscle reveals a common aging signature PLOS GENETICS Zahn, J. M., Sonu, R., Vogel, H., Crane, E., Mazan-Mamczarz, K., Rabkin, R., Davis, R. W., Becker, K. G., Owen, A. B., Kim, S. K. 2006; 2 (7): 1058-1069


    We analyzed expression of 81 normal muscle samples from humans of varying ages, and have identified a molecular profile for aging consisting of 250 age-regulated genes. This molecular profile correlates not only with chronological age but also with a measure of physiological age. We compared the transcriptional profile of muscle aging to previous transcriptional profiles of aging in the kidney and the brain, and found a common signature for aging in these diverse human tissues. The common aging signature consists of six genetic pathways; four pathways increase expression with age (genes in the extracellular matrix, genes involved in cell growth, genes encoding factors involved in complement activation, and genes encoding components of the cytosolic ribosome), while two pathways decrease expression with age (genes involved in chloride transport and genes encoding subunits of the mitochondrial electron transport chain). We also compared transcriptional profiles of aging in humans to those of the mouse and fly, and found that the electron transport chain pathway decreases expression with age in all three organisms, suggesting that this may be a public marker for aging across species.

    View details for DOI 10.1371/journal.pgen.0020115

    View details for Web of Science ID 000239494800016

    View details for PubMedID 16789832

  • Direct electrical detection of DNA synthesis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Pourmand, N., Karhanek, M., Persson, H. H., Webb, C. D., Lee, T. H., Zahradnikova, A., Davis, R. W. 2006; 103 (17): 6466-6470


    Rapid, sequence-specific DNA detection is essential for applications in medical diagnostics and genetic screening. Electrical biosensors that use immobilized nucleic acids are especially promising in these applications because of their potential for miniaturization and automation. Current DNA detection methods based on sequencing by synthesis rely on optical readouts; however, a direct electrical detection method for this technique is not available. We report here an approach for direct electrical detection of enzymatically catalyzed DNA synthesis by induced surface charge perturbation. We discovered that incorporation of a complementary deoxynucleotide (dNTP) into a self-primed single-stranded DNA attached to the surface of a gold electrode evokes an electrode surface charge perturbation. This event can be detected as a transient current by a voltage-clamp amplifier. Based on current understanding of polarizable interfaces, we propose that the electrode detects proton removal from the 3'-hydroxyl group of the DNA molecule during phosphodiester bond formation.

    View details for DOI 10.1073/pnas.0601184103

    View details for Web of Science ID 000237151000011

    View details for PubMedID 16614066

    View details for PubMedCentralID PMC1458907

  • A high-resolution map of transcription in the yeast genome PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA David, L., Huber, W., Granovskaia, M., Toedling, J., Palm, C. J., Bofkin, L., Jones, T., Davis, R. W., Steinmetz, L. M. 2006; 103 (14): 5320-5325


    There is abundant transcription from eukaryotic genomes unaccounted for by protein coding genes. A high-resolution genome-wide survey of transcription in a well annotated genome will help relate transcriptional complexity to function. By quantifying RNA expression on both strands of the complete genome of Saccharomyces cerevisiae using a high-density oligonucleotide tiling array, this study identifies the boundary, structure, and level of coding and noncoding transcripts. A total of 85% of the genome is expressed in rich media. Apart from expected transcripts, we found operon-like transcripts, transcripts from neighboring genes not separated by intergenic regions, and genes with complex transcriptional architecture where different parts of the same gene are expressed at different levels. We mapped the positions of 3' and 5' UTRs of coding genes and identified hundreds of RNA transcripts distinct from annotated genes. These nonannotated transcripts, on average, have lower sequence conservation and lower rates of deletion phenotype than protein coding genes. Many other transcripts overlap known genes in antisense orientation, and for these pairs global correlations were discovered: UTR lengths correlated with gene function, localization, and requirements for regulation; antisense transcripts overlapped 3' UTRs more than 5' UTRs; UTRs with overlapping antisense tended to be longer; and the presence of antisense associated with gene function. These findings may suggest a regulatory role of antisense transcription in S. cerevisiae. Moreover, the data show that even this well studied genome has transcriptional complexity far beyond current annotation.

    View details for DOI 10.1073/pnas.0601091103

    View details for Web of Science ID 000236636400017

    View details for PubMedID 16569694

    View details for PubMedCentralID PMC1414796

  • A haplotype framework for cystic fibrosis mutations in Iran JOURNAL OF MOLECULAR DIAGNOSTICS Elahi, E., Khodadad, A., Kupershmidt, I., Ghasemi, F., Alinasab, B., Naghizadeh, R., Eason, R. G., Amini, M., Esmaili, M., Dooki, M. R., Sanati, M. H., Davis, R. W., Ronaghi, M., Thorstenson, Y. R. 2006; 8 (1): 119-127


    This is the first comprehensive profile of cystic fibrosis transmembrane conductance regulator (CFTR) mutations and their corresponding haplotypes in the Iranian population. All of the 27 CFTR exons of 60 unrelated Iranian CF patients were sequenced to identify disease-causing mutations. Eleven core haplotypes of CFTR were identified by genotyping six high-frequency simple nucleotide polymorphisms. The carrier frequency of 2.5 in 100 (1 in 40) was estimated from the frequency of heterozygous patients and suggests that contrary to popular belief, cystic fibrosis may be a common, under-diagnosed disease in Iran. A heterogeneous mutation spectrum was observed at the CFTR locus in 60 cystic fibrosis (CF) patients from Iran. Twenty putative disease-causing mutations were identified on 64 (53%) of the 120 chromosomes. The five most common Iranian mutations together represented 37% of the expected mutated alleles. The most frequent mutation, DeltaF508 (p.F508del), represented only 16% of the expected mutated alleles. The next most frequent mutations were c.1677del2 (p.515fs) at 7.5%, c.4041C>G (p.N1303K) at 5.6%, c.2183AA>G (p.684fs) at 5%, and c.3661A>T (p.K1177X) at 2.5%. Three of the five most frequent Iranian mutations are not included in a commonly used panel of CF mutations, underscoring the importance of identifying geographic-specific mutations in this population.

    View details for DOI 10.2353/jmoldx.2006.050063

    View details for Web of Science ID 000235245100016

    View details for PubMedID 16436643

  • Modeling of the bioactivated nanopore devices. Conference proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference Talasaz, A. H., Liu, Y., Ronaghi, M., Davis, R. W. 2006; 1: 1830-1833


    This paper presents the modeling of the electrical properties of bioactivated nanopores, customized nanopore devices with a biological macromolecule attached in the pore as the probe. These devices are capable of detecting and analyzing interactions between the attached biomolecule and the molecules in the analyte at a single molecule level.

    View details for PubMedID 17946483

  • Development of a new device for high throughput isolation of live circulating tumor cells. 29th Annual San Antonio Breast Cancer Symposium Talasaz, A. A., Powell, A. A., Mindrinos, M., Carlson, R. W., Pease, F. W., Davis, R. W., Jeffrey, S. S. SPRINGER. 2006: S213–S213
  • Modeling of the bioactivated nanopore devices 28th Annual International Conference of the IEEE-Engineering-in-Medicine-and-Biology-Society Talasaz, A. H., Liu, Y., Rodaghi, M., Davis, R. W. IEEE. 2006: 3403–3406
  • Cell trapping in activated micropores for functional analysis 28th Annual International Conference of the IEEE-Engineering-in-Medicine-and-Biology-Society Talasaz, A. H., Powell, A. A., Stahl, P., Ronaghi, M., Jeffrey, S. S., Mindrinos, M., Davis, R. W. IEEE. 2006: 1397–1400
  • Cell trapping in activated micropores for functional analysis. Conference proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference Talasaz, A. H., Powell, A. A., Stahl, P., Ronaghi, M., Jeffrey, S. S., Mindrinos, M., Davis, R. W. 2006; 1: 1838-1841


    This paper presents a novel device which provides the opportunity to perform high-throughput biochemical assays on different individual cells. In particular, the proposed device is suited to screen the rare cells in biological samples for early stage cancer diagnosis and explore their biochemical functionality. In the process, single cells are precisely positioned and captured in activated micropores. To show the performance of the proposed device, cultured yeast cells and human epithelial circulating tumor cells are successfully captured.

    View details for PubMedID 17945673

  • Quantitative trait loci mapped to single-nucleotide resolution in yeast NATURE GENETICS Deutschbauer, A. M., Davis, R. W. 2005; 37 (12): 1333-1340


    Identifying the genetic variation underlying quantitative trait loci remains problematic. Consequently, our molecular understanding of genetically complex, quantitative traits is limited. To address this issue directly, we mapped three quantitative trait loci that control yeast sporulation efficiency to single-nucleotide resolution in a noncoding regulatory region (RME1) and to two missense mutations (TAO3 and MKT1). For each quantitative trait locus, the responsible polymorphism is rare among a diverse set of 13 yeast strains, suggestive of genetic heterogeneity in the control of yeast sporulation. Additionally, under optimal conditions, we reconstituted approximately 92% of the sporulation efficiency difference between the two genetically distinct parents by engineering three nucleotide changes in the appropriate yeast genome. Our results provide the highest resolution to date of the molecular basis of a quantitative trait, showing that the interaction of a few genetic variants can have a profound phenotypic effect.

    View details for DOI 10.1038/ng1674

    View details for Web of Science ID 000233657300012

    View details for PubMedID 16273108

  • A network-based analysis of systemic inflammation in humans NATURE Calvano, S. E., Xiao, W. Z., Richards, D. R., Felciano, R. M., Baker, H. V., Cho, R. J., Chen, R. O., Brownstein, B. H., Cobb, J. P., Tschoeke, S. K., Miller-Graziano, C., Moldawer, L. L., Mindrinos, M. N., Davis, R. W., Tompkins, R. G., Lowry, S. F. 2005; 437 (7061): 1032-1037


    Oligonucleotide and complementary DNA microarrays are being used to subclassify histologically similar tumours, monitor disease progress, and individualize treatment regimens. However, extracting new biological insight from high-throughput genomic studies of human diseases is a challenge, limited by difficulties in recognizing and evaluating relevant biological processes from huge quantities of experimental data. Here we present a structured network knowledge-base approach to analyse genome-wide transcriptional responses in the context of known functional interrelationships among proteins, small molecules and phenotypes. This approach was used to analyse changes in blood leukocyte gene expression patterns in human subjects receiving an inflammatory stimulus (bacterial endotoxin). We explore the known genome-wide interaction network to identify significant functional modules perturbed in response to this stimulus. Our analysis reveals that the human blood leukocyte response to acute systemic inflammation includes the transient dysregulation of leukocyte bioenergetics and modulation of translational machinery. These findings provide insight into the regulation of global leukocyte activities as they relate to innate immune system tolerance and increased susceptibility to infection in humans.

    View details for DOI 10.1038/nature03985

    View details for Web of Science ID 000232496100047

    View details for PubMedID 16136080

  • Multiplexed variation scanning for 1,000 amplicons in hundreds of patients using mismatch repair detection (MRD) on tag arrays PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Faham, M., Zheng, J. B., Moorhead, M., Fakhrai-Rad, H., Namsaraev, E., Wong, K., Wang, Z. Y., Chow, S. G., Lee, L., Suyenaga, K., Reichert, J., Boudreau, A., EBERLE, J., Bruckner, C., Jain, M., Karlin-Neumann, G., Jones, H. B., Willis, T. D., Buxbaum, J. D., Davis, R. W. 2005; 102 (41): 14717-14722


    Identification of the genetic basis of common disease may require comprehensive sequence analysis of coding regions and regulatory elements in patients and controls to find genetic effects caused by rare or heterogeneous mutations. In this study, we demonstrate how mismatch repair detection on tag arrays can be applied in a case-control study. Mismatch repair detection allows >1,000 amplicons to be screened for variations in a single laboratory reaction. Variation scanning in 939 amplicons, mostly in coding regions within a linkage peak, was done for 372 patients and 404 controls. In total, >180 Mb of DNA was scanned. Several variants more prevalent in patients than in controls were identified. This study demonstrates an approach to the discovery of susceptibility genes for common disease: large-scale direct sequence comparison between patients and controls. We believe this approach can be scaled up to allow sequence comparison in the whole-genome coding regions among large sets of cases and controls at a reasonable cost in the near future.

    View details for DOI 10.1073/pnas.0506677102

    View details for Web of Science ID 000232603600044

    View details for PubMedID 16203980

  • Significance analysis of time course microarray experiments PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Storey, J. D., Xiao, W. Z., Leek, J. T., Tompkins, R. G., Davis, R. W. 2005; 102 (36): 12837-12842


    Characterizing the genome-wide dynamic regulation of gene expression is important and will be of much interest in the future. However, there is currently no established method for identifying differentially expressed genes in a time course study. Here we propose a significance method for analyzing time course microarray studies that can be applied to the typical types of comparisons and sampling schemes. This method is applied to two studies on humans. In one study, genes are identified that show differential expression over time in response to in vivo endotoxin administration. By using our method, 7,409 genes are called significant at a 1% false-discovery rate level, whereas several existing approaches fail to identify any genes. In another study, 417 genes are identified at a 10% false-discovery rate level that show expression changing with age in the kidney cortex. Here it is also shown that as many as 47% of the genes change with age in a manner more complex than simple exponential growth or decay. The methodology proposed here has been implemented in the freely distributed and open-source edge software package.

    View details for DOI 10.1073/pnas.0504609102

    View details for Web of Science ID 000231716700036

    View details for PubMedID 16141318

    View details for PubMedCentralID PMC1201697

  • A novel method for STR-based DNA profiling using microarrays JOURNAL OF FORENSIC SCIENCES Kemp, J. T., Davis, R. W., White, R. L., Wang, S. X., Webb, C. D. 2005; 50 (5): 1109-1113


    We describe a novel method for rapidly identifying and distinguishing between different DNA sequences using short tandem repeat (STR) analysis and DNA microarrays. The method can be used to deduce identity, length, and number of STRs of the target molecule. We refer to this technique as the "variable-length probe array" method for STR profiling (VLPA). The method involves hybridization of the unknown STR target sequence to a DNA microarray displaying complementary probes that vary in length to cover the range of possible STRs. A post-hybridization enzymatic digestion of the DNA hybrids is then used to selectively remove labeled single-stranded regions of DNA from the microarray surface. The number of repeats in the unknown target is then deduced based on the pattern of target DNA that remains hybridized to the array. This DNA profiling technique is useful for performing forensic analysis to uniquely identify individual humans or other species.

    View details for Web of Science ID 000231660300013

    View details for PubMedID 16225215

  • Genome-wide requirements for resistance to functionally distinct DNA-damaging agents PLOS GENETICS Lee, W., Onge, R. P., Proctor, M., Flaherty, P., Jordan, M. I., Arkin, A. P., Davis, R. W., Nislow, C., Giaever, G. 2005; 1 (2): 235-246


    The mechanistic and therapeutic differences in the cellular response to DNA-damaging compounds are not completely understood, despite intense study. To expand our knowledge of DNA damage, we assayed the effects of 12 closely related DNA-damaging agents on the complete pool of approximately 4,700 barcoded homozygous deletion strains of Saccharomyces cerevisiae. In our protocol, deletion strains are pooled together and grown competitively in the presence of compound. Relative strain sensitivity is determined by hybridization of PCR-amplified barcodes to an oligonucleotide array carrying the barcode complements. These screens identified genes in well-characterized DNA-damage-response pathways as well as genes whose role in the DNA-damage response had not been previously established. High-throughput individual growth analysis was used to independently confirm microarray results. Each compound produced a unique genome-wide profile. Analysis of these data allowed us to determine the relative importance of DNA-repair modules for resistance to each of the 12 profiled compounds. Clustering the data for 12 distinct compounds uncovered both known and novel functional interactions that comprise the DNA-damage response and allowed us to define the genetic determinants required for repair of interstrand cross-links. Further genetic analysis allowed determination of epistasis for one of these functional groups.

    View details for DOI 10.1371/journal.pgen.0010024

    View details for Web of Science ID 000234714200011

    View details for PubMedID 16121259

  • Bioluminescence regenerative cycle (BRC) system: Theoretical considerations for nucleic acid quantification assays BIOPHYSICAL CHEMISTRY Hassibi, A., Contag, C., Vlad, M. O., Hafezi, M., Lee, T. H., Davis, R. W., Pourmand, N. 2005; 116 (3): 175-185


    A novel application of bioluminescence for nucleic acid quantification, the bioluminescence regenerative cycle (BRC), is described in theoretical terms and supported by preliminary experimental data. In the BRC system, pyrophosphate (PPi) molecules are released during biopolymerization and are counted and correlated to DNA copy number. The enzymes ATP-sulfurylase and firefly luciferase are employed to generate photons quantitatively from PPi. Enzymatic unity-gain positive feedback is implemented to amplify photon generation and to compensate for decay in light intensity by self-regulation. The cumulative total of photons can be orders of magnitude higher than in typical chemiluminescent processes. A system level theoretical model is developed, taking into account the kinetics of the regenerative cycle, contamination, and detector noise. Data and simulations show that the photon generation process achieves steady state for the time range of experimental measurements. Based on chain reaction theory, computations show that BRC is very sensitive to variations in the efficiencies of the chemical reactions involved and less sensitive to variations in the quantum yield of the process. We show that BRC can detect attomolar quantities of DNA (10(-18) mol), and that the useful dynamic range is five orders of magnitude. Sensitivity is not constrained by detector performance but rather by background bioluminescence caused by contamination by either PPi or ATP (adenosine triphosphate).

    View details for DOI 10.1016/j.bpc.2005.04.002

    View details for Web of Science ID 000230717400001

    View details for PubMedID 15882922

  • Microbes on the human vaginal epithelium PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hyman, R. W., Fukushima, M., Diamond, L., Kumm, J., Giudice, L. C., Davis, R. W. 2005; 102 (22): 7952-7957


    Using solely a gene-based procedure, PCR amplification of the 16S ribosomal RNA gene coupled with very deep sequencing of the amplified products, the microbes on 20 human vaginal epithelia of healthy women have been identified and quantitated. The Lactobacillus content on these 20 healthy vaginal epithelia was highly variable, ranging from 0% to 100%. For four subjects, Lactobacillus was (virtually) the only bacterium detected. However, that Lactobacillus was far from clonal and was a mixture of species and strains. Eight subjects presented complex mixtures of Lactobacillus and other microbes. The remaining eight subjects had no Lactobacillus. Instead, Bifidobacterium, Gardnerella, Prevotella, Pseudomonas, or Streptococcus predominated.

    View details for DOI 10.1073/pnas.0503236102

    View details for Web of Science ID 000229531000032

    View details for PubMedID 15911771

  • Quantitative proteome analysis of human plasma following in vivo lipopolysaccharide administration using O-16/O-18 labeling and the accurate mass and time tag approach MOLECULAR & CELLULAR PROTEOMICS Qian, W. J., Monroe, M. E., Liu, T., Jacobs, J. M., Anderson, G. A., Shen, Y. F., Moore, R. J., ANDERSON, D. J., Zhang, R., Calvano, S. E., Lowry, S. F., Xiao, W. Z., Moldawer, L. L., Davis, R. W., Tompkins, R. G., Camp, D. G., Smith, R. D. 2005; 4 (5): 700-709


    Identification of novel diagnostic or therapeutic biomarkers from human blood plasma would benefit significantly from quantitative measurements of the proteome constituents over a range of physiological conditions. Herein we describe an initial demonstration of proteome-wide quantitative analysis of human plasma. The approach utilizes postdigestion trypsin-catalyzed 16O/18O peptide labeling, two-dimensional LC-FTICR mass spectrometry, and the accurate mass and time (AMT) tag strategy to identify and quantify peptides/proteins from complex samples. A peptide accurate mass and LC elution time AMT tag data base was initially generated using MS/MS following extensive multidimensional LC separations to provide the basis for subsequent peptide identifications. The AMT tag data base contains >8,000 putative identified peptides, providing 938 confident plasma protein identifications. The quantitative approach was applied without depletion of high abundance proteins for comparative analyses of plasma samples from an individual prior to and 9 h after lipopolysaccharide (LPS) administration. Accurate quantification of changes in protein abundance was demonstrated by both 1:1 labeling of control plasma and the comparison between the plasma samples following LPS administration. A total of 429 distinct plasma proteins were quantified from the comparative analyses, and the protein abundances for 25 proteins, including several known inflammatory response mediators, were observed to change significantly following LPS administration.

    View details for DOI 10.1074/mcp.M500045-MCP200

    View details for Web of Science ID 000228932800010

    View details for PubMedID 15753121

    View details for PubMedCentralID PMC1829297

  • Mechanisms of haploinsufficiency revealed by genome-wide profiling in yeast GENETICS Deutschbauer, A. M., Jaramillo, D. F., Proctor, M., Kumm, J., Hillenmeyer, M. E., Davis, R. W., Nislow, C., Giaever, G. 2005; 169 (4): 1915-1925


    Haploinsufficiency is defined as a dominant phenotype in diploid organisms that are heterozygous for a loss-of-function allele. Despite its relevance to human disease, neither the extent of haploinsufficiency nor its precise molecular mechanisms are well understood. We used the complete set of Saccharomyces cerevisiae heterozygous deletion strains to survey the genome for haploinsufficiency via fitness profiling in rich (YPD) and minimal media to identify all genes that confer a haploinsufficient growth defect. This assay revealed that approximately 3% of all approximately 5900 genes tested are haploinsufficient for growth in YPD. This class of genes is functionally enriched for metabolic processes carried out by molecular complexes such as the ribosome. Much of the haploinsufficiency in YPD is alleviated by slowing the growth rate of each strain in minimal media, suggesting that certain gene products are rate limiting for growth only in YPD. Overall, our results suggest that the primary mechanism of haploinsufficiency in yeast is due to insufficient protein production. We discuss the relevance of our findings in yeast to human haploinsufficiency disorders.

    View details for DOI 10.1534/genetics.104.036871

    View details for Web of Science ID 000229263700013

    View details for PubMedID 15716499

  • Application of genome-wide expression analysis to human health and disease PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Cobb, J. P., Mindrinos, M. N., Miller-Graziano, C., Calvano, S. E., Baker, H. V., Xiao, W. Z., Laudanski, K., Brownstein, B. H., Elson, C. M., Hayden, D. L., Herndon, D. N., Lowry, S. F., Maier, R. V., Schoenfeld, D. A., Moldawer, L. L., Davis, R. W., Tompkins, R. G. 2005; 102 (13): 4801-4806


    The application of genome-wide expression analysis to a large-scale, multicentered program in critically ill patients poses a number of theoretical and technical challenges. We describe here an analytical and organizational approach to a systematic evaluation of the variance associated with genome-wide expression analysis specifically tailored to study human disease. We analyzed sources of variance in genome-wide expression analyses performed with commercial oligonucleotide arrays. In addition, variance in gene expression in human blood leukocytes caused by repeated sampling in the same subject, among different healthy subjects, among different leukocyte subpopulations, and the effect of traumatic injury, were also explored. We report that analytical variance caused by sample processing was acceptably small. Blood leukocyte gene expression in the same individual over a 24-h period was remarkably constant. In contrast, genome-wide expression varied significantly among different subjects and leukocyte subpopulations. Expectedly, traumatic injury induced dramatic changes in apparent gene expression that were greater in magnitude than the analytical noise and interindividual variance. We demonstrate that the development of a nation-wide program for gene expression analysis with careful attention to analytical details can reduce the variance in the clinical setting to a level where patterns of gene expression are informative among different healthy human subjects, and can be studied with confidence in human disease.

    View details for DOI 10.1073/pnas.0409768102

    View details for Web of Science ID 000228074000027

    View details for PubMedID 15781863

    View details for PubMedCentralID PMC555033

  • Profiling early infection responses: Pseudomonas aeruginosa eludes host defenses by suppressing antimicrobial peptide gene expression PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Apidianakis, Y., Mindrinos, M. N., Xiao, W. Z., Lau, G. W., Baldini, R. L., Davis, R. W., Rahme, L. G. 2005; 102 (7): 2573-2578


    Insights into the host factors and mechanisms mediating the primary host responses after pathogen presentation remain limited, due in part to the complexity and genetic intractability of host systems. Here, we employ the model Drosophila melanogaster to dissect and identify early host responses that function in the initiation and progression of Pseudomonas aeruginosa pathogenesis. First, we use immune potentiation and genetic studies to demonstrate that flies mount a heightened defense against the highly virulent P. aeruginosa strain PA14 when first inoculated with strain CF5, which is avirulent in flies; this effect is mediated via the Imd and Toll signaling pathways. Second, we use whole-genome expression profiling to assess and compare the Drosophila early defense responses triggered by the PA14 vs. CF5 strains to identify genes whose expression patterns are different in susceptible vs. resistant host-pathogen interactions, respectively. Our results identify pathogenesis- and defense-specific genes and uncover a previously undescribed mechanism used by P. aeruginosa in the initial stages of its host interaction: suppression of Drosophila defense responses by limiting antimicrobial peptide gene expression. These results provide insights into the genetic factors that mediate or restrict pathogenesis during the early stages of the bacterial-host interaction to advance our understanding of P. aeruginosa-human infections.

    View details for DOI 10.1073/pnas.0409588102

    View details for Web of Science ID 000227073100059

    View details for PubMedID 15695583

    View details for PubMedCentralID PMC549001

  • Single DNA molecule detection using nanopipettes and nanoparticles NANO LETTERS Karhanek, M., Kemp, J. T., Pourmand, N., Davis, R. W., Webb, C. D. 2005; 5 (2): 403-407


    Single DNA molecules labeled with nanoparticles can be detected by blockades of ionic current as they are translocated through a nanopipette tip formed by a pulled glass capillary. The nanopipette detection technique can provide not only tools for detection and identification of single DNA and protein molecules but also deeper insight and understanding of stochastic interactions of various biomolecules with their environment.

    View details for DOI 10.1021/nl0480464

    View details for Web of Science ID 000227100500038

    View details for PubMedID 15794633

  • Elevated evolutionary rates in the laboratory strain of Saccharomyces cerevisiae PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Gu, Z. L., David, L., Petrov, D., Jones, T., Davis, R. W., Steinmetz, L. M. 2005; 102 (4): 1092-1097


    By using the maximum likelihood method, we made a genome-wide comparison of the evolutionary rates in the lineages leading to the laboratory strain (S288c) and a wild strain (YJM789) of Saccharomyces cerevisiae and found that genes in the laboratory strain tend to evolve faster than in the wild strain. The pattern of elevated evolution suggests that relaxation of selection intensity is the dominant underlying reason, which is consistent with recurrent bottlenecks in the S. cerevisiae laboratory strain population. Supporting this conclusion are the following observations: (i) the increases in nonsynonymous evolutionary rate occur for genes in all functional categories; (ii) most of the synonymous evolutionary rate increases in S288c occur in genes with strong codon usage bias; (iii) genes under stronger negative selection have a larger increase in nonsynonymous evolutionary rate; and (iv) more genes with adaptive evolution were detected in the laboratory strain, but they do not account for the majority of the increased evolution. The present discoveries suggest that experimental and possible industrial manipulations of the laboratory strain of yeast could have had a strong effect on the genetic makeup of this model organism. Furthermore, they imply an evolution of laboratory model organisms away from their wild counterparts, questioning the relevancy of the models especially when extensive laboratory cultivation has occurred. In addition, these results shed light on the evolution of livestock and crop species that have been under human domestication for years.

    View details for DOI 10.1073/pnas.0409159102

    View details for Web of Science ID 000226617900026

    View details for PubMedID 15647350

  • A transcriptional profile of aging in the human kidney PLOS BIOLOGY Rodwell, G. E., Sonu, R., Zahn, J. M., Lund, J., Wilhelmy, J., Wang, L. L., Xiao, W. Z., Mindrinos, M., Crane, E., Segal, E., Myers, B. D., Brooks, J. D., Davis, R. W., Higgins, J., Owen, A. B., Kim, S. K. 2004; 2 (12): 2191-2201


    In this study, we found 985 genes that change expression in the cortex and the medulla of the kidney with age. Some of the genes whose transcripts increase in abundance with age are known to be specifically expressed in immune cells, suggesting that immune surveillance or inflammation increases with age. The age-regulated genes show a similar aging profile in the cortex and the medulla, suggesting a common underlying mechanism for aging. Expression profiles of these age-regulated genes mark not only age, but also the relative health and physiology of the kidney in older individuals. Finally, the set of aging-regulated kidney genes suggests specific mechanisms and pathways that may play a role in kidney degeneration with age.

    View details for DOI 10.1371/journal.pbio.0020427

    View details for Web of Science ID 000226099600020

    View details for PubMedID 15562319

    View details for PubMedCentralID PMC532391

  • Characterization of synthetic DNA bar codes in Saccharomyces cerevisiae gene-deletion strains PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Eason, R. G., Pourmand, N., Tongprasit, W., Herman, Z. S., Anthony, K., Jejelowo, O., Davis, R. W., Stolc, V. 2004; 101 (30): 11046-11051


    Incorporation of strain-specific synthetic DNA tags into yeast Saccharomyces cerevisiae gene-deletion strains has enabled identification of gene functions by massively parallel growth rate analysis. However, it is important to confirm the sequences of these tags, because mutations introduced during construction could lead to significant errors in hybridization performance. To validate this experimental system, we sequenced 11,812 synthetic 20-mer molecular bar codes and adjacent sequences (>1.8 megabases synthetic DNA) by pyrosequencing and Sanger methods. At least 31% of the genome-integrated 20-mer tags contain differences from those originally synthesized. However, these mutations result in anomalous hybridization in only a small subset of strains, and the sequence information enables redesign of hybridization probes for arrays. The robust performance of the yeast gene-deletion dual oligonucleotide bar-code design in array hybridization validates the use of molecular bar codes in living cells for tracking their growth phenotype.

    View details for DOI 10.1073/pnas.0403672101

    View details for Web of Science ID 000223000200038

    View details for PubMedID 15258289

  • SNP discovery in pooled samples with mismatch repair detection GENOME RESEARCH Fakhrai-Rad, H., Zheng, J. B., Willis, T. D., Wong, K., Suyenaga, K., Moorhead, M., EBERLE, J., Thorstenson, Y. R., Jones, T., Davis, R. W., Namsaraev, E., Faham, M. 2004; 14 (7): 1404-1412


    A targeted discovery effort is required to identify low frequency single nucleotide polymorphisms (SNPs) in human coding and regulatory regions. We here describe combining mismatch repair detection (MRD) with dideoxy terminator sequencing to detect SNPs in pooled DNA samples. MRD enriches for variant alleles in the pooled sample, and sequencing determines the nature of the variants. By using a genomic DNA pool as a template, approximately 100 fragments were amplified and subsequently combined and subjected en masse to the MRD procedure. The variant-enriched pool from this one MRD reaction is enriched for the population variants of all the tested fragments. Each fragment was amplified from the variant-enriched pool and sequenced, allowing the discovery of alleles with frequencies as low as 1% in the initial population. Our results support that MRD-based SNP discovery can be used for large-scale discovery of SNPs at low frequencies in a population.

    View details for DOI 10.1101/gr.2373904

    View details for Web of Science ID 000222434200022

    View details for PubMedID 15231755

  • The diploid genome sequence of Candida albicans PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Jones, T., Federspiel, N. A., Chibana, H., Dungan, J., Kalman, S., Magee, B. B., Newport, G., Thorstenson, Y. R., Agabian, N., Magee, P. T., Davis, R. W., Scherer, S. 2004; 101 (19): 7329-7334


    We present the diploid genome sequence of the fungal pathogen Candida albicans. Because C. albicans has no known haploid or homozygous form, sequencing was performed as a whole-genome shotgun of the heterozygous diploid genome in strain SC5314, a clinical isolate that is the parent of strains widely used for molecular analysis. We developed computational methods to assemble a diploid genome sequence in good agreement with available physical mapping data. We provide a whole-genome description of heterozygosity in the organism. Comparative genomic analyses provide important clues about the evolution of the species and its mechanisms of pathogenesis.

    View details for DOI 10.1073/pnas.0401648101

    View details for Web of Science ID 000221559100024

    View details for PubMedID 15123810

  • Chemogenomic profiling: Identifying the functional interactions of small molecules in yeast PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Giaever, G., Flaherty, P., Kumm, J., Proctor, M., Nislow, C., Jaramillo, D. F., Chu, A. M., Jordan, M. I., Arkin, A. P., Davis, R. W. 2004; 101 (3): 793-798


    We demonstrate the efficacy of a genome-wide protocol in yeast that allows the identification of those gene products that functionally interact with small molecules and result in the inhibition of cellular proliferation. Here we present results from screening 10 diverse compounds in 80 genome-wide experiments against the complete collection of heterozygous yeast deletion strains. These compounds include anticancer and antifungal agents, statins, alverine citrate, and dyclonine. In several cases, we identified previously known interactions; furthermore, in each case, our analysis revealed novel cellular interactions, even when the relationship between a compound and its cellular target had been well established. In addition, we identified a chemical core structure shared among three therapeutically distinct compounds that inhibit the ERG24 heterozygous deletion strain, demonstrating that cells may respond similarly to compounds of related structure. The ability to identify on-and-off target effects in vivo is fundamental to understanding the cellular response to small-molecule perturbants.

    View details for Web of Science ID 000188555400020

    View details for PubMedID 14718668

  • A multi-enzyme model for pyrosequencing NUCLEIC ACIDS RESEARCH Agah, A., Aghajan, M., Mashayekhi, F., Amini, S., Davis, R. W., Plummer, J. D., Ronaghi, M., Griffin, P. B. 2004; 32 (21)


    Pyrosequencing is a DNA sequencing technique based on sequencing-by-synthesis enabling rapid real-time sequence determination. This technique employs four enzymatic reactions in a single tube to monitor DNA synthesis. Nucleotides are added iteratively to the reaction and in case of incorporation, pyrophosphate (PPi) is released. PPi triggers a series of reactions resulting in production of light, which is proportional to the amount of DNA and number of incorporated nucleotides. Generated light is detected and recorded by a detector system in the form of a peak signal, which reflects the activity of all four enzymes in the reaction. We have developed simulations to model the kinetics of the enzymes. These simulations provide a full model for the Pyrosequencing four-enzyme system, based on which the peak height and shape can be predicted depending on the concentrations of enzymes and substrates. Simulation results are shown to be compatible with experimental data. Based on these simulations, the rate-limiting steps in the chain can be determined, and K(M) and kcat of all four enzymes in Pyrosequencing can be calculated.

    View details for DOI 10.1093/nar/gnh159

    View details for Web of Science ID 000225676700001

    View details for PubMedID 15576673

  • A genome-wide study of gene activity reveals developmental signaling pathways in the preimplantation mouse embryo DEVELOPMENTAL CELL Wang, Q. T., Piotrowska, K., Ciemerych, M. A., Milenkovic, L., Scott, M. P., Davis, R. W., Zernicka-Goetz, M. 2004; 6 (1): 133-144


    The preimplantation development of the mammalian embryo encompasses a series of critical events: the transition from oocyte to embryo, the first cell divisions, the establishment of cellular contacts, the first lineage differentiation-all the first subtle steps toward a future body plan. Here, we use microarrays to explore gene activity during preimplantation development. We reveal robust and dynamic patterns of stage-specific gene activity that fall into two major phases, one up to the 2-cell stage (oocyte-to-embryo transition) and one after the 4-cell stage (cellular differentiation). The mouse oocyte and early embryo express components of multiple signaling pathways including those downstream of Wnt, BMP, and Notch, indicating that conserved regulators of cell fate and pattern formation are likely to function at the earliest embryonic stages. Overall, these data provide a detailed temporal profile of gene expression that reveals the richness of signaling processes in early mammalian development.

    View details for Web of Science ID 000222442700013

    View details for PubMedID 14723853

  • Contributions of ATM mutations to familial breast and ovarian cancer CANCER RESEARCH Thorstenson, Y. R., Roxas, A., Kroiss, R., Jenkins, M. A., Yu, K. M., Bachrich, T., Muhr, D., Wayne, T. L., Chu, G., Davis, R. W., Wagner, T. M., Oefner, P. J. 2003; 63 (12): 3325-3333


    This study addresses the prevalence of ATM mutations and the association with breast cancer in Austrian families selected for a history of breast or ovarian cancer or both [hereditary breast and ovarian cancer (HBOC)]. In 270 HBOC families previously screened for BRCA1 and BRCA2 mutations, 137 different sequence alterations of ATM were identified. Seven of these were mutations presumed to cause ataxia telangiectasia based on their effect on the ATM protein, including five that caused a protein truncation and two missense mutations in the catalytic kinase domain of the highly conserved COOH terminus of the protein. The seven mutations were found in 10 families (3.7%). In addition, one missense variant, L1420F, was observed in 13 HBOC families (4.8%) but was not observed in any of the 122 healthy volunteers with no history of breast cancer. In addition, the variant segregated with breast cancer in some of the families, suggesting that it may be pathogenic for breast cancer. Sixty-two additional variants of potential significance were observed in 65 HBOC families, but not in healthy controls. These variants included 24 sequence alterations with possible effects on splicing or protein-protein interactions. This study indicates that there is a significant prevalence of ATM mutations in breast and ovarian cancer families and adds to a growing body of evidence that ATM mutations confer increased susceptibility to breast cancer.

    View details for Web of Science ID 000183586800049

    View details for PubMedID 12810666

  • Multiplexed genotyping with sequence-tagged molecular inversion probes NATURE BIOTECHNOLOGY Hardenbol, P., Baner, J., Jain, M., Nilsson, M., Namsaraev, E. A., Karlin-Neumann, G. A., Fakhrai-Rad, H., Ronaghi, M., Willis, T. D., Landegren, U., Davis, R. W. 2003; 21 (6): 673-678


    We report on the development of molecular inversion probe (MIP) genotyping, an efficient technology for large-scale single nucleotide polymorphism (SNP) analysis. This technique uses MIPs to produce inverted sequences, which undergo a unimolecular rearrangement and are then amplified by PCR using common primers and analyzed using universal sequence tag DNA microarrays, resulting in highly specific genotyping. With this technology, multiplex analysis of more than 1,000 probes in a single tube can be done using standard laboratory equipment. Genotypes are generated with a high call rate (95%) and high accuracy (>99%) as determined by independent sequencing.

    View details for DOI 10.1038/nbt821

    View details for Web of Science ID 000183220800026

    View details for PubMedID 12730666

  • Determination of hepatitis C virus genotype by Pyrosequencing JOURNAL OF VIROLOGICAL METHODS Elahi, E., Pourmand, N., Chaung, R., Rofoogaran, A., Boisver, J., Samimi-Rad, K., Davis, R. W., Ronaghi, M. 2003; 109 (2): 171-176


    A simple sequencing-based assay is described for genotyping of hepatitis C virus (HCV). RT-PCR was employed to amplify a 237-nucleotide-long fragment from the 5' untranslated region (UTR) of the genome using one biotinylated and one normal primer. Subsequent to capture of the PCR products on streptavidin-coated beads, single-stranded DNA separation, and hybridization of sequencing primer, Pyrosequencing was performed. The genotype of 98 samples out of which 77 samples were from American veterans and 21 samples were from Iran was determined. The samples from the American veterans contained six different subtypes, while five subtypes were found in Iranian samples. For rapid population-specific HCV subtyping, a multiplex assay was developed. This study demonstrates the suitability of this technology for low-cost, high throughput and accurate microbial genotyping.

    View details for DOI 10.1016/S0166-0934(03)00068-5

    View details for Web of Science ID 000183092800009

    View details for PubMedID 12711060

  • Genotyping African Haplotypes in ATM using a co-spotted single-base extension assay HUMAN MUTATION Jain, M., Thorstenson, Y. R., Faulkner, D. M., Pourmand, N., Jones, T., Au, M., Oefner, P. J., White, K. P., Davis, R. W. 2003; 22 (3): 214-221


    Human genetic analysis, including population genetic studies, increasingly calls for cost-effective, high-throughput methods for the rapid screening of single nucleotide polymorphisms (SNPs) across many individuals. The modified single-base extension assay described here (arrayed SBE) is a highly accurate and robust method for SNP genotyping that can deliver genotypes at 3.5 cents each, following PCR. Specifically, amino-modified probe/target pairs were prehybridized, then co-spotted in a microarray format prior to enzymatic addition of allele-specific nucleotides. Probe/target identity was determined solely by its physical location on the array rather than by hybridization to a complementary target, resulting in a call rate of 99-100%. These innovations result in an inexpensive, accurate assay with exceptional signal-to-noise ratios, depending on the glass surface employed. Comparison of glass slides from three different manufacturers indicated that aldehyde-based Zyomyx slides provided superior performance for this assay. Arrayed SBE was applied to study the geographic distribution of three African-specific haplotypes in the human ATM gene. Four selectively neutral markers, which define the haplotypes H5, H6, and H7, were screened in a total of 415 individuals. Region-specific haplotype frequencies were consistent with patterns of human migration across and outside of Africa, suggesting a possible haplotype origin in East Africa. Arrayed SBE was a robust tool for this analysis that could be applied to any situation requiring the genotyping of a few SNPs in many individuals.

    View details for DOI 10.1002/humu.10250

    View details for Web of Science ID 000185171100005

    View details for PubMedID 12938086

  • Parallel phenotypic analysis of sporulation and postgermination growth in Saccharomyces cerevisiae PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Deutschbauer, A. M., Williams, R. M., Chu, A. M., Davis, R. W. 2002; 99 (24): 15530-15535


    We have quantitatively monitored the sporulation and germination efficiencies of approximately 4,200 yeast deletion strains in parallel by using a molecular bar coding strategy. In a single study, we doubled the number of genes functionally implicated in sporulation to approximately 400, identifying both positive and negative regulators. Our set of 261 sporulation-deficient genes illustrates the importance of autophagy, carbon utilization, and transcriptional machinery during sporulation. These general cellular factors are more likely to exhibit fitness defects when deleted and less likely to be transcriptionally regulated than sporulation-specific genes. Our postgermination screening assay identified recombinationchromosome segregation genes, aneuploid strains, and possible germination-specific factors. Finally, our results facilitate a genome-wide comparison of expression pattern and mutant phenotype for a developmental process and suggest that 16% of genes differentially expressed during sporulation confer altered efficiency of spore production or defective postgermination growth when disrupted.

    View details for DOI 10.1073/pnas.202604399

    View details for Web of Science ID 000179530000052

    View details for PubMedID 12432101

  • Sequence of Plasmodium falciparum chromosome 12 NATURE Hyman, R. W., Fung, E., Conway, A., Kurdi, O., Mao, J., MIRANDA, M., Nakao, B., Rowley, D., Tamaki, T., Wang, F., Davis, R. W. 2002; 419 (6906): 534-537


    The human malaria parasite Plasmodium falciparum is responsible for the death of more than a million people every year. To stimulate basic research on the disease, and to promote the development of effective drugs and vaccines against the parasite, the complete genome of P. falciparum clone 3D7 has been sequenced, using a chromosome-by-chromosome shotgun strategy. Here we report the nucleotide sequence of the third largest of the parasite's 14 chromosomes, chromosome 12, which comprises about 10% of the 23-megabase genome. As the most (A + T)-rich (80.6%) genome sequenced to date, the P. falciparum genome presented severe problems during the assembly of primary sequence reads. We discuss the methodology that yielded a finished and fully contiguous sequence for chromosome 12. The biological implications of the sequence data are more thoroughly discussed in an accompanying Article (ref. 3).

    View details for DOI 10.1038/nature01102

    View details for Web of Science ID 000178348400050

    View details for PubMedID 12368869

  • Yeast tRNA as carrier in the isolation of microscale RNA for global amplification and expression profiling BIOTECHNIQUES Wang, Q. T., Xiao, W. Z., Mindrinos, M., Davis, R. W. 2002; 33 (4): 788-?


    The characterization of global gene expression patterns of microscale samples is important in many areas of biological and clinical research. The choice of carrier is critical for the efficient isolation and successful amplification of RNA at the nanogram level. Here we show that recovery of nanograms of RNA is significantly higher when carrier linear polyacrylamide is supplemented with carrier tRNA. Reverse transcription and in vitro transcription reactions remain efficient and specific in the presence of carrier tRNA. Finally, comparison of GeneChip array hybridization patterns demonstrates that the presence of carrier tRNA does not cause detectable distortion in global amplification. Taken together, tRNA is a superior carrier for the isolation and global amplification of microscale RNA.

    View details for Web of Science ID 000178641900013

    View details for PubMedID 12398187

  • Gene expression during the life cycle of Drosophila melanogaster SCIENCE Arbeitman, M. N., Furlong, E. E., Imam, F., JOHNSON, E., Null, B. H., Baker, B. S., Krasnow, M. A., Scott, M. P., Davis, R. W., White, K. P. 2002; 297 (5590): 2270-2275


    Molecular genetic studies of Drosophila melanogaster have led to profound advances in understanding the regulation of development. Here we report gene expression patterns for nearly one-third of all Drosophila genes during a complete time course of development. Mutations that eliminate eye or germline tissue were used to further analyze tissue-specific gene expression programs. These studies define major characteristics of the transcriptional programs that underlie the life cycle, compare development in males and females, and show that large-scale gene expression data collected from whole animals can be used to identify genes expressed in particular tissues and organs or genes involved in specific biological and biochemical processes.

    View details for Web of Science ID 000178222000050

    View details for PubMedID 12351791

  • Systematic screen for human disease genes in yeast NATURE GENETICS Steinmetz, L. M., Scharfe, C., Deutschbauer, A. M., Mokranjac, D., Herman, Z. S., Jones, T., Chu, A. M., Giaever, G., Prokisch, H., Oefner, P. J., Davis, R. W. 2002; 31 (4): 400-404


    High similarity between yeast and human mitochondria allows functional genomic study of Saccharomyces cerevisiae to be used to identify human genes involved in disease. So far, 102 heritable disorders have been attributed to defects in a quarter of the known nuclear-encoded mitochondrial proteins in humans. Many mitochondrial diseases remain unexplained, however, in part because only 40-60% of the presumed 700-1,000 proteins involved in mitochondrial function and biogenesis have been identified. Here we apply a systematic functional screen using the pre-existing whole-genome pool of yeast deletion mutants to identify mitochondrial proteins. Three million measurements of strain fitness identified 466 genes whose deletions impaired mitochondrial respiration, of which 265 were new. Our approach gave higher selection than other systematic approaches, including fivefold greater selection than gene expression analysis. To apply these advantages to human disorders involving mitochondria, human orthologs were identified and linked to heritable diseases using genomic map positions.

    View details for DOI 10.1038/ng929

    View details for Web of Science ID 000177147100016

    View details for PubMedID 12134146

  • Functional profiling of the Saccharomyces cerevisiae genome NATURE Giaever, G., Chu, A. M., Ni, L., CONNELLY, C., Riles, L., Veronneau, S., Dow, S., Lucau-Danila, A., Anderson, K., Andre, B., Arkin, A. P., Astromoff, A., El Bakkoury, M., Bangham, R., Benito, R., Brachat, S., Campanaro, S., Curtiss, M., Davis, K., Deutschbauer, A., Entian, K. D., Flaherty, P., Foury, F., Garfinkel, D. J., Gerstein, M., Gotte, D., Guldener, U., Hegemann, J. H., Hempel, S., Herman, Z., Jaramillo, D. F., Kelly, D. E., Kelly, S. L., Kotter, P., LaBonte, D., Lamb, D. C., Lan, N., Liang, H., Liao, H., Liu, L., Luo, C. Y., Lussier, M., Mao, R., Menard, P., Ooi, S. L., Revuelta, J. L., Roberts, C. J., Rose, M., Ross-Macdonald, P., Scherens, B., Schimmack, G., Shafer, B., Shoemaker, D. D., Sookhai-Mahadeo, S., Storms, R. K., Strathern, J. N., Valle, G., Voet, M., Volckaert, G., Wang, C. Y., Ward, T. R., Wilhelmy, J., Winzeler, E. A., Yang, Y. H., Yen, G., Youngman, E., Yu, K. X., Bussey, H., Boeke, J. D., Snyder, M., Philippsen, P., Davis, R. W., Johnston, M. 2002; 418 (6896): 387-391


    Determining the effect of gene deletion is a fundamental approach to understanding gene function. Conventional genetic screens exhibit biases, and genes contributing to a phenotype are often missed. We systematically constructed a nearly complete collection of gene-deletion mutants (96% of annotated open reading frames, or ORFs) of the yeast Saccharomyces cerevisiae. DNA sequences dubbed 'molecular bar codes' uniquely identify each strain, enabling their growth to be analysed in parallel and the fitness contribution of each gene to be quantitatively assessed by hybridization to high-density oligonucleotide arrays. We show that previously known and new genes are necessary for optimal growth under six well-studied conditions: high salt, sorbitol, galactose, pH 8, minimal medium and nystatin treatment. Less than 7% of genes that exhibit a significant increase in messenger RNA expression are also required for optimal growth in four of the tested conditions. Our results validate the yeast gene-deletion collection as a valuable resource for functional genomics.

    View details for DOI 10.1038/nature00935

    View details for Web of Science ID 000177009700029

    View details for PubMedID 12140549

  • Transcriptional response of Saccharomyces cerevisiae to DNA-damaging agents does not identify the genes that protect against these agents PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Birrell, G. W., BROWN, J. A., Wu, H. I., Giaever, G., Chu, A. M., Davis, R. W., Brown, J. M. 2002; 99 (13): 8778-8783


    The recent completion of the deletion of all of the nonessential genes in budding yeast has provided a powerful new way of determining those genes that affect the sensitivity of this organism to cytotoxic agents. We have used this system to test the hypothesis that genes whose transcription is increased after DNA damage are important for the survival to that damage. We used a pool of 4,627 diploid strains each with homozygous deletion of a nonessential gene to identify those genes that are important for the survival of yeast to four DNA-damaging agents: ionizing radiation, UV radiation, and exposure to cisplatin or to hydrogen peroxide. In addition we measured the transcriptional response of the wild-type parental strain to the same DNA-damaging agents. We found no relationship between the genes necessary for survival to the DNA-damaging agents and those genes whose transcription is increased after exposure. These data show that few, if any, of the genes involved in repairing the DNA lesions produced in this study, including double-strand breaks, pyrimidine dimers, single-strand breaks, base damage, and DNA cross-links, are induced in response to toxic doses of the agents that produce these lesions. This finding suggests that the enzymes necessary for the repair of these lesions are at sufficient levels within the cell. The data also suggest that the nature of the lesions produced by DNA-damaging agents cannot easily be deduced from gene expression profiling.

    View details for DOI 10.1073/pnas.132275199

    View details for Web of Science ID 000176478200057

    View details for PubMedID 12077312

  • Multiplex pyrosequencing NUCLEIC ACIDS RESEARCH Pourmand, N., Elahi, E., Davis, R. W., Ronaghi, M. 2002; 30 (7)


    We describe here the development of a new and simple single-tube multiplex Pyrosequencing assay. Genomic DNA or cDNA was employed to PCR amplify region(s) using biotinylated and normal primer(s). Subsequent to capture of PCR products on streptavidin-coated beads, single-stranded DNA separation and hybridization of multiple sequencing primers, Pyrosequencing was performed. The obtained pyrogram resulted in a unique pattern in which the intensity of the signal determined the number of incorporated nucleotide(s). Here, we demonstrate the use of this multiplex Pyrosequencing for single nucleotide polymorphisms genotyping and microbial typing.

    View details for Web of Science ID 000174654200032

    View details for PubMedID 11917037

  • Dissecting the architecture of a quantitative trait locus in yeast NATURE Steinmetz, L. M., Sinha, H., Richards, D. R., Spiegelman, J. I., Oefner, P. J., McCusker, J. H., Davis, R. W. 2002; 416 (6878): 326-330


    Most phenotypic diversity in natural populations is characterized by differences in degree rather than in kind. Identification of the actual genes underlying these quantitative traits has proved difficult. As a result, little is known about their genetic architecture. The failures are thought to be due to the different contributions of many underlying genes to the phenotype and the ability of different combinations of genes and environmental factors to produce similar phenotypes. This study combined genome-wide mapping and a new genetic technique named reciprocal-hemizygosity analysis to achieve the complete dissection of a quantitative trait locus (QTL) in Saccharomyces cerevisiae. A QTL architecture was uncovered that was more complex than expected. Functional linkages both in cis and in trans were found between three tightly linked quantitative trait genes that are neither necessary nor sufficient in isolation. This arrangement of alleles explains heterosis (hybrid vigour), the increased fitness of the heterozygote compared with homozygotes. It also demonstrates a deficiency in current approaches to QTL dissection with implications extending to traits in other organisms, including human genetic diseases.

    View details for Web of Science ID 000174482200044

    View details for PubMedID 11907579

  • Identification and preliminary characterization of mouse Adam33 BMC GENETICS Gunn, T. M., Azarani, A., Kim, P. H., Hyman, R. W., Davis, R. W., Barsh, G. S. 2002; 3


    The metalloprotease-disintegrin family, or ADAM, proteins, are implicated in cell-cell interactions, cell fusion, and cell signaling, and are widely distributed among metazoan phyla. Orthologous relationships have been defined for a few ADAM proteins including ADAM10 (Kuzbanian), and ADAM17 (TACE), but evolutionary relationships are not clear for the majority of family members. Human ADAM33 refers to a testis cDNA clone that does not contain a complete open reading frame, but portions of the predicted protein are similar to Xenopus laevis ADAM13.In a 48 kb region of mouse DNA adjacent to the Attractin gene on mouse chromosome 2, we identified sequences very similar to human ADAM33. A full-length mouse cDNA was identified by a combination of gene prediction programs and RT-PCR, and the probable full-length human cDNA was identified by comparison to human genomic sequence in the homologous region on chromosome 20p13. Mouse ADAM33 is 44% identical to Xenopus laevis ADAM13, however a phylogenetic alignment and consideration of functional domains suggests that the two genes are not orthologous. Mouse Adam33 is widely expressed, most highly in the adult brain, heart, kidney, lung and testis.While mouse ADAM33 is similar to Xenopus ADAM13 in sequence, further examination of its embryonic expression pattern, catalytic activity and protein interactions will be required to assess the functional relationship between these two proteins. Adam33 is expressed in the mouse adult brain and could play a role in complex processes that require cell-cell communication.

    View details for Web of Science ID 000179726900001

    View details for PubMedID 11897009

  • A genome-wide screen in Saccharomyces cerevisiae for genes affecting UV radiation sensitivity PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Birrell, G. W., Giaever, G., Chu, A. M., Davis, R. W., Brown, J. M. 2001; 98 (22): 12608-12613


    The recent completion of the deletion of essentially all of the ORFs in yeast is an important new resource for identifying the phenotypes of unknown genes. Each ORF is replaced with a cassette containing unique tag sequences that allow rapid parallel analysis of strains in a pool by using hybridization to a high-density oligonucleotide array. We examined the utility of this system to identify genes conferring resistance to UV irradiation by using a pool of 4,627 individual homozygous deletion strains (representing deletions of all nonessential genes). We identified most of the nonessential genes previously shown to be involved in nucleotide excision repair, in cell cycle checkpoints, in homologous recombination, and in postreplication repair after UV damage. We also identified and individually confirmed, by replacing the genes, three new genes, to our knowledge not previously reported to confer UV sensitivity when deleted. Two of these newly identified genes have human orthologs associated with cancer, demonstrating the potential of this system to uncover human genes affecting sensitivity to DNA-damaging agents and genes potentially involved in cancer formation.

    View details for Web of Science ID 000171806100058

    View details for PubMedID 11606770

  • Nucleotide sequence and predicted functions of the entire Sinorhizobium meliloti pSymA megaplasmid PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Barnett, M. J., Fisher, R. F., Jones, T., Komp, C., Abola, A. P., Barloy-Hubler, F., Bowser, L., Capela, D., Galibert, F., Gouzy, J., Gurjal, M., Hong, A., Huizar, L., Hyman, R. W., Kahn, D., Kahn, M. L., Kalman, S., Keating, D. H., Palm, C., Peck, M. C., Surzycki, R., Wells, D. H., Yeh, K. C., Davis, R. W., Federspiel, N. A., Long, S. R. 2001; 98 (17): 9883-9888


    The symbiotic nitrogen-fixing soil bacterium Sinorhizobium meliloti contains three replicons: pSymA, pSymB, and the chromosome. We report here the complete 1,354,226-nt sequence of pSymA. In addition to a large fraction of the genes known to be specifically involved in symbiosis, pSymA contains genes likely to be involved in nitrogen and carbon metabolism, transport, stress, and resistance responses, and other functions that give S. meliloti an advantage in its specialized niche.

    View details for Web of Science ID 000170539600076

    View details for PubMedID 11481432

  • Global analysis of ATM polymorphism reveals significant functional constraint AMERICAN JOURNAL OF HUMAN GENETICS Thorstenson, Y. R., Shen, P. D., Tusher, V. G., Wayne, T. L., Davis, R. W., Chu, G., Oefner, P. J. 2001; 69 (2): 396-412


    ATM, the gene that is mutated in ataxia-telangiectasia, is associated with cerebellar degeneration, abnormal proliferation of small blood vessels, and cancer. These clinically important manifestations have stimulated interest in defining the sequence variation in the ATM gene. Therefore, we undertook a comprehensive survey of sequence variation in ATM in diverse human populations. The protein-encoding exons of the gene (9,168 bp) and the adjacent intron and untranslated sequences (14,661 bp) were analyzed in 93 individuals from seven major human populations. In addition, the coding sequence was analyzed in one chimpanzee, one gorilla, one orangutan, and one Old World monkey. In human ATM, 88 variant sites were discovered by denaturing high-performance liquid chromatography, which is 96%-100% sensitive for detection of DNA sequence variation. ATM was compared to 14 other autosomal genes for nucleotide diversity. The noncoding regions of ATM had diversity values comparable to other genes, but the coding regions had very low diversity, especially in the last 29% of the protein sequence. A test of the neutral evolution hypothesis, through use of the Hudson/Kreitman/Aguadé statistic, revealed that this region of the human ATM gene was significantly constrained relative to that of the orangutan, the Old World monkey, and the mouse, but not relative to that of the chimpanzee or the gorilla. ATM displayed extensive linkage disequilibrium, consistent with suppression of meiotic recombination at this locus. Seven haplotypes were defined. Two haplotypes accounted for 82% of all chromosomes analyzed in all major populations; two others carrying the same D126E missense polymorphism accounted for 33% of chromosomes in Africa but were never observed outside of Africa. The high frequency of this polymorphism may be due either to a population expansion within Africa or to selective pressure.

    View details for Web of Science ID 000170108200014

    View details for PubMedID 11443540

  • Genomic evidence for a complete sexual cycle in Candida albicans PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Tzung, K. W., Williams, R. M., Scherer, S., Federspiel, N., Jones, T., Hansen, N., Bivolarevic, V., Huizar, L., Komp, C., Surzycki, R., Tamse, R., Davis, R. W., Agabian, N. 2001; 98 (6): 3249-3253


    Candida albicans is a diploid fungus that has become a medically important opportunistic pathogen in immunocompromised individuals. We have sequenced the C. albicans genome to 10.4-fold coverage and performed a comparative genomic analysis between C. albicans and Saccharomyces cerevisiae with the objective of assessing whether Candida possesses a genetic repertoire that could support a complete sexual cycle. Analyzing over 500 genes important for sexual differentiation in S. cerevisiae, we find many homologues of genes that are implicated in the initiation of meiosis, chromosome recombination, and the formation of synaptonemal complexes. However, others are striking in their absence. C. albicans seems to have homologues of all of the elements of a functional pheromone response pathway involved in mating in S. cerevisiae but lacks many homologues of S. cerevisiae genes for meiosis. Other meiotic gene homologues in organisms ranging from filamentous fungi to Drosophila melanogaster and Caenorhabditis elegans were also found in the C. albicans genome, suggesting potential alternative mechanisms of genetic exchange.

    View details for Web of Science ID 000167521300058

    View details for PubMedID 11248064

  • Transcriptional regulation and function during the human cell cycle NATURE GENETICS Cho, R. J., Huang, M. X., Campbell, M. J., Dong, H. L., Steinmetz, L., Sapinoso, L., Hampton, G., Elledge, S. J., Davis, R. W., Lockhart, D. J. 2001; 27 (1): 48-54


    We report here the transcriptional profiling of the cell cycle on a genome-wide scale in human fibroblasts. We identified approximately 700 genes that display transcriptional fluctuation with a periodicity consistent with that of the cell cycle. Systematic analysis of these genes revealed functional organization within groups of coregulated transcripts. A diverse set of cytoskeletal reorganization genes exhibit cell-cycle-dependent regulation, indicating that biological pathways are redirected for the execution of cell division. Many genes involved in cell motility and remodeling of the extracellular matrix are expressed predominantly in M phase, indicating a mechanism for balancing proliferative and invasive cellular behavior. Transcripts upregulated during S phase displayed extensive overlap with genes induced by DNA damage; cell-cycle-regulated transcripts may therefore constitute coherent programs used in response to external stimuli. Our data also provide clues to biological function for hundreds of previously uncharacterized human genes.

    View details for Web of Science ID 000166187900014

    View details for PubMedID 11137997

  • Prevalence of small inversions in yeast gene order evolution PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Seoighe, C., Federspiel, N., Jones, T., Hansen, N., Bivolarovic, V., Surzycki, R., Tamse, R., Komp, C., Hulzar, L., Davis, R. W., Scherer, S., Tait, E., Shaw, D. J., Harris, D., Murphy, L., Oliver, K., Taylor, K., Rajandream, M. A., Barrell, B. G., Wolfe, K. H. 2000; 97 (26): 14433-14437


    Gene order evolution in two eukaryotes was studied by comparing the Saccharomyces cerevisiae genome sequence to extensive new data from whole-genome shotgun and cosmid sequencing of Candida albicans. Gene order is substantially different between these two yeasts, with only 9% of gene pairs that are adjacent in one species being conserved as adjacent in the other. Inversion of small segments of DNA, less than 10 genes long, has been a major cause of rearrangement, which means that even where a pair of genes has been conserved as adjacent, the transcriptional orientations of the two genes relative to one another are often different. We estimate that about 1,100 single-gene inversions have occurred since the divergence between these species. Other genes that are adjacent in one species are in the same neighborhood in the other, but their precise arrangement has been disrupted, probably by multiple successive multigene inversions. We estimate that gene adjacencies have been broken as frequently by local rearrangements as by chromosomal translocations or long-distance transpositions. A bias toward small inversions has been suggested by other studies on animals and plants and may be general among eukaryotes.

    View details for Web of Science ID 000165993700081

    View details for PubMedID 11087826

  • Y chromosome sequence variation and the history of human populations NATURE GENETICS Underhill, P. A., Shen, P. D., Lin, A. A., Jin, L., Passarino, G., Yang, W. H., Kauffman, E., Bonne-Tamir, B., Bertranpetit, J., Francalacci, P., Ibrahim, M., Jenkins, T., Kidd, J. R., Mehdi, S. Q., Seielstad, M. T., WELLS, R. S., Piazza, A., Davis, R. W., Feldman, M. W., Cavalli-Sforza, L. L., Oefner, P. J. 2000; 26 (3): 358-361


    Binary polymorphisms associated with the non-recombining region of the human Y chromosome (NRY) preserve the paternal genetic legacy of our species that has persisted to the present, permitting inference of human evolution, population affinity and demographic history. We used denaturing high-performance liquid chromatography (DHPLC; ref. 2) to identify 160 of the 166 bi-allelic and 1 tri-allelic site that formed a parsimonious genealogy of 116 haplotypes, several of which display distinct population affinities based on the analysis of 1062 globally representative individuals. A minority of contemporary East Africans and Khoisan represent the descendants of the most ancestral patrilineages of anatomically modern humans that left Africa between 35,000 and 89,000 years ago.

    View details for Web of Science ID 000165176500029

    View details for PubMedID 11062480

  • Population genetic implications from sequence variation in four Y chromosome genes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Shen, P. D., Wang, F., Underhill, P. A., Franco, C., Yang, W. H., Roxas, A., Sung, R., Lin, A. A., Hyman, R. W., Vollrath, D., Davis, R. W., Cavalli-Sforza, L. L., Oefner, P. J. 2000; 97 (13): 7354-7359


    Some insight into human evolution has been gained from the sequencing of four Y chromosome genes. Primary genomic sequencing determined gene SMCY to be composed of 27 exons that comprise 4,620 bp of coding sequence. The unfinished sequencing of the 5' portion of gene UTY1 was completed by primer walking, and a total of 20 exons were found. By using denaturing HPLC, these two genes, as well as DBY and DFFRY, were screened for polymorphic sites in 53-72 representatives of the five continents. A total of 98 variants were found, yielding nucleotide diversity estimates of 2.45 x 10(-5), 5. 07 x 10(-5), and 8.54 x 10(-5) for the coding regions of SMCY, DFFRY, and UTY1, respectively, with no variant having been observed in DBY. In agreement with most autosomal genes, diversity estimates for the noncoding regions were about 2- to 3-fold higher and ranged from 9. 16 x 10(-5) to 14.2 x 10(-5) for the four genes. Analysis of the frequencies of derived alleles for all four genes showed that they more closely fit the expectation of a Luria-Delbrück distribution than a distribution expected under a constant population size model, providing evidence for exponential population growth. Pairwise nucleotide mismatch distributions date the occurrence of population expansion to approximately 28,000 years ago. This estimate is in accord with the spread of Aurignacian technology and the disappearance of the Neanderthals.

    View details for Web of Science ID 000087811600077

    View details for PubMedID 10861003

  • Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. International Conference of the American-Lung-Association/American-Thoracic-Society Brower, R. G., Matthay, M. A., Morris, A., Schoenfeld, D., Thompson, B. T., Wheeler, A., Wiedemann, H. P., Arroliga, A. C., Fisher, C. J., Komara, J. J., Perez-Trepichio, P., Parsons, P. E., Wolkin, R., Welsh, C., Fulkerson, W. J., MacIntyre, N., Mallatratt, L., Sebastian, M., McConnell, R., Wilcox, C., Govert, J., Thompson, D., Clemmer, T., Davis, R., Orme, J., Weaver, L., Grissom, C., Eskelson, M., Young, M., Gooder, V., McBride, K., Lawton, C., d'Hulst, J., Peerless, J. R., Smith, C., Brownlee, J., Pluss, W., Kallet, R., LUCE, J. M., Gottlieb, J., Elmer, M., Girod, A., Park, P., Daniel, B., Gropper, M., Abraham, E., Piedalue, F., Glodowski, J., Lockrem, J., McIntyre, R., Reid, K., Stevens, C., Kalous, D., Silverman, H. J., Shanholtz, C., Corral, W., Toews, G. B., Arnoldi, D., Bartlett, R. H., Dechert, R., Watts, C., Lanken, P. N., Anderson, H., Finkel, B., Hanson, C. W., Barton, R., Mone, M., Hudson, L. D., Lee, C., Carter, G., Maier, R. V., Steinberg, K. P., Bernard, G., Stroud, M., Swindell, B., Stone, L., Collins, L., Mogan, S., Ancukiewicz, M., Hayden, D., Molay, F., Ringwood, N., Wenzlow, G., Kazeroonian, A. S., Gail, D. B., Bosken, C. H., Randall, P., Waclawiw, M., Spragg, R. G., Boyett, J., Kelley, J., Leeper, K., Secundy, M. G., Slutsky, A., Hyers, T. M., Emerson, S. S., Garcia, J. G., Marini, J. J., Pingleton, S. K., Shasby, M. D., Sibbald, W. J. MASSACHUSETTS MEDICAL SOC. 2000: 1301–8
  • High-density arrays and insights into genome function BIOTECHNOLOGY & GENETIC ENGINEERING REVIEWS, VOL 17 Steinmetz, L. M., Davis, R. W. 2000; 17: 109-146

    View details for Web of Science ID 000167911600005

    View details for PubMedID 11255664

  • DAtA: database of Arabidopsis thaliana annotation NUCLEIC ACIDS RESEARCH Palm, C. J., Federspiel, N. A., Davis, R. W. 2000; 28 (1): 102-103


    The Database of Arabidopsis thaliana Annotation (D At A) was created to enable easy access to and analysis of all the Arabidopsis genome project annotation. The database was constructed using the completed A.thaliana genomic sequence data currently in GenBank. An automated annotation process was used to predict coding sequences for GenBank records that do not include annotation. D At A also contains protein motifs and protein similarities derived from searches of the proteins in D At A with motif databases and the non-redundant protein database. The database is routinely updated to include new GenBank submissions for Arabidopsis genomic sequences and new Blast and protein motif search results. A web interface to D At A allows coding sequences to be searched by name, comment, blast similarity or motif field. In addition, browse options present lists of either all the protein names or identified motifs present in the sequenced A.thaliana genome. The database can be accessed at http://baggage.

    View details for Web of Science ID 000084896300027

    View details for PubMedID 10592193

  • Functional analysis of the yeast genome by precise deletion and parallel phenotypic characterization. Novartis Foundation symposium Winzeler, E. A., Liang, H., Shoemaker, D. D., Davis, R. W. 2000; 229: 105-109

    View details for PubMedID 11084935

  • Genome-wide mapping with biallelic markers in Arabidopsis thaliana NATURE GENETICS Cho, R. J., Mindrinos, M., Richards, D. R., Sapolsky, R. J., Anderson, M., Drenkard, E., Dewdney, L., Reuber, T. L., Stammers, M., Federspiel, N., Theologis, A., Yang, W. H., Hubbell, E., Au, M., Chung, E. Y., Lashkari, D., Lemieux, B., Dean, C., Lipshutz, R. J., Ausubel, F. M., Davis, R. W., Oefner, P. J. 1999; 23 (2): 203-207


    Single-nucleotide polymorphisms, as well as small insertions and deletions (here referred to collectively as simple nucleotide polymorphisms, or SNPs), comprise the largest set of sequence variants in most organisms. Positional cloning based on SNPs may accelerate the identification of human disease traits and a range of biologically informative mutations. The recent application of high-density oligonucleotide arrays to allele identification has made it feasible to genotype thousands of biallelic SNPs in a single experiment. It has yet to be established, however, whether SNP detection using oligonucleotide arrays can be used to accelerate the mapping of traits in diploid genomes. The cruciferous weed Arabidopsis thaliana is an attractive model system for the construction and use of biallelic SNP maps. Although important biological processes ranging from fertilization and cell fate determination to disease resistance have been modelled in A. thaliana, identifying mutations in this organism has been impeded by the lack of a high-density genetic map consisting of easily genotyped DNA markers. We report here the construction of a biallelic genetic map in A. thaliana with a resolution of 3.5 cM and its use in mapping Eds16, a gene involved in the defence response to the fungal pathogen Erysiphe orontii. Mapping of this trait involved the high-throughput generation of meiotic maps of F2 individuals using high-density oligonucleotide probe array-based genotyping. We developed a software package called InterMap and used it to automatically delimit Eds16 to a 7-cM interval on chromosome 1. These results are the first demonstration of biallelic mapping in diploid genomes and establish means for generalizing SNP-based maps to virtually any genetic organism.

    View details for Web of Science ID 000082827500021

    View details for PubMedID 10508518

  • Functional characterization of the S-cerevisiae genome by gene deletion and parallel analysis SCIENCE Winzeler, E. A., Shoemaker, D. D., Astromoff, A., Liang, H., Anderson, K., Andre, B., Bangham, R., Benito, R., Boeke, J. D., Bussey, H., Chu, A. M., CONNELLY, C., Davis, K., Dietrich, F., Dow, S. W., El Bakkoury, M., Foury, F., Friend, S. H., Gentalen, E., Giaever, G., Hegemann, J. H., Jones, T., Laub, M., Liao, H., Liebundguth, N., Lockhart, D. J., Lucau-Danila, A., Lussier, M., M'Rabet, N., Menard, P., Mittmann, M., Pai, C., Rebischung, C., Revuelta, J. L., Riles, L., Roberts, C. J., Ross-Macdonald, P., Scherens, B., Snyder, M., Sookhai-Mahadeo, S., Storms, R. K., Veronneau, S., Voet, M., Volckaert, G., Ward, T. R., Wysocki, R., Yen, G. S., Yu, K. X., Zimmermann, K., Philippsen, P., Johnston, M., Davis, R. W. 1999; 285 (5429): 901-906


    The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.

    View details for Web of Science ID 000081860900053

    View details for PubMedID 10436161

  • An automated sample preparation system for large-scale DNA sequencing GENOME RESEARCH Marziali, A., Willis, T. D., Federspiel, N. A., Davis, R. W. 1999; 9 (5): 457-462


    Recent advances in DNA sequencing technologies, both in the form of high lane-density gels and automated capillary systems, will lead to an increased requirement for sample preparation systems that operate at low cost and high throughput. As part of the development of a fully automated sequencing system, we have developed an automated subsystem capable of producing 10,000 sequence-ready ssDNA templates per day from libraries of M13 plaques at a cost of $0.29 per sample. This Front End has been in high throughput operation since June, 1997 and has produced > 400,000 high-quality DNA templates.

    View details for Web of Science ID 000080347900008

    View details for PubMedID 10330125

  • Comparative genomes of Chlamydia pneumoniae and C-trachomatis NATURE GENETICS Kalman, S., Mitchell, W., Marathe, R., Lammel, C., Fan, L., Hyman, R. W., Olinger, L., Grimwood, L., Davis, R. W., Stephens, R. S. 1999; 21 (4): 385-389


    Chlamydia are obligate intracellular eubacteria that are phylogenetically separated from other bacterial divisions. C. trachomatis and C. pneumoniae are both pathogens of humans but differ in their tissue tropism and spectrum of diseases. C. pneumoniae is a newly recognized species of Chlamydia that is a natural pathogen of humans, and causes pneumonia and bronchitis. In the United States, approximately 10% of pneumonia cases and 5% of bronchitis cases are attributed to C. pneumoniae infection. Chronic disease may result following respiratory-acquired infection, such as reactive airway disease, adult-onset asthma and potentially lung cancer. In addition, C. pneumoniae infection has been associated with atherosclerosis. C. trachomatis infection causes trachoma, an ocular infection that leads to blindness, and sexually transmitted diseases such as pelvic inflammatory disease, chronic pelvic pain, ectopic pregnancy and epididymitis. Although relatively little is known about C. trachomatis biology, even less is known concerning C. pneumoniae. Comparison of the C. pneumoniae genome with the C. trachomatis genome will provide an understanding of the common biological processes required for infection and survival in mammalian cells. Genomic differences are implicated in the unique properties that differentiate the two species in disease spectrum. Analysis of the 1,230,230-nt C. pneumoniae genome revealed 214 protein-coding sequences not found in C. trachomatis, most without homologues to other known sequences. Prominent comparative findings include expansion of a novel family of 21 sequence-variant outer-membrane proteins, conservation of a type-III secretion virulence system, three serine/threonine protein kinases and a pair of parologous phospholipase-D-like proteins, additional purine and biotin biosynthetic capability, a homologue for aromatic amino acid (tryptophan) hydroxylase and the loss of tryptophan biosynthesis genes.

    View details for Web of Science ID 000079439700019

    View details for PubMedID 10192388

  • Distribution of haplotypes from a chromosome 21 region distinguishes multiple prehistoric human migrations Jin, L., Underhill, P. A., Doctor, V., Davis, R. W., Shen, P. D., Cavalli-Sforza, L. L., Oefner, P. J. NATL ACAD SCIENCES. 1999: 3796–3800


    Despite mounting genetic evidence implicating a recent origin of modern humans, the elucidation of early migratory gene-flow episodes remains incomplete. Geographic distribution of haplotypes may show traces of ancestral migrations. However, such evolutionary signatures can be erased easily by recombination and mutational perturbations. A 565-bp chromosome 21 region near the MX1 gene, which contains nine sites frequently polymorphic in human populations, has been found. It is unaffected by recombination and recurrent mutation and thus reflects only migratory history, genetic drift, and possibly selection. Geographic distribution of contemporary haplotypes implies distinctive prehistoric human migrations: one to Oceania, one to Asia and subsequently to America, and a third one predominantly to Europe. The findings with chromosome 21 are confirmed by independent evidence from a Y chromosome phylogeny. Loci of this type will help to decipher the evolutionary history of modern humans.

    View details for Web of Science ID 000079507900082

    View details for PubMedID 10097117

  • The mouse mahogany locus encodes a transmembrane form of human attractin NATURE Gunn, T. M., Miller, K. A., He, L., Hyman, R. V., Davis, R. W., Azarani, A., Schlossman, S. F., Duke-Cohan, J. S., Barsh, G. S. 1999; 398 (6723): 152-156


    Agouti protein and agouti-related protein are homologous paracrine signalling molecules that normally regulate hair colour and body weight, respectively, by antagonizing signalling through melanocortin receptors. Expression of Agouti is normally limited to the skin, but rare alleles from which Agouti is expressed ubiquitously, such as lethal yellow, have pleiotropic effects that include a yellow coat, obesity, increased linear growth, and immune defects. The mahogany (mg) mutation suppresses the effects of lethal yellow on pigmentation and body weight, and results of our previous genetic studies place mg downstream of transcription of Agouti but upstream of melanocortin receptors. Here we use positional cloning to identify a candidate gene for mahogany, Mgca. The predicted protein encoded by Mgca is a 1,428-amino-acid, single-transmembrane-domain protein that is expressed in many tissues, including pigment cells and the hypothalamus. The extracellular domain of the Mgca protein is the orthologue of human attractin, a circulating molecule produced by activated T cells that has been implicated in immune-cell interactions. These observations provide new insight into the regulation of energy metabolism and indicate a molecular basis for crosstalk between melanocortin-receptor signalling and immune function.

    View details for Web of Science ID 000079135200045

    View details for PubMedID 10086356

  • Genomic profiling of drug sensitivities via induced haploinsufficiency NATURE GENETICS Giaever, G., Shoemaker, D. D., Jones, T. W., Liang, H., Winzeler, E. A., Astromoff, A., Davis, R. W. 1999; 21 (3): 278-283


    Lowering the dosage of a single gene from two copies to one copy in diploid yeast results in a heterozygote that is sensitized to any drug that acts on the product of this gene. This haploinsufficient phenotype thereby identifies the gene product of the heterozygous locus as the likely drug target. We exploited this finding in a genomic approach to drug-target identification. Genome sequence information was used to generate molecularly tagged heterozygous yeast strains that were pooled, grown competitively in drug and analysed for drug sensitivity using high-density oligonucleotide arrays. Individual heterozygous strain analysis verified six known drug targets. Parallel analysis identified the known target and two hypersensitive loci in a mixed culture of 233 strains in the presence of the drug tunicamycin. Our discovery that both drug target and hypersensitive loci exhibit drug-induced haploinsufficiency may have important consequences in pharmacogenomics and variable drug toxicity observed in human populations.

    View details for Web of Science ID 000078977900020

    View details for PubMedID 10080179

  • On the in vivo function of the RecA ATPase JOURNAL OF MOLECULAR BIOLOGY Campbell, M. J., Davis, R. W. 1999; 286 (2): 437-445


    The Escherichia coli RecA protein is the prototype of the RecA/RAD51/DMC1 family of strand transferases acting in genetic recombination. The E96D mutant was previously isolated in a screen for toxic recA mutants and was found to constitutively derepress the SOS genes and inhibit chromosome segregation in E. coli. Here, we have found that the E96D mutation lowers the RecA kcat value for ATP hydrolysis 100-fold. Use of this mutant reveals that the ATPase and branch migration activities of RecA are not necessarily required for catalyzing in vivo recombinational pairing and LexA cleavage. In addition to its effect on ATP hydrolysis, the mutation causes ATP to more strongly promote the transition to the biologically active, extended conformation of the RecA enzyme. The enhanced ATP binding is apparently the cause for a broader nucleic acid ligand specificity. The use of RNA and double-stranded DNA as cofactors for LexA cleavage could give rise to the inappropriate, constitutive derepression of the SOS genes. This underscores the need for the ATP affinity to be optimized so that RecA becomes selectively activated only during DNA repair and recombination through binding single-stranded DNA.

    View details for Web of Science ID 000078760000011

    View details for PubMedID 9973562

  • Toxic mutations in the recA gene of E. coli prevent proper chromosome segregation JOURNAL OF MOLECULAR BIOLOGY Campbell, M. J., Davis, R. W. 1999; 286 (2): 417-435


    The recA gene of Escherichia coli is the prototype of the recA/RAD51/DMC1/uvsX gene family of strand transferases involved in genetic recombination. In order to find mutations in the recA gene important in catalytic turnover, a genetic screen was conducted for dominant lethal mutants. Eight single amino acid substitution mutants were found to prevent proper chromosome segregation and to kill cells in the presence or absence of an inducible SOS system. All mutants catalyzed some level of recombination and constitutively stimulated LexA cleavage. The mutations occur at the monomer-monomer interface of the RecA polymer or at residues important in ATP hydrolysis, implicating these residues in catalytic turnover. Based on an analysis of the E96D mutant, a model is presented in which slow RecA-DNA dissociation prevents chromosome segregation, engendering lexA-independent, lethal filamentation of cells.

    View details for Web of Science ID 000078760000010

    View details for PubMedID 9973561

  • An arrayable flow-through microcentrifuge for high-throughput instrumentation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Marziali, A., Willis, T. D., Davis, R. W. 1999; 96 (1): 61-66


    A compact, flow-through centrifugation system has been developed specifically for high-throughput centrifugation of large numbers of samples. The instrument is based on multiple high-speed rotors that also serve as sample holders. The small size of the rotors allows them to be arrayed in a standard 96-well microtiter plate spacing, making this device ideal for highly parallel automated instrumentation. Though initially designed for cell separation in DNA sequencing protocols, the flow-through centrifuge can be used to replace conventional centrifugation in most processes involving small samples. Techniques for recovery of both the supernatant and the pellet have been developed, as well as techniques for sample mixing, and cleaning of the reusable rotors. This paper discusses the design and performance of the flow-through centrifuge applied to cell separation and resuspension and to DNA purification and concentration.

    View details for Web of Science ID 000078004400014

    View details for PubMedID 9874772

  • Whole genome genetic-typing in yeast using high-density oligonucleotide arrays PARASITOLOGY Winzeler, E. A., Lee, B., McCusker, J. H., Davis, R. W. 1999; 118: S73-S80


    Genome sequence information in combination with new technologies has allowed researchers to approach genetic problems in new ways. High-density oligonucleotide arrays were used to probe the genome content of the yeast Saccharomyces cerevisiae. We show that these arrays, containing oligonucleotides complementary to the sequenced strain of S. cerevisiae, can be used to identify open reading frames that are missing or present in higher or lower copy number in related isolates of S. cerevisiae. We apply this method to the characterization of the genome of a strain derived from a clinical isolate of S. cerevisiae. Our results show that the telomeres are the regions with the most variability between the two strains.

    View details for Web of Science ID 000082170500009

    View details for PubMedID 10466139

  • Fluorescence-based expression monitoring using microarrays EXPRESSION OF RECOMBINANT GENES IN EUKARYOTIC SYSTEMS Winzeler, E. A., Schena, M., Davis, R. W. 1999; 306: 3-?

    View details for Web of Science ID 000083053500001

    View details for PubMedID 10432445

  • Imaging of apoptosis (programmed cell death) with Tc-99m annexin V JOURNAL OF NUCLEAR MEDICINE Blankenberg, F. G., Katsikis, P. D., Tait, J. F., Davis, R. E., Naumovski, L., Ohtsuki, K., Kopiwoda, S., Abrams, M. J., Strauss, H. W. 1999; 40 (1): 184-191


    Apoptosis (programmed cell death) is a critical element in normal physiology and in many disease processes. Phosphatidylserine (PS), one component of cell membrane phospholipids, is normally confined to the inner leaflet of the plasma membrane. Early in the course of apoptosis, this phospholipid is rapidly exposed on the cell's outer surface. Annexin V, an endogenous human protein, has a high affinity for membrane-bound PS. This protein has been labeled with fluorescein and has been used to detect apoptosis in vitro. We describe the use of radiolabeled annexin V to detect apoptosis in vivo. The results are compared to histologic and flow cytometric methods to identify cells and tissues undergoing apoptosis.Annexin V was coupled to hydrazinonicotinamide (HYNIC) and radiolabeled with 99mTc. Bioreactivity of 99mTc-HYNIC annexin V was compared with fluorescein isothiocyanate (FITC)-labeled annexin V in cultures of Jurkat T-cell lymphoblasts and in ex vivo thymic cell suspensions undergoing apoptosis in response to different stimuli. In addition, the uptake of FITC annexin V and 99mTc-HYNIC annexin V was studied in heat-treated necrotic Jurkat T-cell cultures. In vivo localization of annexin V was studied in Balb/c mice injected with 99mTc-HYNIC annexin V before and after induction of Fas-mediated hepatocyte apoptosis with intravenously administered antiFas antibody.Membrane-bound radiolabeled annexin V activity linearly correlated to total fluorescence as observed by FITC annexin V flow cytometry in Jurkat T-cell cultures induced to undergo apoptosis in response to growth factor deprivation (N = 10, r2 = 0.987), antiFas antibody (N = 8, r2 = 0.836) and doxorubicin (N = 10, r2 = 0.804); and in ex vivo experiments on thymic cell suspensions with dexamethasone-induced apoptosis from Balb/c mice (N = 6, r2 = 0.989). Necrotic Jurkat T-cell cultures also demonstrated marked increases in radiopharmaceutical (4000-5000-fold) above control values. AntiFas antibody-treated Balb/c mice (N = 6) demonstrated a three-fold rise in hepatic uptake of annexin V (P < 0.0005) above control (N = 10), identified both by imaging and scintillation well counting. The increase in hepatic uptake in antiFas antibody-treated mice correlated to histologic evidence of fulminant hepatic apoptosis.These data suggest that 99mTc-HYNIC annexin V can be used to image apoptotic and necrotic cell death in vivo.

    View details for Web of Science ID 000078114700043

    View details for PubMedID 9935075

  • Direct allelic variation scanning of the yeast genome SCIENCE Winzeler, E. A., Richards, D. R., Conway, A. R., Goldstein, A. L., Kalman, S., McCullough, M. J., McCusker, J. H., Stevens, D. A., Wodicka, L., Lockhart, D. J., Davis, R. W. 1998; 281 (5380): 1194-1197


    As more genomes are sequenced, the identification and characterization of the causes of heritable variation within a species will be increasingly important. It is demonstrated that allelic variation in any two isolates of a species can be scanned, mapped, and scored directly and efficiently without allele-specific polymerase chain reaction, without creating new strains or constructs, and without knowing the specific nature of the variation. A total of 3714 biallelic markers, spaced about every 3.5 kilobases, were identified by analyzing the patterns obtained when total genomic DNA from two different strains of yeast was hybridized to high-density oligonucleotide arrays. The markers were then used to simultaneously map a multidrug-resistance locus and four other loci with high resolution (11 to 64 kilobases).

    View details for Web of Science ID 000075531200054

    View details for PubMedID 9712584

  • An automated hydrodynamic process for controlled, unbiased DNA shearing GENOME RESEARCH Thorstenson, Y. R., Hunicke-Smith, S. P., Oefner, P. J., Davis, R. W. 1998; 8 (8): 848-855


    An automated, inexpensive, easy-to-use, and reproducible technique for controlled, random DNA fragmentation has been developed. The technique is based on point-sink hydrodynamics that result when a DNA sample is forced through a small hole by a syringe pump. Commercially available components are used to reduce the cost and complexity of the instrument. The design is optimized to reduce the volume of sample required and to speed processing time. Shearing of the samples can be completely automated by computer control. Ninety percent of sheared DNA fragments fall within a twofold size distribution that is highly reproducible. Three parameters are critical: the flow geometry, the flow rate, and a minimum number of iterations. Shearing is reproducible over a wide range of temperatures, DNA concentrations, and initial DNA size. The cloning efficiency of the sheared DNA is very good even without end repair, the distribution of assembled sequences is random, and there is no sequence bias at the ends of sheared fragments that have been cloned. The instrument, called the Point-sink Shearer (PtS), has already been exported successfully to many other laboratories.

    View details for Web of Science ID 000075674400012

    View details for PubMedID 9724331

  • Microarrays: biotechnology's discovery platform for functional genomics TRENDS IN BIOTECHNOLOGY Schena, M., Heller, R. A., Theriault, T. P., Konrad, K., Lachenmeier, E., Davis, R. W. 1998; 16 (7): 301-306


    Advances in microarray technology enable massive parallel mining of biological data, with biological chips providing hybridization-based expression monitoring, polymorphism detection and genotyping on a genomic scale. Microarrays containing sequences representative of all human genes may soon permit the expression analysis of the entire human genome in a single reaction. These 'genome chips' will provide unprecedented access to key areas of human health, including disease prognosis and diagnosis, drug discovery, toxicology, aging, and mental illness. Microarray technology is rapidly becoming a central platform for functional genomics.

    View details for Web of Science ID 000074546900005

    View details for PubMedID 9675914

  • A genome-wide transcriptional analysis of the mitotic cell cycle MOLECULAR CELL Cho, R. J., Campbell, M. J., Winzeler, E. A., Steinmetz, L., Conway, A., Wodicka, L., Wolfsberg, T. G., Gabrielian, A. E., Landsman, D., Lockhart, D. J., Davis, R. W. 1998; 2 (1): 65-73


    Progression through the eukaryotic cell cycle is known to be both regulated and accompanied by periodic fluctuation in the expression levels of numerous genes. We report here the genome-wide characterization of mRNA transcript levels during the cell cycle of the budding yeast S. cerevisiae. Cell cycle-dependent periodicity was found for 416 of the 6220 monitored transcripts. More than 25% of the 416 genes were found directly adjacent to other genes in the genome that displayed induction in the same cell cycle phase, suggesting a mechanism for local chromosomal organization in global mRNA regulation. More than 60% of the characterized genes that displayed mRNA fluctuation have already been implicated in cell cycle period-specific biological roles. Because more than 20% of human proteins display significant homology to yeast proteins, these results also link a range of human genes to cell cycle period-specific biological functions.

    View details for Web of Science ID 000075174500007

    View details for PubMedID 9702192

  • Parallel analysis of genetic selections using whole genome oligonucleotide arrays PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Cho, R. J., Fromont-Racine, M., Wodicka, L., Feierbach, B., Stearns, T., Legrain, P., Lockhart, D. J., Davis, R. W. 1998; 95 (7): 3752-3757


    Thousands of genes have recently been sequenced in organisms ranging from Escherichia coli to human. For the majority of these genes, however, available sequence does not define a biological role. Efficient functional characterization of these genes requires strategies for scaling genetic analyses to the whole genome level. Plasmid-based library selections are an established approach to the functional analysis of uncharacterized genes and can help elucidate biological function by identifying, for example, physical interactors for a gene and genetic enhancers and suppressors of mutant phenotypes. The application of these selections to every gene in a eukaryotic genome, however, is generally limited by the need to manipulate and sequence hundreds of DNA plasmids. We present an alternative approach in which identification of nucleic acids is accomplished by direct hybridization to high-density oligonucleotide arrays. Based on the complete sequence of Saccharomyces cerevisiae, high-density arrays containing oligonucleotides complementary to every gene in the yeast genome have been designed and synthesized. Two-hybrid protein-protein interaction screens were carried out for S. cerevisiae genes implicated in mRNA splicing and microtubule assembly. Hybridization of labeled DNA derived from positive clones is sufficient to characterize the results of a screen in a single experiment, allowing rapid determination of both established and previously unknown biological interactions. These results demonstrate the use of oligonucleotide arrays for the analysis of two-hybrid screens. This approach should be generally applicable to the analysis of a range of genetic selections.

    View details for Web of Science ID 000072848500079

    View details for PubMedID 9520439

  • Functional analysis of the yeast genome CURRENT OPINION IN GENETICS & DEVELOPMENT Winzeler, E. A., Davis, R. W. 1997; 7 (6): 771-776


    The release of the complete genome sequence of the yeast Saccharomyces cerevisiae has ushered in a new phase of genome research in which sequence function will be assigned. The goal is to determine the biological function of each of the > 6,000 open reading frames in the yeast genome. Innovative approaches have been developed that exploit the sequence data and yield information about gene expression levels, protein levels, subcellular localization and gene function for the entire genome.

    View details for Web of Science ID 000071734800005

    View details for PubMedID 9468786

  • Yeast microarrays for genome wide parallel genetic and gene expression analysis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA LASHKARI, D. A., DeRisi, J. L., McCusker, J. H., Namath, A. F., Gentile, C., Hwang, S. Y., Brown, P. O., Davis, R. W. 1997; 94 (24): 13057-13062


    We have developed high-density DNA microarrays of yeast ORFs. These microarrays can monitor hybridization to ORFs for applications such as quantitative differential gene expression analysis and screening for sequence polymorphisms. Automated scripts retrieved sequence information from public databases to locate predicted ORFs and select appropriate primers for amplification. The primers were used to amplify yeast ORFs in 96-well plates, and the resulting products were arrayed using an automated micro arraying device. Arrays containing up to 2,479 yeast ORFs were printed on a single slide. The hybridization of fluorescently labeled samples to the array were detected and quantitated with a laser confocal scanning microscope. Applications of the microarrays are shown for genetic and gene expression analysis at the whole genome level.

    View details for Web of Science ID A1997YJ45600066

    View details for PubMedID 9371799

  • Detection of numerous Y chromosome biallelic polymorphisms by denaturing high-performance liquid chromatography GENOME RESEARCH Underhill, P. A., Jin, L., Lin, A. A., Mehdi, S. Q., Jenkins, T., Vollrath, D., Davis, R. W., CAVALLISFORZA, L. L., Oefner, P. J. 1997; 7 (10): 996-1005


    Y chromosome haplotypes are particularly useful in deciphering human evolutionary history because they accentuate the effects of drift, migration, and range expansion. Significant acceleration of Y biallelic marker discovery and subsequent typing involving heteroduplex detection has been achieved by implementing an innovative and cost-efficient method called denaturing high-performance liquid chromatography (DHPLC). The power of the method resides in its sensitivity and ability to rapidly compare amplified sequences in an automated manner. We have determined the allelic states of 22 Y polymorphisms; 19 of which are unreported, in 718 diverse extant chromosomes; established haplotype frequencies; and deduced a phylogeny. All major geographic regions, including Eurasia, are characterized by mutations reflecting episodes of genetic drift and expansion. Most biallelic markers are localized regionally. However, some show wider dispersal and designate older, core haplotypes. One transversion defines a major haplogroup that distinguishes a previously unknown deep, apparently non-African branch. It provides evidence of an ancient bottleneck event. It is now possible to anticipate the inevitable detailed reconstruction of human Y chromosome genealogy based on several tens to even hundreds of these important polymorphisms.

    View details for Web of Science ID A1997YC51500009

    View details for PubMedID 9331370

  • Whole genome analysis: Experimental access to all genome sequenced segments through larger-scale efficient oligonucleotide synthesis and PCR PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA LASHKARI, D. A., McCusker, J. H., Davis, R. W. 1997; 94 (17): 8945-8947


    The recent ability to sequence whole genomes allows ready access to all genetic material. The approaches outlined here allow automated analysis of sequence for the synthesis of optimal primers in an automated multiplex oligonucleotide synthesizer (AMOS). The efficiency is such that all ORFs for an organism can be amplified by PCR. The resulting amplicons can be used directly in the construction of DNA arrays or can be cloned for a large variety of functional analyses. These tools allow a replacement of single-gene analysis with a highly efficient whole-genome analysis.

    View details for Web of Science ID A1997XR76500008

    View details for PubMedID 9256414

  • Application of DNA typing methods and genetic analysis to epidemiology and taxonomy of Saccharomyces isolates JOURNAL OF CLINICAL MICROBIOLOGY Clemons, K. V., Park, P. S., McCusker, J. H., McCullough, M. J., Davis, R. W., Stevens, D. A. 1997; 35 (7): 1822-1828


    We have previously described differences in phenotype and virulence among clinical and nonclinical isolates of Saccharomyces. To further characterize these isolates, a comparison of restriction fragment length polymorphism (RFLP) patterns and genetic analysis were done. The cellular DNA of each of 49 clinical and 11 nonclinical isolates of Saccharomyces was digested with the endonuclease EcoRI, and the resultant fragments were separated by electrophoresis. Sixty isolates were grouped on the basis of the presence (group B) or absence (group A) of a 3-kb band. Group A contained 43 isolates (35 clinical and 8 nonclinical isolates) in 31 discernible subgroups, and group B had 17 isolates (14 clinical and 3 nonclinical isolates) in 10 subgroups. Interestingly, six of eight known vaginal isolates were group B, with four of those six being identical. Virulence of isolates was associated with membership in group A (P = 0.03). Comparison of known members of sibling species within the genus Saccharomyces, which cannot be distinguished by standard biochemical tests, showed that S. paradoxus, S. bayanus, and S. cerevisiae could be differentiated by RFLP analysis. Genetic analysis of the isolates forming viable spores showed that most group A isolates were diploid and members of the species S. cerevisiae. Those group A and B isolates unable to form viable spores may be diploid hybrids between Saccharomyces species. The group B isolates that formed viable spores were tetraploid and may also be interspecific hybrids. Overall, clinical isolates of Saccharomyces were very heterogeneous and exhibited little clonality. RFLP pattern analysis could be a useful method of demonstrating transmission in patients with infection or between environmental sources and patients.

    View details for Web of Science ID A1997XE59100036

    View details for PubMedID 9196202

  • The nucleotide sequence of Saccharomyces cerevisiae chromosome V NATURE Dietrich, F. S., Mulligan, J., Hennessy, K., Yelton, M. A., Allen, E., Araujo, R., Aviles, E., Berno, A., Brennan, T., Carpenter, J., Chen, E., Cherry, J. M., Chung, E., Duncan, M., Guzman, E., Hartzell, G., HunickeSmith, S., Hyman, R. W., Kayser, A., Komp, C., Lashkari, D., Lew, H., LIN, D., MOSEDALE, D., Nakahara, K., Namath, A., Norgren, R., Oefner, P., Oh, C., Petel, F. X., Roberts, D., Sehl, P., Schramm, S., Shogren, T., Smith, V., Taylor, P., Wei, Y., Botstein, D., Davis, R. W. 1997; 387 (6632): 78-81


    Here we report the sequence of 569,202 base pairs of Saccharomyces cerevisiae chromosome V. Analysis of the sequence revealed a centromere, two telomeres and 271 open reading frames (ORFs) plus 13 tRNAs and four small nuclear RNAs. There are two Tyl transposable elements, each of which contains an ORF (included in the count of 271). Of the ORFs, 78 (29%) are new, 81 (30%) have potential homologues in the public databases, and 112 (41%) are previously characterized yeast genes.

    View details for Web of Science ID A1997XB54600008

    View details for PubMedID 9169868

  • Discovery and analysis of inflammatory disease-related genes using cDNA microarrays PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Heller, R. A., Schena, M., Chai, A., Shalon, D., Bedilion, T., Gilmore, J., Woolley, D. E., Davis, R. W. 1997; 94 (6): 2150-2155


    cDNA microarray technology is used to profile complex diseases and discover novel disease-related genes. In inflammatory disease such as rheumatoid arthritis, expression patterns of diverse cell types contribute to the pathology. We have monitored gene expression in this disease state with a microarray of selected human genes of probable significance in inflammation as well as with genes expressed in peripheral human blood cells. Messenger RNA from cultured macrophages, chondrocyte cell lines, primary chondrocytes, and synoviocytes provided expression profiles for the selected cytokines, chemokines, DNA binding proteins, and matrix-degrading metalloproteinases. Comparisons between tissue samples of rheumatoid arthritis and inflammatory bowel disease verified the involvement of many genes and revealed novel participation of the cytokine interleukin 3, chemokine Gro alpha and the metalloproteinase matrix metallo-elastase in both diseases. From the peripheral blood library, tissue inhibitor of metalloproteinase 1, ferritin light chain, and manganese superoxide dismutase genes were identified as expressed differentially in rheumatoid arthritis compared with inflammatory bowel disease. These results successfully demonstrate the use of the cDNA microarray system as a general approach for dissecting human diseases.

    View details for Web of Science ID A1997WP33400014

    View details for PubMedID 9122163

  • Automation for the Arabidopsis genome sequencing project TRENDS IN PLANT SCIENCE Marziali, A., Federspiel, N., Davis, R. 1997; 2 (2): 71-74
  • CDP1, a novel Saccharomyces cerevisiae gene required for proper nuclear division and chromosome segregation GENETICS Foreman, P. K., Davis, R. W. 1996; 144 (4): 1387-1397


    To identify new gene products involved in chromosome segregation, we isolated Saccharomyces cerevisiae mutants that require centromere binding factor I (Cbf1p) for viability. One Cbf1p-dependent mutant (denoted cdp1-1) was selected for further analysis. The CDP1 gene encodes a novel 125-kD protein that is notably similar to previously identified mouse, human and Caenorhabditis elegans proteins. CDP1 delta and cdp1-1 mutant cells were temperature sensitive for growth. At the permissive temperature, cdp1-1 and cdp1 delta cells lost chromosomes at a frequencies approximately 20-fold and approximately 110-fold higher than wild-type cells, respectively. These mutants also displayed unusually long and numerous bundles of cytoplasmic microtubules as revealed by immunofluorescent staining. In addition, we occasionally observed improperly oriented mitotic spindles, residing entirely within one of the cells. Presumably as a result of undergoing nuclear division with improperly oriented spindles, a large percentage of cdp1 cells had accumulated multiple nuclei. While cdp1 mutant cells were hypersensitive to the microtubule-disrupting compound thiabendazole, they showed increased resistance to the closely related compound benomyl relative to wild-type cells. Taken together, these results suggest that Cdp1p plays a role in governing tubulin dynamics within the cell and may interact directly with microtubules or tubulin.

    View details for Web of Science ID A1996VY93100007

    View details for PubMedID 8978028

  • Quantitative phenotypic analysis of yeast deletion mutants using a highly parallel molecular bar-coding strategy NATURE GENETICS Shoemaker, D. D., LASHKARI, D. A., Morris, D., Mittmann, M., Davis, R. W. 1996; 14 (4): 450-456


    A quantitative and highly parallel method for analysing deletion mutants has been developed to aid in determining the biological function of thousands of newly identified open reading frames (ORFs) in Saccharomyces cerevisiae. This approach uses a PCR targeting strategy to generate large numbers of deletion strains. Each deletion strain is labelled with a unique 20-base tag sequence that can be detected by hybridization to a high-density oligonucleotide array. The tags serve as unique identifiers (molecular bar codes) that allow analysis of large numbers of deletion strains simultaneously through selective growth conditions. Hybridization experiments show that the arrays are specific, sensitive and quantitative. A pilot study with 11 known yeast genes suggests that the method can be extended to include all of the ORFs in the yeast genome, allowing whole genome analysis with a single selective growth condition and a single hybridization.

    View details for Web of Science ID A1996VV73000021

    View details for PubMedID 8944025

  • Efficient random subcloning of DNA sheared in a recirculating point-sink flow system NUCLEIC ACIDS RESEARCH Oefner, P. J., HUNICKESMITH, S. P., Chiang, L., Dietrich, F., Mulligan, J., Davis, R. W. 1996; 24 (20): 3879-3886


    Based on a high-performance liquid chromatographic pump, we have built a device that allows recirculation of DNA through a 63-microm orifice with ensuing fractionation to a minimum fragment size of approximately 300 base pairs. Residence time of the DNA fragments in the converging flow created by a sudden contraction was found to be sufficiently long to allow extension of the DNA molecules into a highly extended conformation and, hence, breakage to occur at midpoint. In most instances, 30 passages sufficed to obtain a narrow size distribution, with >90% of the fragments lying within a 2-fold size distribution. The shear rate required to achieve breakage was found to be inversely proportional to the 1.0 power of the molecular weight. Compared with a restriction digest, up to 40% of all fragments could be cloned directly, with only marginal improvements in cloning efficiency having been observed upon prior end repair with Klenow, T4 polymerase or T4 polynucleotide kinase. Sequencing revealed a fairly random distribution of the fragments.

    View details for Web of Science ID A1996VP70200001

    View details for PubMedID 8918787

  • Parallel human genome analysis: Microarray-based expression monitoring of 1000 genes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Schena, M., Shalon, D., Heller, R., Chai, A., Brown, P. O., Davis, R. W. 1996; 93 (20): 10614-10619


    Microarrays containing 1046 human cDNAs of unknown sequence were printed on glass with high-speed robotics. These 1.0-cm2 DNA "chips" were used to quantitatively monitor differential expression of the cognate human genes using a highly sensitive two-color hybridization assay. Array elements that displayed differential expression patterns under given experimental conditions were characterized by sequencing. The identification of known and novel heat shock and phorbol ester-regulated genes in human T cells demonstrates the sensitivity of the assay. Parallel gene analysis with microarrays provides a rapid and efficient method for large-scale human gene discovery.

    View details for Web of Science ID A1996VL33300017

    View details for PubMedID 8855227



    A high-capacity system was developed to monitor the expression of many genes in parallel. Microarrays prepared by high-speed robotic printing of complementary DNAs on glass were used for quantitative expression measurements of the corresponding genes. Because of the small format and high density of the arrays, hybridization volumes of 2 microliters could be used that enabled detection of rare transcripts in probe mixtures derived from 2 micrograms of total cellular messenger RNA. Differential expression measurements of 45 Arabidopsis genes were made by means of simultaneous, two-color fluorescence hybridization.

    View details for Web of Science ID A1995TA37400041

    View details for PubMedID 7569999



    An automated oligonucleotide synthesizer has been developed that can simultaneously and rapidly synthesize up to 96 different oligonucleotides in a 96-well microtiter format using phosphoramidite synthesis chemistry. A modified 96-well plate is positioned under reagent valve banks, and appropriate reagents are delivered into individual wells containing the growing oligonucleotide chain, which is bound to a solid support. Each well has a filter bottom that enables the removal of spent reagents while retaining the solid support matrix. A seal design is employed to control synthesis environment and the entire instrument is automated via computer control. Synthesis cycle times for 96 couplings are < 11 min, allowing a plate of 96 20-mers to be synthesized in < 5 hr. Oligonucleotide synthesis quality is comparable to commercial machines, with average coupling efficiencies routinely > 98% across the entire 96-well plate. No significant well-to-well variations in synthesis quality have been observed in > 6000 oligonucleotides synthesized to date. The reduced reagent usage and increased capacity allow the overall synthesis cost to drop by at least a factor of 10. With the development of this instrument, it is now practical and cost-effective to synthesize thousands to tens of thousands of oligonucleotides.

    View details for Web of Science ID A1995RP74800064

    View details for PubMedID 7644513


    View details for Web of Science ID A1995QR21500020

    View details for PubMedID 7598894



    We have previously determined the relative virulence of isolates of Saccharomyces cerevisiae on the basis of differences in proliferation and resistance to clearance in CD-1 mice. These infections were not fatal. To further characterize S. cerevisiae pathogenesis, we studied a virulent clinical isolate, YJM128, and an avirulent nonclinical isolate, Y55, in C5-deficient mice. DBA/2N mice were infected intravenously with YJM128 or Y55, and temporal burdens of yeast cells in various organs were determined. After infection with 10(7) CFU, Y55 increased by 13-fold and YJM128 increased by 20-fold in the brain from day 0 to 3. In addition, YJM128 increased by 4-fold in the kidneys, whereas Y55 decreased by 16-fold. Both isolates declined in number in other organs. In all studies, 90% of mice infected with 10(7) CFU of YJM128 died between days 2 and 7, whereas no mice infected with equivalent numbers of Y55 died. No mice died after infection with 10(6) CFU of Y55 or YJM128. The importance of C5 was confirmed by studies using B10.D2/oSnJ (C5-) mice and their congenic C5+ counterparts. Again, the C5- mice were most susceptible to infection with S. cerevisiae, with 63% infected with YJM128 dying by day 7; no C5+ mice died. No Y55-infected mice died, and mean burdens in the brain at day 14 were sevenfold lower in C5+ mice than in C5- mice. Seven of 10 other S. cerevisiae isolates were also more virulent in DBA/2N than CD-1 mice, causing > or = 40% mortality. These data indicate that C5 is a critical factor in host resistance against S. cerevisiae infections and further confirm the pathogenic potential of some isolates of S. cerevisiae.

    View details for Web of Science ID A1995QC60300016

    View details for PubMedID 7822013



    Saccharomyces cerevisiae isolates have been shown previously to exhibit a high degree of variation in their ability to proliferate and persist in CD-1 mice (K.V. Clemons, J.H. McCusker, R. W. Davis, and D.A. Stevens, J. Infect. Dis. 169:859-867, 1994). Isolate origin was not a firm predictor of virulence phenotype, since the virulence phenotypes of clinical and nonclinical isolates ranged from virulent to avirulent and from intermediate to avirulent, respectively. Therefore, it was important to determine if there was any association between putative virulence traits and virulence that might help explain the variation in virulence phenotypes. S. cerevisiae isolates spanning a range of virulence phenotypes in experimental infections were examined for putative virulence traits: the ability to grow at supraoptimal temperatures (42, 39, and 37 degrees C), gelatin liquefaction, casein utilization, and pseudohyphal formation. Gelatin liquefaction appeared to be unrelated to pseudohyphal formation on casein or to virulence. Significant differences in the ability to grow at 39 and 42 degrees C were observed when the virulent and intermediate classes were compared with the avirulent class. Less extreme but still significant differences in pseudohyphal formation were observed when the virulent and intermediate classes were compared with the avirulent class. Therefore, two virulence traits, similar to those identified in other pathogenic fungi, the ability to grow at elevated temperatures and pseudohyphal formation, have been identified in S. cerevisiae.

    View details for Web of Science ID A1994PT32900033

    View details for PubMedID 7960125



    The Arabidopsis mutant ttg lacks both trichomes (epidermal hairs) and anthocyanin pigments. Trichomes and anthocyanins are restored by the constitutive expression of the maize transcriptional regulator (R). The expression of an R-glucocorticoid receptor chimeric protein results in a steroid hormone-dependent, conditional allele of R that functions in whole Arabidopsis plants. The response of the chimeric protein to pulses of hormone was used to define the pattern and timing of trichome formation on the developing leaf epidermis. Each adaxial epidermal leaf cell appears to have an equal probability of differentiating into a trichome; there is a temporal zone of decision for trichome cell fate that proceeds as a wave from the tip to the base of developing leaves.

    View details for Web of Science ID A1994PN27200035

    View details for PubMedID 7939683



    Homeobox genes are present in both plants and animals. Homeobox-leucine zipper genes, however, have been identified thus far only in the small mustard plant Arabidopsis thaliana. This observation suggests that homeobox-leucine zipper genes evolved after the divergence of plants and animals, perhaps to mediate specific regulatory events. To better understand this gene family, we isolated several sequences containing the homeobox-leucine zipper motif and carried out a comparative analysis of nine homeobox-leucine zipper genes (HAT1, HAT2, HAT3, HAT4, HAT5, HAT7, HAT9, HAT14, and HAT22). Gene structures, sequence comparisons, and chromosomal locations suggest a simple model for the evolution of these genes. The model postulates that a primordial homeobox gene acquired a leucine zipper by exon capture. The nascent homeobox-leucine zipper gene then appears to have undergone a series of gene duplication and chromosomal translocation events, leading to the formation of the HAT gene family. This work has general implications for the evolution of regulatory genes.

    View details for Web of Science ID A1994PE38800018

    View details for PubMedID 7915839



    A series of cloning vectors, designated YCpIF, was constructed to facilitate the conditional synthesis of epitope-tagged, truncated and chimeric proteins in Saccharomyces cerevisiae. These vectors contain a translation start codon upstream from a multiple cloning site (MCS) in each of the three reading frames. Protein synthesis is under the control of the GAL1 promoter, which drives transcription when cells are grown on galactose-containing medium, but not when they are grown on glucose-containing medium. Different versions of the vectors contain four different commonly used selectable markers. In addition, YCpIF15, YCpIF16 and YCpIF17 contain a sequence encoding an epitope from influenza virus hemagglutinin upstream from the MCS. These vectors facilitate the addition of this epitope tag to the N terminus of any protein. The epitope is recognized by a commercially available monoclonal antibody.

    View details for Web of Science ID A1994NW55000010

    View details for PubMedID 7517907



    Two Arabidopsis thaliana genes, QRT1 and QRT2, are required for pollen separation during normal development. In qrt mutants, the outer walls of the four meiotic products of the pollen mother cell are fused, and pollen grains are released in tetrads. Pollen is viable and fertile, and the cytoplasmic pollen contents are discrete. Pollination with a single tetrad usually yields four seeds, and genetic analysis confirmed that marker loci segregate in a 2:2 ratio within these tetrads. These mutations allow tetrad analysis to be performed in Arabidopsis and define steps in pollen cell wall development.

    View details for Web of Science ID A1994NP22100042

    View details for PubMedID 8197459



    Saccharomyces cerevisiae isolates from human patients have been genetically analyzed. Some of the characteristics of these isolates are very different from laboratory and industrial strains of S. cerevisiae and, for this reason, stringent genetic tests have been used to confirm their identity as S. cerevisiae. Most of these clinical isolates are able to grow at 42 degrees, a temperature that completely inhibits the growth of most other S. cerevisiae strains. This property can be considered a virulence trait and may help explain the presence of these isolates in human hosts. The ability to grow at 42 degrees is shown to be polygenic with primarily additive effects between loci. S. cerevisiae will be a useful model for the evolution and genetic analysis of fungal virulence and the study of polygenic traits.

    View details for Web of Science ID A1994NB82100004

    View details for PubMedID 8013903



    The FLP/FRT site-specific recombination system of Saccharomyces cerevisiae was expressed in stably transformed tobacco plants. The FLP protein efficiently catalyzes recombination between two directly repeated FLP recombination target (FRT) sites, deleting the sequence between them. In the constructs tested here, this deletion places the CaMV 35S promoter adjacent to a hygromycin resistance marker; transcriptional activation of the marker allows direct selection of recombination events. After crossing plants containing an integrated FLP expression construct with plants containing a FLP substrate, F1 seedlings can be selected directly for hygromycin resistance, indicating that recombination occurs at, or very early after zygote formation. Molecular analysis confirmed the expected recombination product.

    View details for Web of Science ID A1994NC62100004

    View details for PubMedID 8152416



    Centromere binding factor 1 (Cbf1p or CP1) binds to the CDEI region of Saccharomyces cerevisiae centromeres and is a member of the basic helix-loop-helix (bHLH) class of proteins. Deletion of the gene encoding Cbf1p results in an increased frequency of chromosome loss, hypersensitivity to low levels of microtubule disrupting drugs (such as thiabendazole and benomyl) and methionine auxotrophy. By polymerase chain reaction-based random mutagenesis of the CBF1 gene we have obtained a number of mutant alleles that make full-length protein with impaired function. The mutations in these alleles are clustered in or just downstream from the bHLH domain. Among the alleles obtained was a class that was more compromised for transcriptional activation and a class that was more compromised for chromosome loss and thiabendazole hypersensitivity. These results indicate that at least some aspects of the role of Cbf1p in chromosome segregation and transcriptional activation are distinct. In contrast, increased chromosome loss and thiabendazole hypersensitivity were not separated in any of the alleles, suggesting that these phenotypes reflect the same mechanistic defect. These observations are consistent with a model that suggests that one role of Cbf1p in chromosome segregation may be to improve the efficiency with which contact between the kinetochore and spindle microtubules is established or maintained.

    View details for Web of Science ID A1993LZ43200005

    View details for PubMedID 8243994



    Plants distinguish among the pollen grains that land on the stigma, permitting only compatible pollen to fertilize egg cells. To investigate these cell-cell interactions, Arabidopsis mutations that affect pollen-pistil communication were isolated. A male-sterile mutation that disrupts pollen-pistil interactions by eliminating the extracellular pollen coat (tryphine) is described here. Stigma cells that contact the mutant pollen produce callose, a carbohydrate synthesized in response to foreign pollen. The mutant pollen fails to germinate because it does not absorb water from the stigma, yet germinates in vitro, indicating it is viable. The defect is also conditional; high humidity results in pollen hydration and successful fertilization. Analysis of mature, mutant pollen indicated that it is deficient in long-chain lipids and has none of the lipoidic tryphine normally present on its surface. Immature mutant pollen grains have aberrant tryphine that disappears during pollen development. The sterile plants also lack stem waxes, and pollen from other wax-defective (eceriferum) mutants with reduced fertility has few of the lipid droplets normally present in tryphine. These results demonstrate that tryphine is critical for pollen-stigma interactions and suggest that tryphine lipids are required for fertilization, either by directly signaling the stigma or by stabilizing other tryphine components.

    View details for Web of Science ID A1993LF50900005

    View details for PubMedID 8504936



    The HAT4 gene from the plant Arabidopsis thaliana encodes a homeo domain protein that contains a leucine zipper motif. Homeo domain-leucine zipper (HD-Zip) proteins have not been found in animal systems, suggesting that HAT4 may define a new family of transcription factors that regulate higher plant development. To explore this possibility, functional studies of HAT4 were carried out in yeast and in transgenic plants. Point mutants of HAT4 isolated in yeast define functionally critical residues within the HD-Zip domain, many of which correspond to highly conserved positions in known homeo domains and leucine zippers. Transgenic plants bearing constructs that alter HAT4 expression exhibit a series of interesting developmental phenotypes, including changes in morphology and developmental rate. Thus, the HAT4 gene of Arabidopsis encodes an HD-Zip protein that functions as a novel developmental regulator.

    View details for Web of Science ID A1993KR21800004

    View details for PubMedID 8449400



    This work reports the identification, characterization, and nucleotide sequence of STE20, a newly discovered gene involved in the Saccharomyces cerevisiae mating response pathway, to date one of the best understood signal transduction pathways. STE20 encodes a putative serine/threonine-specific protein kinase with a predicted molecular mass of 102 kDa. Its expression pattern is similar to that of several other protein kinases in the mating response pathway. Deletion of the kinase domain of STE20 causes sterility in both haploid mating types. This sterility can be partially suppressed by high-level production of STE12 but is not suppressible by high levels of STE4 or a dominant STE11 truncation allele. A truncation allele of STE20 was isolated that can activate the mating response pathway in the absence of exogenous mating pheromone. This allele causes dominant growth arrest that cannot be suppressed by deletions of STE4, STE5, STE7, STE11, or STE12. The allele is able to suppress the mating defect of a strain in which the STE20 kinase domain has been deleted, but not the mating defects of strains carrying mutations in STE4, STE5, STE7, STE11, or STE12.

    View details for Web of Science ID A1993KH51600020

    View details for PubMedID 8421676



    Anthocyanin pathway-specific transcriptional activators R and C1 from the monocot maize were expressed in two dicots, Arabidopsis thaliana and Nicotiana tabacum. Expression of R caused augmented anthocyanin pigmentation in both plant species and augmented trichome (hair) production in Arabidopsis. Alone, C1 had no effect. Hybrid transgenic Arabidopsis expressing both C1 and R produced anthocyanins in root, petal, and stamen tissues that normally never express anthrocyanins. When R was expressed in the transparent testa glabrous (without anthocyanins and trichomes) mutant of Arabidopsis, the deficiency was complemented and both anthocyanins and trichomes were restored.

    View details for Web of Science ID A1992KB96400033

    View details for PubMedID 1465611



    In Saccharomyces cerevisiae, numerous genes have been identified by selection from high-copy-number libraries based on "multicopy suppression" or other phenotypic consequences of overexpression. Although fruitful, this approach suffers from two major drawbacks. First, high copy number alone may not permit high-level expression of tightly regulated genes. Conversely, other genes expressed in proportion to dosage cannot be identified if their products are toxic at elevated levels. This work reports construction of a genomic DNA expression library for S. cerevisiae that circumvents both limitations by fusing randomly sheared genomic DNA to the strong, inducible yeast GAL1 promoter, which can be regulated by carbon source. The library obtained contains 5 x 10(7) independent recombinants, representing a breakpoint at every base in the yeast genome. This library was used to examine aberrant gene expression in S. cerevisiae. A screen for dominant activators of yeast mating response identified eight genes that activate the pathway in the absence of exogenous mating pheromone, including one previously unidentified gene. One activator was a truncated STE11 gene lacking approximately 1000 base pairs of amino-terminal coding sequence. In two different clones, the same GAL1 promoter-proximal ATG is in-frame with the coding sequence of STE11, suggesting that internal initiation of translation there results in production of a biologically active, truncated STE11 protein. Thus this library allows isolation based on dominant phenotypes of genes that might have been difficult or impossible to isolate from high-copy-number libraries.

    View details for Web of Science ID A1992KA90300102

    View details for PubMedID 1454852



    When the recA protein (RecA) of Escherichia coli promotes strand exchange between single-stranded DNA (ssDNA) circles and linear double-stranded DNAs (dsDNA) with complementary 5' or 3' ends a polarity is observed. This property of RecA depends on ATP hydrolysis and the ssDNA that is displaced in the reaction since no polarity is observed in the presence of the non-hydrolyzable ATP analog, ATP gamma S, or in the presence of single-strand specific exonucleases. Based on these results a model is presented in which both the 5' and 3' complementary ends of the linear dsDNA initiate pairing with the ssDNA circle but only one end remains stably paired. According to this model, the association/dissociation of RecA in the 5' to 3' direction on the displaced strand determines the polarity of strand exchange by favoring or blocking its reinvasion into the newly formed dsDNA. Reinvasion is favored when the displaced strand is coated with RecA whereas it is blocked when it lacks RecA, remains covered by single-stranded DNA binding protein or is removed by a single-strand specific exonuclease. The requirement for ATP hydrolysis is explained if the binding of RecA to the displaced strand occurs via the dissociation and/or transfer of RecA, two functions that depend on ATP hydrolysis. The energy for strand exchange derives from the higher binding constant of RecA for the newly formed dsDNA as compared with that for ssDNA and not from ATP hydrolysis.

    View details for Web of Science ID A1992JN25900005

    View details for PubMedID 1326055



    Homeobox genes encode a large family of homeodomain proteins in animal systems. To test whether such genes are also abundant in higher plants, degenerate oligonucleotides complementary to sequences encoding the recognition helix (helix three) of the homeodomain were used to screen genomic and cDNA libraries from the plant Arabidopsis thaliana. Analysis of 8 of the 41 cDNAs isolated revealed that each encodes a presumptive homeodomain; interestingly, most of these clones also contain a leucine zipper motif tightly linked to the homeodomain. It is concluded that Arabidopsis encodes a large family of homeodomain proteins, including members that contain a homeodomain/leucine-zipper (HD-Zip) motif.

    View details for Web of Science ID A1992HR85300046

    View details for PubMedID 1349174



    Promoters that allow the selective induction of gene expression in vivo constitute an important methodology in eukaryotic organisms such as yeast and the fruit fly, but to date no such system has been described for higher plants. Given the fact that mammalian steroid receptors can function as hormone-dependent inducers of gene expression in heterologous systems, the feasibility of using mammalian steroid hormones as selective inducers of plant gene expression was investigated. Here it is shown that the glucocorticoid receptor expressed in plant cells is capable of activating a test gene linked to glucocorticoid response elements, providing the transfected plant cells are treated with glucocorticoid hormone. Nanomolar concentrations of glucocorticoids are sufficient to induce gene expression more than 150-fold, without causing detectable alterations in the physiology of the cultured plant cells. These findings indicate that glucocorticoid induction of steroid-responsive promoters should provide a general method for regulating gene expression in plant cells and imply that such a system might ultimately function in whole plants such as Arabidopsis thaliana.

    View details for Web of Science ID A1991GT48000010

    View details for PubMedID 1961707



    The use of proline as a nitrogen source causes hypersensitivity to 5-fluoro-orotic acid (5FOA) and allows up to 40-fold less of this drug to be used to select for the loss of URA3 function in Saccharomyces cerevisiae. 5FOA hypersensitivity is presumably due to the absence of nitrogen catabolite repression when proline is substituted for (NH4)2SO4 as a nitrogen source. There are two constraints to the use of the proline-5FOA combination: (1) S288c genetic background strains are hypersensitive to 5FOA when grown in proline as a nitrogen source but at least one other genetic background is resistant to low levels of 5FOA under these conditions. (2) The addition of some nutritional supplements confers phenotypic resistance to the 5FOA-proline combination.

    View details for Web of Science ID A1991GF65000007

    View details for PubMedID 1767588



    In reactions between linear single-stranded DNAs (ssDNAs) and circular double-stranded DNAs (dsDNAs), stable joint molecule formation promoted by the recA protein (RecA) requires negative superhelicity, a homologous end, and an RecA-ssDNA complex. Linear ssDNAs with 3'-end homology react more efficiently than linear ssDNAs with 5'-end homology. This 3'-end preference is explained by the finding that 3'-ends are more effectively coated by RecA than 5'-ends, as judged by exonuclease VII protection, and are thus more reactive. The ability of linear ssDNAs with 5'-end homology to react is improved by the presence of low concentrations of exonuclease VII. In reactions between ssDNAs and linear dsDNAs with end homology, stable joint molecule formation occurs more efficiently when the homology is at the 3'-end rather than at the 5'-end of the complementary strand. In addition, linear dsDNAs with homology at the 3'-end of the complementary strand react more efficiently with linear ssDNAs with 3'-end homology than with linear ssDNAs with 5'-end homology. The ability of linear ssDNAs with 5'-end homology to react, in the absence of single-stranded DNA-binding protein, is improved by adding 33-46 nucleotides of heterologous sequence to the 5'-end of the linear ssDNA. The poor reactivity of linear ssDNAs with 5'-end homology is explained by a lack of RecA at the 5'-ends of linear ssDNAs, which is a consequence of the polar association and dissociation of RecA.

    View details for Web of Science ID A1991FP08600021

    View details for PubMedID 2037569


    View details for Web of Science ID A1991FP31900035

    View details for PubMedID 2041753



    The centromere and its binding proteins constitute the kinetochore structure of metaphase chromosomes, which is crucial for the high accuracy of the chromosome segregation process. Isolation and analysis of the gene encoding a centromere binding protein from the yeast S. cerevisiae, CBF1, are described in this paper. DNA sequence analysis of the CBF1 gene reveals homology with the transforming protein myc and a family of regulatory proteins known as the helix-loop-helix (HLH) proteins. Disruption of the CBF1 gene caused a decrease in the growth rate, an increase in the rate of chromosome loss/nondisjunction, and hypersensitivity to the antimitotic drug thiabendazole. Unexpectedly, the cbf1 null mutation concomitantly resulted in a methionine auxotrophic phenotype, which suggests that CBF1, like other HLH proteins in higher eukaryotic cells, participates in the regulation of gene expression.

    View details for Web of Science ID A1990DC93400007

    View details for PubMedID 2185892



    The recA protein (RecA) promotes DNA pairing and strand exchange optimally in the presence of single-stranded binding protein (SSB). Under these conditions, 3' homologous ends are essential for stable joint molecule formation between linear single-stranded DNA (ssDNA) and supercoiled DNA (i.e. 3' ends are 50-60 times more reactive than 5' ends). Linear ssDNAs with homology at the 5' end do not participate in pairing. In the absence of SSB, the strand exchange reaction is less efficient; however, linear ssDNAs with 3' end homology are still 5- to 10-fold more reactive than those with 5' end homology. The preference for a 3' homologous end in the absence of SSB suggests that this is an intrinsic property of RecA-promoted strand exchange. The preferential reactivity of 3' homologous ends is likely to be a consequence of the polarity of polymerization of RecA on ssDNA. Specifically, since RecA polymerizes in the 5'----3' direction, 3' ends are more likely to be coated with RecA and, hence, will be more reactive than 5' ends.

    View details for Web of Science ID A1990CZ67700065

    View details for PubMedID 2182632



    Cold-sensitive mutations in the SPB genes (spb1-spb7) of Saccharomyces cerevisiae suppress the inhibition of translation initiation resulting from deletion of the poly(A)-binding protein gene (PAB1). The SPB4 protein belongs to a family of adenosine triphosphate (ATP)-dependent RNA helicases. The aberrant production of 25S ribosomal RNA (rRNA) occurring in spb4-1 mutants or the deletion of SPB2 (RPL46) permits the deletion of PAB1. These data suggest that mutations affecting different steps of 60S subunit formation can allow PAB-independent translation, and they indicate that further characterization of the spb mutations could lend insight into the biogenesis of the ribosome.

    View details for Web of Science ID A1990CQ97300040

    View details for PubMedID 2408148



    In response to water spray, subirrigation, wind, touch, wounding, or darkness, Arabidopsis regulates the expression of at least four touch-induced (TCH) genes. Ten to thirty minutes after stimulation, mRNA levels increase up to 100-fold. Arabidopsis plants stimulated by touch develop shorter petioles and bolts. This developmental response is known as thigmomorphogenesis. TCH 1 cDNA encodes the putative Arabidopsis calmodulin differing in one amino acid from wheat calmodulin. Sequenced regions of TCH 2 and TCH 3 contain 44% and 70% amino acid identities to calmodulin, respectively. The regulation of this calmodulin-related gene family in Arabidopsis suggests that calcium ions and calmodulin are involved in transduction of signals from the environment, enabling plants to sense and respond to environmental changes.

    View details for Web of Science ID A1990CP23700003

    View details for PubMedID 2302732

  • GENE TARGETING AT THE HUMAN CD4 LOCUS BY EPITOPE ADDITION GENES & DEVELOPMENT Jasin, M., Elledge, S. J., Davis, R. W., Berg, P. 1990; 4 (2): 157-166


    Homologous recombination at the CD4 locus in a human T-cell line has been achieved by an approach called epitope addition. The endogenous CD4 gene provided transcription, translation, and leader sequences to a crippled introduced Thy-1 gene, resulting in the expression of murine Thy-1 epitopes on the surface of the human cells. Thy-1+ cells were selected using the Fluorescence Activated Cell Sorter (FACS). An estimated 700-fold enrichment for homologous versus nonhomologous integration events was obtained, such that 70% of cells scoring positive for Thy-1 were derived from gene targeting. Three of the Thy-1+ cell lines expressed protein only from the targeted allele; thus, these cells were functionally CD4-.

    View details for Web of Science ID A1990CN76300001

    View details for PubMedID 1692556


    View details for Web of Science ID A1989CX06400010

    View details for PubMedID 2698080



    Depletion of the essential poly(A) binding protein (PAB) in S. cerevisiae by promoter inactivation or by the utilization of a temperature-sensitive mutation (pab1-F364L) results in the inhibition of translation initiation and poly(A) tail shortening. Reversion analysis of pab1-F364L yielded seven independent, extragenic cold-sensitive mutations (spb1-spb7) that also suppress a PAB1 deletion. These mutations allow translation initiation without significantly changing poly(A) tail lengths in the absence of PAB, and they affect the amount of 60S ribosomal subunit. Consistent with this, SPB2 encodes the ribosomal protein L46. These data suggest that the 60S subunit mediates the PAB requirement of translation initiation, thereby ensuring that only intact poly(A)+ mRNA will be translated efficiently in vivo.

    View details for Web of Science ID A1989AP72200008

    View details for PubMedID 2673535



    A yeast artificial chromosome (YAC) library in Saccharomyces cerevisiae consisting of 30,000 clones with an average insert size of 0.1 megabase pair of human DNA has been generated from primary fibroblast DNA. A YAC vector was modified to enable the recovery of both ends of a human DNA insert in plasmids in Escherichia coli and to confer G418 resistance to mammalian cells. A rapid method for yeast colony hybridization was used that exploits the ability of yeast spheroplasts to regenerate in a thin layer of calcium alginate. This method permits direct replica plating and processing of colonies from the primary transformation plate to nitrocellulose filters. Yeast colony hybridization conditions have been established to identify, within a YAC library of human genomic DNA, artificial chromosomes with homology to human DNA probes of unique single-copy sequence. An artificial chromosome with a 0.1-megabase-pair insert from the human Xq28 region has been identified by hybridization to a DNA probe that detects a unique sequence near the 3' end of the factor VIII gene.

    View details for Web of Science ID A1989AH94900049

    View details for PubMedID 2668948



    A centromere-specific DNA-binding protein has been purified to homogeneity by a combination of conventional and sequence-affinity chromatography from the yeast Saccharomyces cerevisiae. This protein (designated CBP-I) has an apparent molecular weight of 16,000. It binds specifically to the CDEI (centromere DNA element I) region of yeast centromere DNA, as shown by the electrophoretic mobility retardation assay and DNase I protection analysis, but does not bind specifically to other regions of yeast centromere DNA such as CDEII and CDEIII. The relative binding affinity of purified CBP-I to five different point mutations of CDEI correlates directly with the previously determined ability of each point mutation to convey centromere function in a mitotic chromosome segregation assay (J. H. Hegemann, J. H. Shero, G. Cottarel, P. Philippsen, and P. Hieter, Mol. Cell. Biol. 8:2523-2535, 1988). This supports the authenticity of CBP-I as a functional component of the yeast kinetochore.

    View details for Web of Science ID A1989U757700027

    View details for PubMedID 2668736



    We describe a genetic selection method designed to facilitate the cloning of genes encoding sequence-specific DNA-binding proteins. The strategy selects for clones expressing particular sequence-specific DNA-binding activities from a library of clones encoding other, nonspecific proteins. Specific DNA-binding sites have been placed near the start of transcription of the strong synthetic conII promoter to create promoters that can be repressed by the corresponding sequence-specific DNA-binding proteins. Transcription from the conII derivatives in the absence of repression interferes with the phenotypic expression of an adjacent drug-resistance gene, aadA. Sequence-specific DNA-binding proteins are shown to repress these promoters and alleviate transcriptional interference of aadA, resulting in drug resistance in cells expressing the appropriate DNA-binding protein.

    View details for Web of Science ID A1989U652300054

    View details for PubMedID 2657725



    We have investigated the effects of two types of DNA-binding proteins on bacterial repression. First, the effects of operator positioning on repression by stationary DNA-binding proteins, the Lac repressor and the Trp repressor, were examined in vivo. Both operator number and positioning play a role in determining in vivo levels of repression. Operators located within a promoter are more efficient regulators than those positioned at the start of transcription. Second, we investigated the effects of DNA-binding protein density on repression using a mobile DNA-binding protein, Escherichia coli RNA polymerase. We employed a transcriptional interference assay using convergent transcriptional units. The strong synthetic promoter conI and its derivatives were observed to interfere with expression of the aadA gene, which confers spectinomycin resistance upon its host. Transcriptional interference by RNA polymerase occurred only in cis and had a strong dependence on polymerase density that was modulated by varying the promoter strengths. A change in the density of approximately fourfold completely abolished the observed transcriptional interference. Several models are discussed to explain the repression patterns observed for stationary and mobile DNA-binding proteins.

    View details for Web of Science ID A1989T437900006

    View details for PubMedID 2523839



    A simple assay called the sectoring shuffle was developed to monitor the mutational state of essential genes cloned into yeast centromeric plasmids. The essence of this assay is the creation of a conditional phenotype, colony color sectoring, for an essential gene in the absence of conditional thermosensitive or cold-sensitive alleles of that gene. This allows the quick determination of the mutational state of a cloned essential gene by observing its effect on the sectoring phenotype of the tester strain. During the course of this work we developed a family of 20 Escherichia coli-yeast shuttle vectors, pUN plasmids, containing ARS1 CEN4 and a variety of selectable markers as well as the SUP11 gene which can act as a color marker in the proper background. These vectors are compact and have been very useful for the sectoring-shuffle assay and for gene analysis in general. This paper describes these vectors, the sectoring shuffle and several applications of sectoring phenotypes.

    View details for Web of Science ID A1988R036800009

    View details for PubMedID 3063604



    We used the his3 recombinational substrates (his3 fragments) to direct large interchromosomal (translocations) and intrachromosomal (deletions and tandem duplications) rearrangements in the yeast Saccharomyces cerevisiae. In strains completely deleted for the wild-type HIS3 gene, his3 fragments, one containing a deletion of 5' amino acid coding sequences and the other containing a deletion of 3' amino acid coding sequences, were first placed at preselected sites by homologous recombination. His+ revertants that arose via spontaneous mitotic recombination between the two his3 fragments were selected. This strategy was used to direct rearrangements in both RAD52+ and rad52 mutant strains. Translocations occurred in the RAD52+ genetic background and were characterized by orthogonal field alternating gel electrophoresis of yeast chromosomal DNA and by standard genetic techniques. An unexpected translocation was also identified in which HIS3 sequences were amplified. Two types of tandem duplications of the GAL(7, 10, 1) locus were also directed, and one type was not observed in rad52 mutants. Recombination mechanisms are discussed to account for these differences.

    View details for Web of Science ID A1988Q294000045

    View details for PubMedID 3054515



    A technique is described for physically positioning any cloned DNA on a native or artificial Saccharomyces cerevisiae chromosome. The technique involves splitting a chromosome at a specific site by transformation with short linear molecules containing the cloned DNA at one end and telomeric sequences at the other. Recombination between the end of the linear molecules and homologous chromosomal sequences gives rise to chromosome fragments comprising all sequences distal or proximal to the mapping site depending on the orientation of the cloned DNA. The recombinant products are recovered by screening for stabilization of a suppressor tRNA on the linear molecules using a colony color assay. The cloned DNA is positioned relative to the chromosome ends by sizing the chromosomal fragments using alternating contour-clamped homogeneous electric field gel electrophoresis. Application of this technique to organisms other than S. cerevisiae and to the analysis of exogenous DNA cloned in yeast is discussed.

    View details for Web of Science ID A1988P781800055

    View details for PubMedID 3045811

    View details for PubMedCentralID PMC281898



    The sequence of nitrate reductase (EC mRNA from the plant Arabidopsis thaliana has been determined. A 3.0-kilobase-long cDNA was isolated from a lambda gt10 cDNA library of Arabidopsis leaf poly(A)+ RNA. The cDNA hybridized to a 3.2-kilobase mRNA whose level increased 15-fold in response to treatment of the plant with nitrate. An open reading frame encoding a 917 amino acid protein was found in the sequence. This protein is very similar to tobacco nitrate reductase, being greater than 80% identical within a section of 450 amino acids. By comparing the Arabidopsis protein sequence with other protein sequences, three functional domains were deduced: (i) a molybdenum-pterin-binding domain that is similar to the molybdenum-pterin-binding domain of rat liver sulfite oxidase, (ii) a heme-binding domain that is similar to proteins in the cytochrome b5 superfamily, and (iii) an FAD-binding domain that is similar to NADH-cytochrome b5 reductase.

    View details for Web of Science ID A1988P362000013

    View details for PubMedID 3393528

  • TANDEM ARRAY OF HUMAN VISUAL PIGMENT GENES AT XQ28 SCIENCE Vollrath, D., Nathans, J., Davis, R. W. 1988; 240 (4859): 1669-1672


    Unequal crossing-over within a head-to-tail tandem array of the homologous red and green visual pigment genes has been proposed to explain the observed variation in green-pigment gene number among individuals and the prevalence of red-green fusion genes among color-blind subjects. This model was tested by probing the structure of the red and green pigment loci with long-range physical mapping techniques. The loci were found to constitute a gene array with an approximately 39-kilobase repeat length. The position of the red pigment gene at the 5' edge of the array explains its lack of variation in copy number. Restriction maps of the array in four individuals who differ in gene number are consistent with a head-to-tail configuration of the genes. These results provide physical evidence in support of the model and help to explain the high incidence of color blindness in the human population.

    View details for Web of Science ID A1988N833700035

    View details for PubMedID 2837827

  • AN ELECTROPHORETIC KARYOTYPE OF NEUROSPORA-CRASSA MOLECULAR AND CELLULAR BIOLOGY Orbach, M. J., Vollrath, D., Davis, R. W., Yanofsky, C. 1988; 8 (4): 1469-1473


    A molecular karyotype of Neurospora crassa was obtained by using an alternating-field gel electrophoresis system which employs contour-clamped homogeneous electric fields. The migration of all seven N. crassa chromosomal DNAs was defined, and five of the seven molecules were separated from one another. The estimated sizes of these molecules, based on their migration relative to Schizosaccharomyces pombe chromosomal DNA molecules, are 4 to 12.6 megabases. The seven linkage groups were correlated with specific chromosomal DNA bands by hybridizing transfers of contour-clamped homogeneous electric field gels with radioactive probes specific to each linkage group. The mobilities of minichromosomal DNAs generated from translocation strains were also examined. The methods used for preparation of chromosomal DNA molecules and the conditions for their separation should be applicable to other filamentous fungi.

    View details for Web of Science ID A1988M717700010

    View details for PubMedID 2967910



    Excellent resolution of chromosomal DNA molecules from Saccharomyces cerevisiae, Candida albicans and Schizosaccharomyces pombe has been obtained using alternating contour-clamped homogeneous electric field (CHEF) gel electrophoresis. The largest of these molecules is greater than 5 Mb in size and is resolved after 130 hours in a 0.6% agarose gel at a field strength of 1.3 V/cm and a switching interval of 1 hour. Separation of concatamers of phage lambda DNA reveals four regions of resolution in alternating CHEF gel electrophoresis. There are two regions of good resolution in which mobility approximates a linear function of molecular weight. These are separated by a region of lower resolution and bounded at high molecular weights by a region of little or no resolution. The four regions are of practical and possibly theoretical importance.

    View details for Web of Science ID A1987K383600015

    View details for PubMedID 2959907



    The poly(A)-binding protein (PAB) gene of Saccharomyces cerevisiae is essential for cell growth. A 66-amino acid polypeptide containing half of a repeated N-terminal domain can replace the entire protein in vivo. Neither an octapeptide sequence conserved among eucaryotic RNA-binding proteins nor the C-terminal domain of PAB is required for function in vivo. A single N-terminal domain is nearly identical to the entire protein in the number of high-affinity sites for poly(A) binding in vitro (one site with an association constant of approximately 2 X 10(7) M-1) and in the size of the binding site (12 A residues). Multiple N-terminal domains afford a mechanism of PAB transfer between poly(A) strands.

    View details for Web of Science ID A1987J716400032

    View details for PubMedID 3313012



    Ribonucleotide reductase catalyzes the first step in the pathway for the production of deoxyribonucleotides needed for DNA synthesis. The gene encoding the small subunit of ribonucleotide reductase was isolated from a Saccharomyces cerevisiae genomic DNA expression library in lambda gt11 by a fortuitous cross-reaction with anti-RecA antibodies. The cross-reaction was due to an identity between the last four amino acids of each protein. The gene has been named RNR2 and is centromere linked on chromosome X. The nucleotide sequence was determined, and the deduced amino acid sequence, 399 amino acids, shows extensive homology with other eucaryotic ribonucleotide reductases. Transplason mutagenesis was used to disrupt the RNR2 gene. A novel assay using colony color sectoring was developed to demonstrate visually that RNR2 is essential for mitotic viability. RNR2 encodes a 1.5-kilobase mRNA whose levels increase 18-fold after treatment with the DNA-damaging agent 4-nitroquinoline 1-oxide. CDC8 was also found to be inducible by DNA damage, but POL1 and URA3 were not inducible by 4-nitroquinoline 1-oxide. The expression of these genes defines a new mode of regulation for enzymes involved in DNA biosynthesis and sharpens our picture of the events leading to DNA repair in eucaryotic cells.

    View details for Web of Science ID A1987J391400015

    View details for PubMedID 3313004



    One of the earliest detectable events during plant-pathogen interaction is a rapid increase in ethylene biosynthesis. This gaseous plant stress hormone may be a signal for plants to activate defense mechanisms against invading pathogens such as bacteria, fungi, and viruses. The effect of ethylene on four plant genes involved in three separate plant defense response pathways was examined; these included (i and ii) genes that encode L-phenylalanine ammonia-lyase (EC and 4-coumarate:CoA ligase [4-coumarate:CoA ligase (AMP-forming), EC], enzymes of the phenylpropanoid pathway, (iii) the gene encoding chalcone synthase, an enzyme of the flavonoid glycoside pathway, and (iv) the genes encoding hydroxyproline-rich glycoprotein, a major protein component(s) of plant cell walls. Blot hybridization analysis of mRNA from ethylene-treated carrot roots reveals marked increases in the levels of phenylalanine ammonia-lyase mRNA, 4-coumarate CoA ligase mRNA, chalcone synthase mRNA, and certain hydroxyproline-rich glycoprotein transcripts. The effect of ethylene on hydroxyproline-rich glycoprotein mRNA accumulation was different from that of wounding. Ethylene induces two hydroxyproline-rich glycoprotein mRNAs (1.8 and 4.0 kilobases), whereas wounding of carrot root leads to accumulation of an additional hydroxyproline-rich mRNA (1.5 kilobases). These results indicate that at least two distinct signals, ethylene and a wound signal, can affect the expression of plant defense-response genes.

    View details for Web of Science ID A1987J472700025

    View details for PubMedID 16593860



    The RecA protein of Escherichia coli is important for genetic recombination in vivo and can promote synapsis and strand exchange in vitro. The DNA pairing and strand exchange reactions have been well characterized in reactions with circular single strands and linear duplexes, but little is known about these two processes using substrates more characteristic of those likely to exist in the cell. Single-stranded linear DNAs were prepared by separating strands of duplex molecules or by cleaving single-stranded circles at a unique restriction site created by annealing a short defined oligonucleotide to the circle. Analysis by gel electrophoresis and electron microscopy revealed that, in the presence of RecA and single-stranded binding proteins, a free 3' homologous end is essential for stable joint molecule formation between linear single-stranded and circular duplex DNA.

    View details for Web of Science ID A1987F976300018

    View details for PubMedID 3543934



    The assimilation of nitrate in plants involves the reduction of nitrate to ammonia in two steps. The first step requires nitrate reductase, a nitrate-inducible enzyme. When seedlings of squash (Cucurbita maxima L.) were treated with nitrate, both nitrate reductase activity and protein were induced in the cotyledons. Poly(A)(+) RNA was prepared from cotyledons of nitrate-treated seedlings and was used to construct a lambdagt11 cDNA library. Using antibodies from mice immunized against purified nitrate reductase from squash, a recombinant lambda phage was isolated that encoded part of the nitrate reductase mRNA. The antigens produced by the recombinant phage were used to affinity purify anti-nitrate reductase antibody from ascites fluid of immunized mice. The purified antibody bound to nitrate reductase protein on immunoblots and immunoprecipitated the enzyme from squash protein extracts. The cDNA insert (1.2 kilobases) hybridized to a 3.2-kilobase RNA that was 120-fold more abundant in nitrate-induced cotyledons compared with the uninduced tissue.

    View details for Web of Science ID A1986E643100012

    View details for PubMedID 16593773



    Due to the paucity of mutations in biochemical pathways in plants, an alternative approach to classical genetics was tested by expressing antisense RNA in plant cells. A series of plasmids was constructed with the bacterial gene for chloramphenicol acetyltransferase (EC linked in either the sense or antisense orientation to several different plant gene promoters. Various ratios of sense and antisense chloramphenicol acetyltransferase gene plasmids were introduced into plant protoplasts by electric field-mediated DNA transfer ("electroporation") and the level of expression in each combination was monitored by chloramphenicol acetyltransferase assays. Transcription of antisense RNA was found to effectively block the expression of target genes. Thus, the observation that antisense RNA inhibits gene expression in bacteria and animal systems has been extended to the plant kingdom. Antisense RNA techniques have immediate practical applications in both basic research and in plant genetic engineering.

    View details for Web of Science ID A1986D540900005

    View details for PubMedID 16593734