Sara L. (Sally) Tobin

All Publications

• Customers or research participants?: Guidance for research practices in commercialization of personal genomics GENETICS IN MEDICINE Tobin, S. L., Cho, M. K., Lee, S. S., Magnus, D. C., Allyse, M., Ormond, K. E., Garrison, N. A. 2012; 14 (10): 833-835

  View details for DOI 10.1038/gim.2012.64

  View details for Web of Science ID 000309645900001

  View details for PubMedID 22699154

• Informational risk, institutional review, and autonomy in the proposed changes to the common rule. IRB Allyse, M., Karkazis, K., Lee, S. S., Tobin, S. L., Greely, H. T., Cho, M. K., Magnus, D. 2012; 34 (3): 17-19

  View details for PubMedID 22830179

• Strangers at the Benchside: Research ethics consultation AMERICAN JOURNAL OF BIOETHICS Cho, M. K., Tobin, S. L., Greely, H. T., McCormick, J., Boyce, A., Magnus, D. 2008; 8 (3): 4-13

  Abstract

  Institutional ethics consultation services for biomedical scientists have begun to proliferate, especially for clinical researchers. We discuss several models of ethics consultation and describe a team-based approach used at Stanford University in the context of these models. As research ethics consultation services expand, there are many unresolved questions that need to be addressed, including what the scope, composition, and purpose of such services should be, whether core competencies for consultants can and should be defined, and how conflicts of interest should be mitigated. We make preliminary recommendations for the structure and process of research ethics consultation, based on our initial experiences in a pilot program.

  View details for DOI 10.1080/15265160802109322

  View details for Web of Science ID 000257030400004

  View details for PubMedID 18570086

• Research Ethics Consultation: The Stanford Experience IRB: Ethics and Human Research Cho MK, Tobin SL, Greely HT, McCormick J, Boyce A, Magnus D 2008; 30 (6)
• Initiating ocular proteomics for cataloging bovine retinal proteins: Microanalytical techniques permit the identification of proteins derived from a novel photoreceptor preparation EXPERIMENTAL EYE RESEARCH Nishizawa, Y., Komori, N., Usukura, J., Jackson, K. W., Tobin, S. L., Matsumoto, H. 1999; 69 (2): 195-212

  Abstract

  Though some mechanisms of photoreception have been well characterized, others remain obscure. Presumably, most, if not all, of the major players in photoreceptor-specific functions are present in large amounts in the photoreceptor layer, and a catalog of these proteins will prove a useful tool for vision researchers. As a first step toward a complete catalog of photoreceptor cells, we have developed a novel method for isolating the photoreceptor cell monolayer from bovine retina. Electron microscopic studies of both the photoreceptor layer and the residual retina from which the photoreceptor layer had been removed, indicate that the preparation contains the photoreceptor outer segments and the majority of the inner segments. Proteins were extracted from the isolated photoreceptor cell layer as well as the rest of the retina with isoelectric focusing lysis buffer, and the protein components were separated by two-dimensional gel electrophoresis. The obtained protein maps reveal several classes of proteins that appear to be expressed more abundantly or specifically in the photoreceptor layer than in the rest of the retina. Four of these protein spots were excised and in-gel digested with trypsin, and the digests were extracted with solvent. The mixture of peptides digested from each protein was analyzed by high performance liquid chromatography interfaced with electrospray ionization tandem quadrupole mass spectrometry or by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Some of the peptides were isolated and their sequences were determined by gas phase Edman degradation. RNA transcripts extracted from the photoreceptor layer or the whole retina were subjected to Northern blot analysis as well as to reverse transcriptase-polymerase chain reaction amplification of probes for the successful selection of cDNA clones. These data permit both the identification of virtually any protein detectable on a two-dimensional gel, and also enable the corresponding cDNA clone to be selected. We have validated this approach by identifying aspartate aminotransferase and creatine kinase from the populations of abundant photoreceptor layer proteins. Both aspartate aminotransferase and creatine kinase are of mitochondrial origin and are thought to play crucial roles in photoreceptor functions by producing glutamate and ATP, respectively. We also identified two photoreceptor layer specific proteins: an acidic and high molecular weight protein, interphotoreceptor retinoid-binding protein, and an acidic and small molecular weight protein, recoverin.The technique presented here will allow vision researchers to discover and identify the proteins that are expressed specifically or abundantly in the photoreceptor cell as well as the proteins that undergo post-translational modification or modulation in expression under a defined biological condition. With the use of this technology, we anticipate that a researcher who knows only the 2-D gel position of a protein of interest can identify the protein, isolate a cDNA clone, and move into molecular genetic studies. Moreover, this streamlined technology will enable one to assemble a catalog of photoreceptor proteins using a minute amount of materials in a short period of time. We believe that such a catalog will serve as a valuable resource for vision investigators and will accelerate the rate of research progress.

  View details for Web of Science ID 000081883700006

  View details for PubMedID 10433856

• Defining dementia: Social and historical background of Alzheimer disease 49th Annual Meeting of the American-Society-of-Human-Genetics Fox, P. J., Kelly, S. E., Tobin, S. L. MARY ANN LIEBERT INC. 1999: 13–19

  Abstract

  Though Alzheimer disease (AD) has been recognized as a distinct entity since 1907, scientific understanding of, and public interest in, the disease remained very limited until the 1970s. The perception of AD as a significant problem has been substantially affected by cultural and demographic changes and by interest group and federal government initiatives. The recognition of AD has transformed senility from an expected stage of life into a "disease." It has also increased fear both of the individual effects of having AD and of the social consequences of AD in the population. Both the biotechnology industry and AD activist organizations will play a role in the social implications of genetic testing for AD.

  View details for Web of Science ID 000087218200003

  View details for PubMedID 10464573

• The genetics of Alzheimer disease and the application of molecular tests 49th Annual Meeting of the American-Society-of-Human-Genetics Tobin, S. L., Chun, N., Powell, T. M., McConnell, L. M. MARY ANN LIEBERT INC. 1999: 37–45

  Abstract

  Two general classes of genes are associated with the development of Alzheimer disease (AD). The first group consists of genes that appear to cause AD when mutated, and the second category is composed of genes that are statistically associated with AD, depending on the inheritance of specific alleles. This paper reviews the current state of knowledge about the genetics of AD, and we then discuss the two molecular tests that are currently commercially available. These include a genetic test for mutations in the presenilin 1 (PS1) gene that can diagnose or predict a subset of early onset familial AD with a high degree of certainty. The value of the genetic test for the apolipoprotein (APOE) allele status is far less clear. Inheritance of the epsilon 4 allele is associated with an increased risk of AD at a population level, but APOE genotyping is inappropriate for prediction of future disease in an individual and offers only a marginal increase in diagnostic certainty when symptomatic individuals are tested. In the future, genetic tests may become more broadly applicable to the diagnosis and prediction of AD. However, the utility of such tests is currently limited to a small subset of individuals because in the vast majority of AD cases no clear genetic or environmental cause has been defined.

  View details for Web of Science ID 000087218200006

  View details for PubMedID 10464576

• Sonchus yellow net rhabdovirus nuclear viroplasms contain polymerase-associated proteins JOURNAL OF VIROLOGY Martins, C. R., Johnson, J. A., Lawrence, D. M., Choi, T. J., Pisi, A. M., Tobin, S. L., Lapidus, D., Wagner, J. D., Ruzin, S., McDonald, K., Jackson, A. O. 1998; 72 (7): 5669-5679

  Abstract

  We have initiated a study of the cytopathology of nucleorhabdoviruses by analyzing the subcellular localization of sonchus yellow net virus (SYNV) genomic and antigenomic RNAs and the encoded polymerase proteins. In situ hybridizations demonstrated that the minus-strand genomic RNA sequences are restricted to the nuclei of infected cells, while the complementary plus-strand antigenomic RNA sequences are present in both the nuclei and the cytoplasm. Immunofluorescence and immunogold labeling experiments also revealed that the nucleocapsid (N) protein and phosphoprotein (M2) are primarily localized to discrete regions within the nuclei and in virus particles that accumulate in perinuclear spaces. The N protein antiserum specifically labeled the nuclear viroplasms, whereas the M2 antiserum was more generally distributed throughout the nuclei. Antibody detection also indicated that the polymerase (L) protein is present in small amounts in the viroplasm. When the N and M2 proteins were expressed individually from the heterologous potato virus X (PVX) vector, both proteins preferentially accumulated in the nuclei. In addition, viroplasm-like inclusions formed in the nuclei of cells infected with the PVX vector containing the N gene. Fusions of the carboxy terminus of beta-glucuronidase to N and M2 resulted in staining of the nuclei of infected cells following expression from the PVX vector. Deletion analyses suggested that multiple regions of the N protein contain signals that are important for nuclear localization.

  View details for Web of Science ID 000074160500042

  View details for PubMedID 9621026

• Phosrestide-1, a peptide derived from the Drosophila photoreceptor protein phosrestin I, is a potent substrate for Ca2+/calmodulin-dependent protein kinase II from rat brain COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY Kahn, E. S., Kinumi, T., Tobin, S. L., Matsumoto, H. 1998; 119 (4): 739-746

  Abstract

  Multifunctional Ca2+/calmodulin-dependent protein kinase type II (CaMK II) plays a crucial role in mediation of cellular responses to rising cytosolic Ca2+ levels. We find that the novel peptide substrate PGTIEKKRSNAMKKMKSIEQHR serves as a highly potent substrate for CaMK II enzymes purified from both Drosophila and rat. The peptide is derived from a photoreceptor-specific protein, phosrestin I, of the Drosophila compound eye and is designated as phosrestide-1. Using saturating substrate concentrations, the enzymes from both species transfer the gamma-phosphoryl group of ATP to phosrestide-1 at a level three to ten times greater than to the commercially available mammalian-derived CaMK II substrates, autocamtide-3 and syntide-2. This indicates a conservation of substrate preferences for CaMK II derived from distantly related species, a dipteran fly and a mammal. Although phosrestide-1 contains two potential serine residues for CaMK II phosphorylation, we find that only the C-terminal serine is phosphorylated by rat CaMK II. However, removal of the upstream sequence containing the N-terminal serine substantially reduced the potency of phosrestide-1 as a CaMK II substrate to a level comparable to that of syntide-2 or autocamtide-3. We also find that a peptide representing the N-terminal segment of phosrestide-1 does not inhibit either CaMK II. Therefore, the enhanced potency of phosrestide-1 as a CaMK II substrate is likely to be due to a preferred conformation of the peptide induced by the N-terminal segment rather than to a specific binding of the enzymes to the N-terminus of the peptide. To the best of our knowledge, phosrestide-1 is the first CaMK II substrate which is designed based on an invertebrate sequence. The high phosphorylation level of phosrestide-1 by CaMK II of mammalian origin may reflect highly conserved CaMK II signaling cascades between vertebrates and invertebrates.

  View details for Web of Science ID 000075828300015

  View details for PubMedID 9787765

• Phosrestin I undergoes the earliest light-induced phosphorylation by a calcium/calmodulin-dependent protein kinase in Drosophila photoreceptors Neuron Matsumoto H, Kurien B, Takagi Y, Kahn E, Kinumi T, Komori N, Yamada T, Hayashi F, Isono K, Pak WL, Tobin SL 1994; 12: 997
• DEVELOPMENTAL EXPRESSION OF THE DROSOPHILA-MELANOGASTER CALMODULIN GENE INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY HANSONPAINTON, O., Randolph, V., Saugstad, J. A., Oh, S. K., Tobin, S. L. 1992; 36 (3): 343-351

  Abstract

  The highly conserved, intracellular calcium binding protein calmodulin is present in all cells at all times. In addition to this constitutive level, the amount of calmodulin is highly regulated according to the tissue or stage of development. Since there are only a few genes or a single gene for this protein in most species, intricate regulatory elements may be necessary to effect its complex regulation. This report adds new information concerning the gene structure and outlines the developmental and spatial regulation of Drosophila melanogaster calmodulin transcripts. The gene contains five exons, including a 49 bp exon in the 5' untranslated region, and spans over 16 kb. Homologues to this small, 5' noncoding exon have not been found in other calmodulin genes. The combined level of the transcripts is developmentally regulated, and the relative amounts of the two transcript size classes (1.65 kb and 1.9 kb) are differentially regulated during development. Primer extension experiments and RNase protection mapping show that both size classes of Drosophila calmodulin transcripts initiate at the same site but undergo alternative termination within the final exon. The spatial distribution of calmodulin transcripts was examined by in situ hybridization to sections of adults and to developmentally staged whole mount embryos. Calmodulin transcripts are evenly distributed early in embryogenesis. In later stages of embryogenesis, higher levels accumulate in the developing nerve cord and other tissues. Elevated levels of calmodulin transcripts are seen quite distinctly in the adult neural tissues and in the photoreceptor region of the compound eye.

  View details for Web of Science ID A1992JV11900001

  View details for PubMedID 1280154

• TRANSCRIPTS OF INDIVIDUAL DROSOPHILA ACTIN GENES ARE DIFFERENTIALLY DISTRIBUTED DURING EMBRYOGENESIS DEVELOPMENTAL GENETICS Tobin, S. L., Cook, P. J., Burn, T. C. 1990; 11 (1): 15-26

  Abstract

  The temporal and spatial patterns of accumulation of transcripts from individual actin genes during Drosophila embryogenesis have been determined by in situ hybridization. We describe the subcloning into transcription vectors of unique DNA fragments derived from the 3' transcribed, but nontranslated region of each actin gene. These fragments then served as templates for the synthesis in vitro of single-stranded, radio-active gene-specific RNA probes. Probe characterization and hybridization to developmental RNA blots are presented, demonstrated the independent developmental accumulation of actin transcripts from each gene. Each gene-specific probe has been hybridized in situ to the transcripts present in embryonic frozen sections. The results of these experiments have demonstrated that transcripts from each actin gene accumulate differentially in developing Drosophila tissues. The 5C and 42A actin genes are cytoplasmic actin genes, with transcripts distributed in all cells and tissues of the developing embryo. Therefore these genes presumably encode the cytoplasmic actins used for functions common to all cells. Transcripts from both cytoplasmic actin genes are evenly distributed in preblastoderm embryos, becoming localized to the periphery at blastoderm formation [5C: Burn et al.: Dev Biol 131:345-355, 1989]. Later in development, levels of these cytoplasmic transcripts vary in specific tissues. While the patterns of localization of 5C actin transcripts have been published [Burn et al.: Dev Biol 131:345-355, 1989], differential neurological localization is presented here; 42A transcripts are localized at higher concentrations in the midgut, the brain, nerve cord, and gonad. Both 87E and 57B transcripts accumulated in the developing larval body wall musculature, but at differing levels and in differing patterns. Transcripts of the 79B and the 88F actin genes were undetectable in embryos. The results of these experiments suggest dedicated contributions of individual actin genes to complex developmental processes.

  View details for Web of Science ID A1990DH91000003

  View details for PubMedID 1694472

• ALTERATION BY HEAT-SHOCK AND IMMUNOLOGICAL CHARACTERIZATION OF DROSOPHILA SMALL NUCLEAR RIBONUCLEOPROTEINS JOURNAL OF CELL BIOLOGY WRIGHTSANDOR, L. G., Reichlin, M., Tobin, S. L. 1989; 108 (6): 2007-2016

  Abstract

  Sera from human patients with systemic lupus erythematosus (SLE) have been shown to react with snRNP particles of both mammals and Drosophila (Mount, S. M. and J. A. Steitz. 1981. Nucleic Acids Res. 9:6351-6368). We have utilized fully characterized monospecific sera and specifically purified antibodies to carry out indirect immunofluorescence experiments with frozen sections of Drosophila embryos. Embryos subjected to severe heat shock before sectioning showed reduced binding of anti-Sm sera. Anti-nRNP sera reacted identically with antigens of heat shocked and non-heat-shocked sections. The reduction in anti-Sm fluorescence was restored by a brief salt wash. These results imply a noncovalent alteration in the conformation of Sm antigens with the administration of heat shock that can revert with exposure to salt. Drosophila antigens have been compared to mammalian standards, showing partial identity with bovine spleen extract (BSE) antigens when reacted with anti-Sm sera. The antigenic relatedness between affinity-purified heat-shocked and non-heat-shocked Drosophila antigens and their mammalian homologues was examined by quantitative ELISA methodology. In all cases, the Drosophila antigens from heat-shocked and non-heat-shocked embryos were identical. We theorize that the heat shock-induced alteration of Sm antigen reverst during extraction. Because the snRNP antigens have been shown to be involved in splicing, and because splicing is inhibited during heat shock (Yost, H. J., and S. Lindquist. 1986. Cell. 45:185-193), our results provide information on the nature and stability of a change in these antigens which may be a central element in control of the heat shock response.

  View details for Web of Science ID A1989AC40500001

  View details for PubMedID 2525559

• TISSUE-SPECIFIC EXPRESSION OF THE 79B ACTIN GENE DURING DROSOPHILA DEVELOPMENT DEVELOPMENTAL BIOLOGY COURCHESNESMITH, C. L., Tobin, S. L. 1989; 133 (2): 313-321

  Abstract

  We report temporal and tissue-specific patterns of expression of the 79B actin gene in Drosophila. The pattern of accumulation of 79B actin transcripts was determined and compared to the expression of a 79B actin promoter fused to the Escherichia coli beta-galactosidase reporter gene. This chimeric gene contained approximately 4 kb of 5' flanking DNA from the 79B actin gene. We found that beta-galactosidase activity in transformants was localized in the same tissues and possessed the same developmental timing as the transcripts of the 79B actin gene. Expression is limited to tubular-type muscles found predominantly in the thorax and leg, including the direct flight muscles, pleurosternal muscles, and muscles of the various leg segments. In addition, the 79B actin gene is expressed in muscles which support the head and abdomen, in the scutellar pulsatile organ, and in abdominal muscles which are present only in male flies.

  View details for Web of Science ID A1989U845400001

  View details for PubMedID 2499492

• ALTERNATIVE 5C-ACTIN TRANSCRIPTS ARE LOCALIZED IN DIFFERENT PATTERNS DURING DROSOPHILA EMBRYOGENESIS DEVELOPMENTAL BIOLOGY Burn, T. C., Vigoreaux, J. O., Tobin, S. L. 1989; 131 (2): 345-355

  Abstract

  The Drosophila actin gene located at cytogenetic position 5C forms at least 9 and perhaps as many as 15 different transcripts with the use of alternative transcriptional start points, differential splicing, and different regions of cleavage/polyadenylation. Each transcript contains one of two alternative 5' exons. We have subcloned unique recombinant DNA probes specific for each separate 5' exon and for three polyadenylation regions into vectors containing T3 and T7 promoters. Single stranded, tritium-labeled RNA probes were generated in vitro from these constructs. These probes have been hybridized in situ to RNA transcripts present in tissue sections from Drosophila embryos. The results of these experiments indicate that transcripts homologous to the two separate 5' exon-specific probes accumulate in strikingly different patterns during Drosophila development. Thus the incorporation of a particular 5' exon into a transcript is correlated with tissue-specific localization of that transcript. In contrast, probes for each of the three polyadenylation regions do not detect any tissue-specific localization of transcripts.

  View details for Web of Science ID A1989T043200008

  View details for PubMedID 2492241

• REGULATION OF LARVAL CUTICLE PROTEIN GENE-EXPRESSION IN DROSOPHILA-MELANOGASTER DEVELOPMENTAL GENETICS Kimbrell, D. A., Tojo, S. J., Alexander, S., Brown, E. E., Tobin, S. L., Fristrom, J. W. 1989; 10 (3): 198-209

  Abstract

  Genes that encode 3rd instar larval cuticle proteins (LCP's) of Drosophila melanogaster are located in at least two chromosomal sites. The genes encoding four of the five predominant LCP's are located in a cluster at the chromosomal region 44D. They are organized in pairs that are transcribed divergently, and expressed with different timing during the third larval instar. Towards understanding the basis of gene regulation within the 44D cluster, we have analyzed genetic variants, including the 2-3 variant, which has an insertion of a copia-like transposable element, H.M.S. Beagle, within the 44D cluster. The Beagle element appears to inactivate the LCP-3 gene by inserting into its TATA box, but also may cause the precocious expression of two other LCP genes, LCP-1 and LCP-f2, in the cluster. The long terminal repeat (LTR) of the Beagle element apparently contains a sequence, perhaps an enhancer-like element, which causes altered expression of these genes. We have also investigated the cis-regulatory elements involved in expression of the LCP-2 gene in wild-type larvae. We have identified two upstream regions that may contain separate cis-regulatory elements. The region between -252 bp and -515 bp may be essential for any expression of LCP-2. Additionally, the region between -515 bp and -795 bp appears to be required for the normal level of expression of the LCP-2 gene.

  View details for Web of Science ID A1989U717200008

  View details for PubMedID 2500284

• STAGE-SPECIFIC SELECTION OF ALTERNATIVE TRANSCRIPTIONAL INITIATION SITES FROM THE 5C ACTIN GENE OF DROSOPHILA-MELANOGASTER GENES & DEVELOPMENT Vigoreaux, J. O., Tobin, S. L. 1987; 1 (10): 1161-1171

  Abstract

  The transcription unit of the 5C actin gene exhibits a complex organization that is unique among the six actin genes of Drosophila melanogaster. Three different mRNA size classes showing distinct patterns of accumulation throughout development are detected on Northern blots. We have determined the structure of the various 5C actin transcripts by exon mapping using strand-specific RNA probes, primer extension analysis, and DNA sequences analysis of both cDNA and genomic clones. All the transcripts share a single protein-coding nucleotide sequence but are heterogeneous in the 5' and 3' untranslated regions. The 5' untranslated region of each transcript consists of either one of two small exons (exon 1 and exon 2) which are alternatively spliced to a single acceptor site 8 bp upstream from the translation initiation codon in exon 3. Results from primer extension analysis suggest that transcription can initiate from either exon 1 or exon 2, and also from a third site within exon 2. We detect an increase in the relative abundance of exon 1-containing transcripts at larval and pupal stages, as well as a change in the proportion of transcripts that initiate at either of the two exon 2 sites. Five polyadenylation sites have been found within three termination/processing regions that define the three size classes of polyadenylated transcripts. The results of our experiments indicate the existence in vivo of all possible combinations of 5' exon with 3' polyadenylation site. However, particular combinations of 5' initiation site and 3' polyadenylation site are preferred at certain developmental stages.

  View details for Web of Science ID A1987L549600012

  View details for PubMedID 3123314

• STRUCTURE AND EXPRESSION OF THE DROSOPHILA CALMODULIN GENE NUCLEIC ACIDS RESEARCH Yamanaka, M. K., Saugstad, J. A., HANSONPAINTON, O., McCarthy, B. J., Tobin, S. L. 1987; 15 (8): 3335-3348

  Abstract

  We have isolated and characterized cDNA and genomic clones representing the calmodulin gene of Drosophila melanogaster. As demonstrated by genomic blots and by reconstruction experiments, the calmodulin gene is represented once in the Drosophila genome. In situ hybridization of cloned probes to the polytene chromosomes of third instar larvae permitted the localization of the gene to region 49A on the left arm of the second chromosome. Two transcripts of 1.65 and 1.9 kb are produced from this gene. The accumulation of calmodulin message was measured at several stages of Drosophila development. The results of these experiments suggest developmental regulation of the gene. Three intervening sequences interrupt the protein coding nucleotides and two of these are located within calmodulin functional domains. The DNA sequence encoding the protein is presented; the derived amino acid sequence is compared to that of other species. The structural similarities of the Drosophila calmodulin gene to calmodulin genes of other species and to other calcium binding protein genes are discussed.

  View details for Web of Science ID A1987H106800009

  View details for PubMedID 3106931

• In vitro transcription of Drosophila actin and 70,000-dalton heat shock protein genes. journal of biological chemistry Nierman, W. C., Miller, A. E., Tobin, S. L., Ingolia, T. D., Sanchez, F., RDEST, U., ZULAUF, E., McCarthy, B. J. 1983; 258 (20): 12618-12623

  Abstract

  Drosophila genomic DNAs containing a chromosomal locus 87C1 70,000-dalton heat shock protein gene, the locus 79B actin gene, and the 88F actin gene have been used as templates in an in vitro HeLa transcription system. RNA polymerase II-dependent transcription initiates from specific sites on the heat shock protein gene and the 79B actin gene. The locations of the transcription start sites were determined by two types of experiments: sizing of RNA runoff transcripts and S1 nuclease mapping of the 5' terminus of the in vitro transcripts. Transcription initiates at or near the in vivo initiation site of the heat shock protein gene and initiates at or near a site 14 nucleotides downstream of the in vivo start site of the 79B actin gene. The addition of the 79B actin, 88F actin, or heat shock protein templates to a HeLa extract transcription reaction precluded the transcription of a second template subsequently added. The exclusion occurs rapidly, within 15 s, is not dependent on transcription, and is only partially resistant to high concentrations of the second added template. We propose that stable protein-promoter complexes play an important role in maintaining exclusive transcription of the first template added in vitro.

  View details for PubMedID 6313667

• INVITRO TRANSCRIPTION OF DROSOPHILA ACTIN AND 70,000-DALTON HEAT-SHOCK PROTEIN GENES JOURNAL OF BIOLOGICAL CHEMISTRY Nierman, W. C., Miller, A. E., Tobin, S. L., Ingolia, T. D., Sanchez, F., RDEST, U., ZULAUF, E., McCarthy, B. J. 1983; 258 (20): 2618-2623

  View details for Web of Science ID A1983RN36300091

• 2 DROSOPHILA ACTIN GENES IN DETAIL - GENE STRUCTURE, PROTEIN-STRUCTURE AND TRANSCRIPTION DURING DEVELOPMENT JOURNAL OF MOLECULAR BIOLOGY Sanchez, F., Tobin, S. L., RDEST, U., ZULAUF, E., McCarthy, B. J. 1983; 163 (4): 533-551

  Abstract

  DNA fragments representing the six Drosophila actin genes have been isolated by recombinant DNA techniques. We have compared the transcriptional characteristics of the actin genes at the cytological loci 79B and 88F. The activity of each gene in vivo was examined using gene-specific probes from transcribed, but non-translated 3' regions of each gene. The genes show similar patterns of transcriptional activity during development until the pupal stage, with two periods showing RNA accumulation at two to three hours and 12 to 15 hours during embryonic development, followed by large increases in the proportion of message from each gene in first and second instar larvae. During pupal development, the 88F gene apparently produces a larger proportion of transcripts than at any other developmental stage, while the transcripts of the 79B gene are reduced to a level lower than in first and second instar larvae. The 5' end of each messenger RNA in larvae has been mapped by nuclease S1 digestion of hybrids between restriction fragments of genes and homologous mRNAs. The two genes display widely differing capacities to serve as templates for transcription in vitro in HeLa cell extracts. The complete DNA sequences of both genes including the flanking regions immediately 3' and 5' to the gene are presented. These data permit comparison of the DNA sequences of these Drosophila actin genes with each other and with the DNA sequence and protein sequence information available for the actins of Drosophila and other organisms. These two genes share the common structural feature of an intervening sequence at amino acid 307, though the sequences within each intron differ greatly. This may be a reflection of a duplication event, followed by divergence of the intervening sequences. We discuss possible correlations between the DNA sequences of each 5' flanking region and the differences in transcriptional characteristics of these two distinct but closely related genes.

  View details for Web of Science ID A1983QB95100002

  View details for PubMedID 6405041

• DEVELOPMENTAL EXPRESSION OF A DROSOPHILA ACTIN GENE ENCODING ACTIN-I NATURE ZULAUF, E., Sanchez, F., Tobin, S. L., RDEST, U., McCarthy, B. J. 1981; 292 (5823): 556-558

  View details for Web of Science ID A1981MA29000049

  View details for PubMedID 6789212

• MULTIPLE ACTIN-RELATED SEQUENCES IN THE DROSOPHILA-MELANOGASTER GENOME CELL Tobin, S. L., ZULAUF, E., Sanchez, F., Craig, E. A., McCarthy, B. J. 1980; 19 (1): 121-131

  Abstract

  In this paper we describe the isolation and characterization of a 7.2 kb D. melanogaster chromosomal DNA fragment (K1) which contains nucleotide sequences complementary to D. melanogaster actin mRNA. Plasmid K1 was identified using a Dictyostelium actin cDNA plasmid, B1, as a probe. D. melanogaster mRNA selected by hybridization with immobilized K1 DNA was translated in vitro to yield products which co-migrate with the D. melanogaster actins I, II and III in two-dimensional gel electrophoresis and bind to DNAase I agarose. A physical map localizing restriction endonuclease cleavage sites in the K1 DNA fragment and the direction of transcription is presented. The position of the coding region has been localized by hybridization with labeled B1 DNA and with labeled poly(A)-containing D. melanogaster RNA. On the basis of hybridization of labeled subfragments of plasmid K1 to restriction endonuclease-cleaved D. melanogaster embryo DNA, we conclude that the nucleotide sequence of the presumptive coding region is responsible for labeling of a pattern of multiple restriction fragments from embryo DNA. The chromosomal locus from which DNA fragment K1 is derived has been localized by in situ hybridization to two closely linked bands in the region 88F. Related DNA sequences corresponding to putative actin genes have also been mapped cytologically. These results support the hypothesis that the genes for actin in D. melanogaster are members of a closely related family of coding sequences.

  View details for Web of Science ID A1980JC60800011

  View details for PubMedID 6244099