New & Noteworthy

Getting Into Yeast’s Genes

September 26, 2013

Yeast has been responsible for a lot of hook ups in its day (think beer goggles and margaritas on the beach).  Now it is payback time.  In a new study, Giraldo-Perez and Goddard have figured out how to make yeast more promiscuous.

No, they don’t get the yeast drunk.  Instead, they found that strains containing VDE, a homing endonuclease gene (HEG), entered meiosis more often than genetically identical strains that lacked VDE.  The yeast that contained this “selfish” gene (well, actually intein) were ready to go haploid more often than those that didn’t.

VDE and its ilk are said to be selfish because they end up getting passed down to more offspring than a certain Austrian monk might have predicted.  When a diploid is heterozygous for an HEG, the homing endonuclease cuts the sister chromosome at the equivalent spot. Then, when the diploid undergoes meiosis, the sister is repaired through recombination causing both chromosomes to contain the VDE gene.  Now instead of two spores containing VDE, all four will.

Giraldo-Perez and Goddard monitored the percentage of sporulating cells over a 30 day period and found that after five days, a higher percentage of diploids homozygous for VDE sporulated compared to diploids heterozygous for or lacking VDE.  The authors contend that under the right conditions, this increased sporulation would allow VDE to spread through a population 20 times faster than it might otherwise.  And the authors found that VDE needs something like this or it might disappear.

Like alcohol, VDE isn’t all lowered inhibitions and good times.  For example, yeast homozygous for VDE grow significantly more slowly than do yeast lacking VDE in YPD, grape juice, vineyard soil, vine bark (heterozygotes fall in between).  This obviously puts yeast carrying VDE at a disadvantage, meaning that if it didn’t have another trick up its sleeve, it would dwindle away to nothing.  That trick is speeding up sporulation. 

The authors weren’t able to determine why this little bit of DNA can have such a profound effect on the growth rate of yeast.  It is almost certainly too little DNA to affect the time it takes the yeast to copy its DNA.  And the endonuclease itself is probably not randomly nicking the chromosomal DNA in the mitotic state, since it is kept out of the nucleus by host encoded karyopherins.

So VDE is a truly a parasitic selfish gene.  It is parasitic because it sucks a little of the life out of a yeast cell.  And it is selfish because way more daughters end up with it than might be predicted.  Sounds like a nice description for many drunk people…

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Have Your Fuel and Eat It Too

September 19, 2013

Back in 2008 and 2011 there were huge spikes in the cost of food that caused riots in various parts of the world.  These things were pretty bad and one of our favorite beast’s best products, ethanol, may have been at least partly to blame.  In an attempt to deal with global warming, governments had created incentives that made it more lucrative to turn food into ethanol to power cars rather than keeping it as food to feed people.  The law of unintended consequences reared its ugly head and caused food prices to rise high enough to be unaffordable by the very poor.   

This situation arose because right now, pretty much the only commercially viable way to make ethanol is to use sugars like those found in sugar cane or starches like those found in corn.  Ultimately this won’t be a problem once scientists learn to coax yeast or other microorganisms to make ethanol out of agricultural waste.  Until then, though, one way to lessen the impact of ethanol production on food supplies might be to engineer a yeast strain that can more efficiently turn sugars into ethanol. 

One of the most inefficient parts of yeast fermentation is that the silly thing converts anywhere from 4-10% of the sugars it gets into glycerol instead of ethanol.   In a new study, Guadalupe-Medina and coworkers have engineered a strain of yeast that produces 60% less glycerol and 8% more ethanol than other commercial strains.  If they can scale this up, it might help us feed both the world’s population and our cars.

It has been known for some time that yeast end up making glycerol during fermentation because of redox-cofactor balancing issues.  In essence, the excess NADH that is made in fermentation reactions is reoxidized by converting part of the sugar into glycerol.  One obvious way to get less glycerol would be to give the yeast some other way to reoxidize its NADH. 

Guadalupe-Medina and coworkers decided to persuade yeast to use carbon dioxide instead of sugars.  Not only would this make sugar use more efficient, but their particular plan would also convert that carbon dioxide into a precursor that could be shunted into the ethanol producing pathway.  Theoretically the yeast should now increase its ethanol production both by wasting less sugar on glycerol and by turning carbon dioxide into ethanol.  And it turns out that this idea actually worked in practice.

The first step was to introduce the Rubisco enzyme into the yeast.  Rubisco (ribulose-1,5-bisphosphate carboxylase oxygenase) is really one of the key enzymes in life…it provides the foundation for almost all life on the planet by fixing carbon dioxide from the air into ribulose-1,5 phosphate.  But that isn’t the important point here.  No, the key point for this work is that in the process of doing this, the enzyme oxidizes NADH.  By putting Rubisco in yeast, the yeast should now be able to reoxidize its NADH without making useless glycerol.

Of course this is easier said than done!  Rubisco is multi-subunit in most beasts and persnickety to boot.  But with a bit of work, they managed to get Saccharomyces cerevisiae to express a working copy of Rubisco.

So they would only have to introduce a single gene, the authors used the single subunit enzyme from T. dentrificans. As expected, this gene alone was not enough.  They knew from previous work that Rubisco would not work in yeast without the help of a couple of E. coli chaperones, groEL and groES.  When they expressed all three genes at the same time, they got Rubisco to fix carbon dioxide in Saccharomyces cerevisiae.  

The next step was to introduce the enzyme phosphoribulokinase (PRK) so that the ribulose-1,5 phosphate could be converted into 3-phosphoglycerate, a precursor in the ethanol pathway.  Luckily this was much easier than Rubisco and worked on the first try.  They had now engineered a Frankenyeast that should be able to make more ethanol and less glycerol.

When they tested the new strain, Guadalupe-Medina and coworkers found they had indeed engineered a more efficient yeast.  In anaerobic chemostat conditions, this yeast made 68% less glycerol and 11% more ethanol than the usual commercial strain.  They obtained similar results, 60% less glycerol and 8% more ethanol, in batch fermentations.  They had succeeded in improving an already awesome beast.

If this strain works on an industrial scale and if commercial producers all used this strain instead of the ones they currently use, the authors calculate we could get an extra 5 billion liters of ethanol added to the 110 billion we are already making.  That might just be enough to tide us over until scientists come up with a way to make ethanol commercially from non-food sources.

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Fred Sherman, 1932-2013

September 18, 2013

The yeast community mourns the loss of Dr. Fred Sherman, who passed away on September 16, 2013. Dr. Sherman was a member of the faculty at the University of Rochester from 1961 until his death. He served as Chair of the Department of Biochemistry and then Chair of the combined Department of Biochemistry and Biophysics from 1982-1999. He performed ground-breaking research on the structure and regulation of genes and the effects of genetic mutations on proteins and was a proponent of the use of baker’s yeast as a genetic model system – a system that is now used at virtually all research centers worldwide, largely due to Dr. Sherman’s efforts and his teaching of many leaders in the field. The importance of his work has been recognized by his appointment to the prestigious National Academy of Sciences in 1985, by his receipt of an Honorary Doctorate from the University of Minnesota in 2002, by his election as a Fellow of the American Association for the Advancement of Science in 2006, and by his receipt of both the George W. Beadle Award and the Lifetime Achievement Award from the Genetics Society of America in 2006. He was continuously funded by NIH for over 45 years.

Dr. Sherman’s family will receive friends on FRIDAY September 20, from 3-7 PM at Michael R. Yackiw Funeral Home, 1650 Empire Blvd., Webster. On SATURDAY, friends may join his family for a graveside service gathering at the Mt. Hope Ave. entrance of Mt. Hope Cemetery at 11 AM. In lieu of flowers, contributions may be directed to a fund to support an annual lecture in Fred’s memory. To donate please mail donations to: Fred Sherman Lecture of the University of Rochester, Box 712, University of Rochester Medical Center, 601 Elmwood Ave., Rochester NY, 14642.

Plans for a future memorial service will be announced at a later date.

Parthenogenesis, Saccharomyces Style

September 10, 2013

Parthenogenesis is one of the cooler things in biology. When a female Komodo dragon can’t find a mate, her eggs simply double their DNA and voila, a whole litter of female Komodo dragons is born. (Interestingly, they aren’t clones of mom…)

Now, this doesn’t work in mammals like us (curse you imprinting!), but something similar can happen in yeast. Given the right conditions and the right mutations, yeast can go from haploid to diploid without all that messy mating.

In a new study out in GENETICS, Schladebeck and Mösch uncover the newest mutation to be shown to cause whole genome duplication (WGD) in haploid Saccharomyces cerevisiae: the whi3 deletion. And this mutant is no slouch…the haploid will go diploid in no time flat if given the right conditions.

Schladebeck and Mösch looked at the stability of the haploid state of the whi3 mutant in both minimal and rich media, either in liquid culture or on solid agar. They generated fresh whi3 deletion strains and then followed them in each of these growth conditions for 72 days, passaging them every two days. 

What they found was that the haploid state was actually pretty stable in liquid culture using minimal media. They found very few diploid cells after 72 days. The same was not true for the other growth conditions.

On solid minimal media and liquid rich medium, there was a complete switch after 72 days. And on solid rich medium, the cells were all diploid after only 14 days. Genome duplication appeared to stop at the diploid level though. Even after 72 days on solid rich media there was no sign of tetraploids.

The authors next set out to figure out why deleting WHI3 had such a profound impact on haploid stability. They have not yet figured out everything that is going on, but they did uncover some interesting clues.

First they looked at the protein Nip100p. They already knew that NIP100 interacted genetically with WHI3, and that a nip100 deletion mutation affected chromatid separation. They found that Nip100p levels were significantly reduced when WHI3 was deleted, and even more so when the whi3 mutant strain was grown on solid rich medium. These are the conditions that most favored the transition from haploid to diploid. This suggests that NIP100 might be a key player in maintaining the haploid state.

The authors also compared transcriptional profiles of the wild type haploid strain, the whi3 deletion in a haploid background, and the whi3 homozygous mutant diploid. One of the findings from these experiments was that most of the genes involved in the yeast cohesion complex were upregulated in the absence of WHI3. Since this complex is required for sister chromatid cohesion, the idea would be that inefficient separation of chromatids in the whi3 mutant would increase the rate of whole genome duplication.

One of the as yet unexplained aspects of all of this is why the diploid state remains stable. There was no difference between the haploid and diploid deletion strains with regard to either Nip100p levels or transcription of cohesion-relate4d genes – the cohesins were upregulated in both and Nip100p was reduced in both.

One idea Schladebeck and Mösch put forth is that the diploid state isn’t inherently stable in this mutant. Instead, they do not see tetraploids simply because tetraploids have decreased viability. They appear but are quickly outcompeted by their diploid sisters.

The discovery about WHI3’s role in controlling ploidy is just one aspect of this new study. The authors also found important new information about the central regulatory role of WHI3 in cell division and biofilm formation.

The finding about ploidy control is important because maintaining haploid and diploid status is obviously a big deal: you don’t want to switch willy nilly from one to the other. And many pathogenic fungi, such as Candida albicans, change the organization of their genomes to adapt to changing growth conditions in their human hosts. They have WHI3 homologs, so these results could lead to better ways to cure fungal infections. Just one more example of how basic research can lead scientists to stumble on unexpected but ultimately important results…

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics