New & Noteworthy

In Yeast, Being Old Can Be a Good Thing

March 13, 2017

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A square slab can make an excellent door stop. Over time, though, the corners can get chipped, making the slab a bit rounded. This new bit of rock makes a less useful doorstop, but a much better wheel! The chipping and aging of the original stone has made it worse in some situations, but better in others.

A new study by Frenk and coworkers in Aging Cell shows that something similar can happen in yeast. Young yeast are much better at utilizing glucose, but older yeast have them beat with galactose (as well as with raffinose and acetate).

One way to think about this is that age turns yeast from a glucose specialist into a sugar generalist. Aging chips away at a yeast cell’s ability to use glucose, but this loss results in a gain in its ability to use galactose.

So at least in the right environment (i.e., when there’s lots of galactose around), with our old friend Saccharomyces cerevisiae, there can be advantages to getting older.

What makes this particularly fascinating is that at least in yeast, this suggests that there may be a positive selection for aging because of the advantage it can give in certain environments. Those yeast who are ageless would compete less well compared to their aging counterparts when their glucose was taken away. The aging process wins out over immortality!

Frenk and coworkers used a relatively simple experimental set up. Take young cells and old cells, mix them together, and see which outcompetes the other using various sugars.

They used yeast that had been aged for 6, 24, and 48 hours in glucose. This is a nice range as 6-hour “old” yeast are fully viable, 24-hour “old” yeast are starting to suffer a bit in the reproductive viability department, and the 48-hour “old” yeast have passed the median lifetime of a yeast cell. Young adult, middle aged, and elderly yeast.

In the first experiment, they compared these yeast to log-phase yeast which the authors refer to as young (vs. the other three which are referred to as aged).

While the 6 hour yeast could hold its own against the young yeast in glucose, the 24-hour and 48-hour yeast grew much more slowly. This is what you would expect, the younger yeast growing faster than the older yeast. The young guns outdoing the older generations.

The situation was different in galactose. Here, the elderly, 48-hour yeast, ran circles around the young yeast. They blew them out of the water.

And it appears to be an age thing. When they compared 6- and 48-hour yeast that were aged in galactose instead of glucose, the more aged yeast still won. So, it wasn’t the shift in environment that caused the difference, it was in the older yeast cells all along.

The change is also not permanent. The offspring of the older yeast weren’t any better at growing in galactose than the younger yeast were. Only the cells that had lived a longer life could use galactose so well.

A concern here is that yeast as old as 48 hours are pretty rare in the wild. But when they changed assays and looked at colony size as opposed to competition, they saw that even 18-hour yeast had an advantage over the young whippersnappers.

This was such a surprising result that they also looked at cell cycle times of individual aged cells and their daughters. The older mother cells cycled faster in galactose than their daughters. And the opposite was true in glucose.

So it really looks like there are advantages to growing older. Things break down a bit, but that breakdown uncovers new talents that had previously lain dormant.

If you’re a yeast, growing old is not a one-way decline into dotage. You gain new abilities that, under the right conditions, let you outcompete your children! The older cells are selected for in the right environments. #APOYG shows us something good about growing older.

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

Categories: Research Spotlight

Tags: aging, evolution of aging, generalist, specialist

Feed a Cold, Starve a Cancer?

March 02, 2017

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You may have heard the old wives’ tale of feed a cold, starve a fever. Turns out that this isn’t particularly good advice (although some studies do suggest that with a fever, you shouldn’t force-feed yourself). It also turns out to have probably originated in the 19th century and not from Chaucer in the 14th as many websites claim.

But while starving a fever is probably never a good idea, starving a cancer can be. Not by following the medical myth that since cancers use a lot of sugar, you can starve them by cutting down on sugar in your diet. Instead you can starve some cancers by denying them the amino acid asparagine (Asn).

On their way to becoming cancerous, acute lymphoblastic leukemia (ALL) cells lose their ability to make Asn. This means that unlike the cells around it, they need to pull Asn from the blood to make their proteins and to survive.

Doctors exploit this weakness by injecting L-asparaginase amidohydralase (L-ASNase) into patients which starves the cancer cell by depleting Asn levels in the blood. The cells around the cancer cells are fine because they can still make Asn.

Right now doctors use L-ASNase from two different bacterial sources: Escherichia coli and Erwinia chrysanthemi. But if a recent study by Costa and coworkers in Scientific Reports holds up, they might want to think about switching to using the Saccharomyces cerevisiae L-ASNase encoded by the ASP1 gene.

An older study had suggested that the yeast enzyme might be too weak to be useful. This new study finds that this is not the case.

The difference between the older study and this one was the purification protocol. The older study purified the native enzyme through multiple chromatography steps while this study used a single affinity chromatography step. The purified yeast and E. coli versions have comparable activity in this study.

They are also comparable in terms of being able to work with very low concentrations of Asn. This is important as Asn levels are very low in the blood.

What makes the yeast enzyme potentially better is that it is much worse at hydrolyzing a second amino acid, glutamine, than are the bacterial versions. This higher specificity for Asn is important because one of the major side effects of the current treatment is neurotoxicity caused by decreased levels of glutamine in the blood. Since the yeast version hydrolyzes glutamine at a lower rate, they predict patients may not suffer as badly from this side effect with the yeast version.

Of course this is all for naught if the yeast enzyme can’t kill cancer cells! Or if it kills cells indiscriminately.

The S. cerevisiae version was nearly as good as the E.coli version in tissue culture. After 72 hours of incubation, both versions had little effect on normal cells (HUVEC), and both were cytotoxic to the L-ASNase-sensitive cell line MOLT-4 with the E. coli version killing 95% of MOLT-4 cells and the yeast version killing 85% of them.

Taken together these results suggest that the S. cerevisiae version may be an alternative to the bacterial versions. It may be able to kill cancer cells with fewer side effects.

But the yeast version is not the only alternative in town. Another group is engineering the E. coli version to lessen its propensity for hydrolyzing glutamine. Either way it looks like certain leukemia patients may be getting an effective cancer treatment with fewer side effects.

Beer, wine, bread, chocolate, and now maybe a treatment for a nasty form of leukemia. Yeast may be humanity’s best friend. #APOYG!

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

Categories: Research Spotlight, Yeast and Human Disease

Tags: Acute lymphocytic leukemia, cancer, Proteins, Recombinant protein therapy

Turning to the Prion Expert: Yeast

February 15, 2017

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If you have a legal problem, you get a lawyer. A medical problem, a doctor. A leaky faucet, a plumber.

And if you are trying to find and figure out if a protein is a prion, you turn to the model organism where it is best understood. Yes, that is our old friend, the yeast Saccharomyces cerevisiae.

Prions became famous in the 1980’s when mad cow disease started to pop up in Britain. These are fascinating proteins that can cause an inheritable disease without affecting a cell’s DNA.

What happens is that prions can undergo a spontaneous conformational change. Now of course, this isn’t all that special. Lots of proteins can exist in different conformations.

But what makes a prion special is that the spontaneous change sets off a chain reaction where pretty much every copy of that prion protein is converted to that second conformation. And every translated prion protein thereafter both in the cell and any cells derived from that cell have that conformation too. So the new conformation along with the new traits it confers is passed on stably.

In mad cow disease, this spontaneous change causes neurological problems eventually resulting in death. But not every prion is so dangerous. Sometimes, as is the case in yeast, they can give new properties that allow survival in a new environment. Now these yeast have a new advantage in the absence of changed DNA.

Up until now prions have pretty much been confined to eukaryotes. That looks to change if a new study in Science by Yuan and Hochschild holds up.

They found that some bacteria have proteins that can and do behave as prions in a laboratory setting. The next step is to determine if they ever do so in the wild. If they do, then this would tell us that functioning prions may have evolved before the Bacteria/Eukaryota split and so be older than scientists previously thought.

The first step in finding a bacterial prion involved combing through a few bacterial genomes. Did I say a few? I meant something like 60,000 of them!

Of course you need the right tool for your search. This is where yeast’s incredibly well characterized set of prions comes in handy.

Yuan and Hochschild used an algorithm trained on known yeast prions to search through the bacterial genomes for proteins that have domains that would be predicted to be able to enter into a prion conformation. Among the proteins they found was the Rho protein in Clostridium botulinum E3 strain Alaska E43. Like the authors, I’ll call this protein Cb-Rho from here on out.

As you might remember, Rho is that famous transcription termination protein found in many different bacteria including E. coli. The E. coli version, however, does not look particularly like a prion nor did the authors find that it acts like one either.

A hallmark of prions is that one of the conformations involves their ability to form amyloid aggregates. These authors used a bacterial assay to show that this happened with Cb-Rho.

They next checked whether the prion portion of the protein behaved as a prion in a yeast cell. They substituted the prion domain from Cb-Rho with that of the one from Sup35p, a yeast prion. This chimeric protein behaved similarly to wild type Sup35p.

They next tested their protein in E. coli. Often the prion conformation of the prion protein results in decreased activity of the protein. This is true in the Sup35p case, for example.

Sup35 acts as a translation release factor – it is an important factor in stopping translation at a stop codon. It is less active in its prion form meaning that there is now more read through of stop codons.

Rho is a transcription termination factor and so it would make sense that the prion conformation of Rho would result in lower levels of transcription termination. This is just what the authors found when they tested Cb-Rho in E. coli.

They used a reporter in which a transcription termination site was placed upstream of the lacZ gene. The idea is that if transcription termination is compromised, more lacZ will get made making the colonies a darker blue. And since termination is not 100% efficient, if Rho is doing its job, the colonies will be light blue.

When they plated out E. coli containing an engineered form of Cb-Rho, they got two classes of colonies, light blue and dark blue. And more importantly, this colony color was inheritable. In other words, light blue colonies gave more light blue colonies and dark blue colonies gave more dark blue colonies.

This isn’t to say that the colony color was a permanent state. It wasn’t. A bit under 1% of the colonies would spontaneously revert to the other form, as you’d expect from a prion protein.

So with the invaluable help of yeast, these authors were able to find and validate a protein from bacteria that can act as a prion. If they find a function for this in the wild, then yeast will have helped us better understand the evolution of life on Earth. Again. #APOYG!

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

Categories: Research Spotlight

Tags: amyloid, bacterial prion, lacZ, protein aggregate

Membrane Snorkeling with Arginine

February 06, 2017

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Snorkeling is a blast. With a small tube stuck out into the air you can explore the wonders of the sea for much longer than you would be able to otherwise.

It is obviously important that the snorkel be long enough to reach out of the water. If it isn’t, you’ll be sucking down a lungful of water in no time.

Something similar can happen with membrane spanning proteins except that in this case, a snorkel is not essential to the protein swimming in the greasy confines of a membrane. Instead, positively charged amino acids like lysine or arginine can be tolerated much more often than you might think because of their structure.

Since membranes are hydrophobic, the amino acids in the part of the protein that span the membrane tend to be hydrophobic as well. Charged amino acids are usually trouble.

A proposed exception is amino acids like arginine and lysine. Their positive charges are each at the end of a long aliphatic chain:

What is thought to happen in that the aliphatic chain of the lysine or arginine snuggles up to the greasy part of the phospholipid and the positive part of these amino acids sticks out of the membrane to the more aqueous environment on the other side. The aliphatic side chain is long enough for the charged group to get out of the hydrophobic part. These amino acids may also work well because they can interact with the net negative charge that some phospholipid head groups have.

This is all very cool but, to date, there is very little in vivo work that supports this idea. Which of course means we need to turn to that workhorse model organism Saccharomyces cerevisiae to get some evidence!

In a new study out in GENETICS, Keskin and coworkers use yeast to provide some in vivo evidence that these positively charged amino acids are well tolerated in the membrane-anchoring domain of the yeast protein Fis1p. While I won’t have the space to go into some of the other fascinating experiments in this study, please read it over to learn more about how proteins are inserted into the mitochondrial outer membrane and the structure of this particular carboxy-terminal anchor.

The authors investigated the 27 amino acid carboxy-terminal anchor of this protein by first individually changing every one of its amino acids into every other amino acid (deep mutational scanning). Well, they didn’t get every possible combination. But 98.9% of them is pretty good!

Next they used a very clever screen to find which of these Fis1p mutants that could not properly insert into the mitochondrial membrane. Basically, they fused transcription activator Gal4p to their mutant library. If a mutant protein cannot insert into the membrane, then it is free to enter the nucleus and activate transcription of either a HIS3 or URA3 reporter.

When they did this they were surprised to see that the positively charged amino acids arginine and lysine were well tolerated in the membrane spanning portion of the protein. As expected, mutants with the negatively charged amino acids aspartic acid or glutamic acid in this region of the protein were not.

Here are these four amino acids:

The key difference here (besides the different charges) is the length of the aliphatic chain, the “snorkel” part of the amino acid. Both of the negatively charged amino acids are too short to be able to stick the charged part of the amino acid out of the membrane layer. They are like a snorkeler trying to snorkel with a tube that’s too short. It won’t work well.

So membrane spanning regions are not as hydrophilic fearing as many scientists and protein prediction programs may think.

It may be that the algorithms used to predict membrane spanning regions of proteins are missing some of them because they are too strict about the presence of charged amino acids like lysine and arginine. Perhaps these programs need to be modified to better allow for the presence of an errant arginine or lysine lurking in a long stretch of hydrophobic amino acids.

Of course we turned to yeast to help us better understand reality so we can come up with better algorithms. We may need to listen to yeast so that the programs can be adjusted to think about more than charge, and focus on the length of the snorkel too. #APOYG!

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

Categories: Research Spotlight

Tags: amino acid snorkeling, deep mutational scanning, membrane insertion, mitochondrial division, mitochondrial protein targeting

Don’t miss Fungal Pathogen Genomics!

January 30, 2017

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The application deadline for the Fungal Pathogen Genomics workshop to be held May 11-17, 2017 at the Wellcome Genome Campus in Hinxton, Cambridge, UK is fast approaching! Be sure to apply by this Friday, February 3!

This exciting new week-long course aims to provide experimental biologists working on fungal organisms with hands-on experience in genomic-scale data analysis; including genome browsers and comparison tools, data mining using resources such as FungiDB, Ensembl/PhytoPathDB, PomBase, SGD/CGD, MycoCosm, analysis of genome annotation, and next generation sequence analysis and visualization (including RNA sequence analysis and variant calling). An important aim is that the participants should understand the origin of data available in public resources and how to analyse it in conjunction with their own.

The course is taught as a collaborative effort between available fungal informatics resources. The majority of this intensive course will be based on hands-on exercises, supplemented by lectures on genomics and bioinformatics techniques and keynote presentations by distinguished guest speakers.

Don’t miss out – apply now!

Categories: Announcements

Not Recycling (RNA) Can be Bad for your Health

January 12, 2017

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Not too long ago, it was common to see people pouring used motor oil into street drains. Or to have people dumping old prescription drugs down their sinks.

Practices like these were (and are) terrible for the environment. Nature simply can’t deal with a buildup of this stuff (click here for some examples of the effects of pharmaceuticals on the environment).

Which is why it is so great that there are now ways to deal with waste like this. We can recycle it or at the very least dispose of it more carefully.

Turns out that things are similar in a cell. When its trash isn’t disposed of properly and/or recycled, the cell can suffer. And if cells suffer, so can the person made up of those cells.

One case where something like this is probably happening is in patients with the neurological disorder Pontocerebellar hypoplasia type 1B (PCH1B). These people have a mutated EXOSC3, an important gene for a cell’s RNA exosome. Presumably, this terrible disease is the result of certain cells not being able to properly clear some of their old RNAs.

In a new study out in GENETICS, Fasken and coworkers use good old Saccharomyces cerevisiae to begin to figure out what might be going on in the cells of these patients. They found that the most severe mutation seems to make it harder for the mutated protein to be part of the RNA exosome. As a result of being left out, the mutant protein is degraded more quickly leading to a buildup of some RNAs.

These sets of experiments were made a bit more complicated by the fact that human EXOSC3 cannot substitute for RRP40, the equivalent gene in yeast. This meant the researchers needed to focus on only those disease-causing mutations that hit the most highly conserved residues: EXOSC3-G31A, D132A and W238R.

Of these three, only the W238R yeast equivalent, rrp40-195R had much of an effect on the yeast. Fasken and coworkers propose that this is because this is the most deleterious of the three mutants.

Yeast harboring rrp40-195R grew more slowly at both 30 and 37 degrees C with the more pronounced effect at the higher temperature. At 37 degrees C, this mutant had higher levels of certain RNAs but not others. The RNA exosome was compromised for some but not all yeast RNAs.

And it wasn’t compromised everywhere. Although the RNA exosome works both in the nucleus and the cytoplasm, this mutant appeared to only be compromised in the nucleus. (Check the paper out for the cool way they figured this out.)

Next, the authors wanted to work out what this mutation did to the protein and the exosome. They were able to show that the mutant protein was more unstable than the wild type version and, interestingly, was even less stable when co-expressed with the wild type protein. They also showed that the mutant protein associated less well with the exosome complex and, again, this was exacerbated if the wild type protein was also present.

A reasonable model here is that RRP40 is more prone to degradation when it is not part of the RNA exosome. If true, then the mutant version of the protein is less stable because it is less often a part of the RNA exosome. And wild type RRP40 outcompetes the mutant protein making the mutant stay out of the complex for even more time.

OK, so they have done some good work showing why this mutant of RRP40 affects the growth of a yeast cell. But what we really want to know is if these results explain what is going on in the cells of people with the disease.

Fasken and coworkers tackled this by looking at the effect of expressing the equivalent mouse Exosc3 mutant in the presence of wild type endogenous Exosc3 in mouse neuronal cells (the types of cells affected in PCH1B patients). They found that just like in yeast cells, the mutant was less stable in mouse neuronal cells.

So it looks like the recycling machinery for RNA is broken in these cells because of an unstable component and that this leads to a buildup of toxic RNAs. But if the yeast experiments hold up, not all RNAs are affected.

It is more like people still being able to recycle their cans and bottles but not their motor oil. Certain parts of the environment like waterways take a hit but other parts are left relatively unscathed.

This makes sense when you think about PCH1B. Only a few cell types are affected by the mutation in the EXOSC3 gene. In other words, most cells can deal with a slightly wonky RNA exosome.

Yeast has again helped researchers better understand a genetic disease. Awesome indeed. #APOYG

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

Categories: Research Spotlight

Tags: EXOSC3, Pontocerebellar hypoplasia type 1B, RRP40

Happy Holidays from SGD!

December 20, 2016

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We want to take this opportunity to wish you and your family, friends and lab mates the best during the upcoming holidays.

Stanford University will be closed for two weeks from Wednesday, December 21, 2016 through Tuesday, January 3, 2017. Regular operations will resume on Wednesday, January 4, 2017.

Although SGD staff members will be taking time off, please rest assured that the website will remain up and running throughout the winter break, and we will attempt to keep connected via email should you have any questions.

Happy Holidays and best wishes for all good things in the coming New Year!

Categories: Announcements

SGD December 2016 Newsletter

December 20, 2016

SGD periodically sends out its newsletter to colleagues designated as contacts in SGD. This December 2016 newsletter is also available on the community wiki. If you would like to receive the SGD newsletter in the future please use the Colleague Submission/Update form to let us know.

Categories: Newsletter

Budding Yeast, a Caffeine Wimp No More

December 16, 2016

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Some people get the jitters from a single espresso while others need a triple shot just to get started in the morning. Some of this is due to caffeine tolerance—a buildup of resistance to the marvelous effects of that wonderfully addictive substance, caffeine. But the rest has to do with genetic differences that affect how well each of us processes caffeine—our caffeine sensitivity.

Our best buddy Saccharomyces cerevisiae is a real wimp when it comes to caffeine. In fact, like a lot of other microorganisms, caffeine actually kills this yeast. S. cerevisiae is indeed a sensitive soul when it comes to caffeine.

In a new study in the Journal of Agricultural and Food Chemistry, Wang and coworkers were able to toughen up budding yeast against caffeine by adding bfr1, a gene from Schizosaccharomyces pombe that encodes the ABC transporter that shunts caffeine out of the cell. And then, using random mutagenesis, they were able to make bfr1 even better at its caffeine-exporting job. Although the yeast don’t get any of the pleasurable effects of caffeine, at least they can now happily grow in cultures that have more caffeine than a strong cup of coffee.

This new attribute could prove to be incredibly useful if caffeine producers ever want to start making caffeine biologically instead of synthetically. You can imagine adding the caffeine pathway from coffee to yeast and having the yeast merrily exporting caffeine to the culture medium where it can be harvested. And who knows, maybe they can have the yeast make caffeine and alcohol at the same time creating the equivalent of a vodka and Red Bull in a single step!

Previous research had shown that bfr1 was an important player in helping S. pombe deal with caffeine. When Wang and coworkers added the gene to S. cerevisiae, this newly engineered yeast could now better tolerate caffeine. For example, whereas wild type yeast barely grew with 8 mg/ml caffeine, the engineered yeast did OK.

These authors next turned to random mutagenesis of the bfr1 gene to screen for mutants that could tolerate even more caffeine. And boy did they win the lottery on this one! A mutant that they named bfr1-B did great even at concentrations of 25 mg/ml caffeine. Now they were getting somewhere.

Bfr1 doesn’t just export caffeine; it actually exports many different compounds. The authors found that bfr1-B was fairly specific for increased resistance to caffeine. For example, when they tested the bfr1-B mutant with theophylline, a structurally similar compound, and atropine, a structurally distinct compound, they found that S. cerevisiae expressing the mutant were, if anything, more sensitive to these compounds. They found what looked like a caffeine-specific mutant. 

When they looked at the mutant, Wang and coworkers found that there were 11 amino acid substitutions scattered across the protein. The next step was to figure out which ones mattered and which ones didn’t.

Using a bit of modeling with the 3-D structure of other ABC transporters, they settled on testing three mutations individually. Two of the mutations, S36 and D340 were in the nucleotide binding domain (NBD) and the third, Y497, was in the transmembrane domain (TMD). The NBD is where ATP binds to the transporter to supply the energy to move caffeine across the membrane. 

Of the three, only D340 in the nucleotide binding domain conferred caffeine resistance. While not as robust as bfr1-B, this mutant allowed yeast expressing it to tolerate caffeine concentrations up to 15 mg/ml, conditions under which cells with wild type bfr1 failed to grow. 

So while this mutation explains a lot of why bfr1-B is so good at dealing with caffeine, it is not the whole story. At least some of those other 10 mutations contribute to how well bfr1-B does with caffeine.

In the end we have a bullet-proof yeast when it comes to caffeine that should prove useful for anyone who wants yeast to synthesize caffeine for them. Of course unlike even the most grizzled 30 year coffee drinker with ideal genetics, the yeast almost certainly gets no joy from its morning Joe. But at least that cup of coffee won’t kill it!

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

Categories: Research Spotlight

Tags: ABC transporter, BFR1, caffeine, Schizosaccharomyces pombe

Sign Up Now! Next SGD Webinar: December 14, 2016

December 12, 2016

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Looking for human disease-related information in SGD? There is so much to find! Active areas of curation at SGD include yeast-human homology, disease associations, alleles and phenotype variants, and functional complementation relationships.

Join our upcoming webinar on December 14th, 9:30 AM PST to learn about homology and disease data in SGD. In this quick 15 minute session, we will demonstrate the best ways to research this information on our website and provide a helpful tutorial on related SGD tools and features. Our webinars are always an excellent opportunity to connect with the SGD team–be sure to bring questions if you have them!

All are welcome to this event. If you are interested attending, please register here: http://bit.ly/SGDwebinar6

This is the sixth episode in the SGD Webinar Series. For more information on the SGD Webinar Series, please visit our wiki page: SGD Webinar Series.

Categories: Announcements, Homologs, Tutorial, Yeast and Human Disease

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