New & Noteworthy

Be Good, For Adaptation’s Sake

December 17, 2012

You may never have herded cows. But in one way or another, you’ve certainly experienced the tragedy of the commons.

When herders get away with cheating, everyone loses. The same thing is often true for yeast.
Image from Wikimedia Commons

This happens when a village shares a pasture that can only feed a certain number of cows. For the system to work, everyone has to cooperate and keep the total number of cows under that limit. But inevitably, one cheater comes along and adds extra cows to his herd. At no immediate cost to himself, he gets all the benefits of the extra cows. But then tragedy kicks in as the pasture is overgrazed until no one can have any cows.

This doesn’t just happen out in the village. We can see it in the overfishing of the oceans, the production of carbon dioxide contributing to global warming, the milking of investors on Wall Street, and many other aspects of modern life. But perhaps surprisingly, we can even see it in cultures of the humble yeast S. cerevisiae.

In a recent issue of the Proceedings of the National Academy of Sciences, Waite and Shou set up a yeast system to look at the factors influencing the tragedy of the commons. In human society, sometimes cheaters cause a collapse, but other times, cooperators get together and exile the cheaters from the village. You might think that since yeast aren’t quite as smart as humans, in yeast “society” the cheaters would always win. However, Waite and Shou found that sometimes the cheaters were marginalized or even driven out, and the cooperators thrived!

To do these experiments, the researchers set up a very clever pasture in miniature. They engineered three strains, each marked with a different-colored fluorescent marker so they could be distinguished from each other.

The two “cooperator” strains needed each other to survive on minimal medium: one required lysine and produced excess adenine, while the other required adenine and produced excess lysine. The “cheater” strain required lysine but it didn’t provide any nutrients. So the cheater needed one of the cooperators to survive, but didn’t contribute anything to the common good.

As we might expect, being cooperative has a cost. The generous production of extra nutrients made the cooperator grow slower than the otherwise identical cheater strain. So you would predict that if you mixed the cooperators and the cheater in equal numbers and grew them together, the cheater would take over and collapse the culture every time.

However, when the researchers mixed all three strains in a 1:1:1 ratio and grew lots of replicate cultures, they found that cheaters didn’t always prosper. Sometimes the nice guys finished first.

Of course some of the cultures did collapse under the influence of the rapacious cheaters. After a while these cultures stopped growing and turned out to be made up of mostly dead or dying cheater cells. The cheaters had taken over the culture, selfishly using up the lysine until eventually there was not enough to continue growing.

But unexpectedly, other replicate cultures were growing much faster, at rates similar to cultures without any cheaters. In these cultures, the two cooperator strains had either dominated the culture or even driven the cheaters extinct!

To explain this, the researchers proposed that the intense selection pressure led to an adaptive race between cooperators and cheaters. In surviving cultures, the rare cooperator with a small advantage had outcompeted the cheaters.

To confirm this, they took a close look at the winning cooperators. They found that the fitness advantage could be inherited, so they used whole-genome resequencing to find out why the cooperators were outcompeting the cheaters. They kept finding mutations in the same five genes.

These genes all made sense, as mutating them would help in an environment with limited amounts of lysine. For example, most of the mutations were found in ECM21 and DOA4. Both of these gene products are important in pathways that break down proteins like permeases. Knocking them out would keep the permeases around longer, making for better lysine uptake. But this newfound advantage did not come without a price.

Dogs playing cards

While cheating is usually a good short term strategy, it doesn’t always work out so well in the long term.
Image from Wikimedia Commons

The researchers tested directly whether the adaptive mutations improved growth in limiting amounts of lysine. Without exception, they did. But almost all these strains grew more slowly in abundant lysine than did their ancestor strains. That explains why these mutant strains only became a significant proportion of the population late in the life of the culture, when lysine levels were very low.

The same mutations can arise in both cooperators and cheaters, of course. But when cheaters become better at growing in low lysine levels, they just become that much better at making themselves extinct. When cooperators get better at growing in low lysine levels, they are better able to keep growing and keep the cheaters at bay.

So the take-home lesson is that cooperation does pay, after all. Especially in a constantly changing environment, cooperators can often win the adaptive race and squelch the cheaters. Maybe we should take a hint from little S. cerevisiae that being kind to each other is not only a nice thing to do, it’s in all of our best interests!

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

Categories: Research Spotlight

Tags: evolution , Saccharomyces cerevisiae , adaptation

SGD Fall 2012 Newsletter

December 14, 2012

SGD sends out its quarterly newsletter to colleagues designated as contacts in SGD. This Fall 2012 newsletter is also available online. If you would like to receive this letter in the future please use the Colleague Submission/Update form to let us know.

Categories: Newsletter

New Strain Sequences Available

December 12, 2012

SGD has incorporated two new S. cerevisiae strain genomes: CEN.PK113-7D and ZTW1. CEN.PK113-7D is a laboratory strain derived from parental strains ENY.WA-1A and MC996A, and is popular for use in systems biology studies. ZTW1 was isolated from corn mash used for industrial bioethanol production in China in 2007. We have also incorporated an updated genomic sequence for W303-derivative K6001, a key model organism for research into aging. The previous version of W303 was an early view courtesy of Markus Ralser. SGD has updated the ORF DNA and protein sequence alignments to include these two additional strains, and the updated W303. We have also included the new genomic, coding, and protein sequences in both the BLAST Search and Pattern Matching (PatMatch) tools. The alignments are accessible via the Analyze Sequence section on the Locus Summary pages. Also available are retrieval and download options.

Categories: Sequence

As Good as the Original

December 07, 2012

Unlike the rest of us, cells are happy with either a copy or the original.

If you got to choose between an original and a copy of that original, you’d undoubtedly choose the original. Because of mistakes during the copying process, the original is bound to be superior.

Cairns was the first scientist to try to apply the same logic to cells like mother cells of the budding yeast S. cerevisiae, or stem cells. The idea is that since these cells make lots of copies of themselves, they might have some mechanism for keeping the original DNA and sending the copies to the new cells. This would protect the original DNA from building up mutations.

While this seems reasonable, the many studies done to date have failed to provide any compelling evidence to support the idea. When scientists look at a cell’s DNA in bulk, they see it randomly dividing between mother and daughter. But this is not the end of the story.

Another idea people have had is that one of the strands of the double helix is kept in its original form in the mother while the other is free to be passed on to the daughter. There is mounting evidence in yeast and mammalian cells that there is some strand-specific selection going on. But in a new study out in GENETICS, Keyes and coworkers show that this selection is not dependent on a strand being an original as opposed to a copy.

If mother cells preferentially keep one of the strands, then the red ones should only end up in the MD and DM cell types in this experiment. The real results were that all four cell populations had the red DNA. (Image from Keyes et al. used with permission from GENETICS)

They use a couple of cool tricks to be able to follow specific strands of DNA in specific cell types in these experiments. First, they engineered a strain that would incorporate BrdU (bromodeoxyuridine) into newly synthesized DNA, so that they could distinguish between the original and copied strands. Second, they used a technique to separate mother cells from daughter cells that involves arresting the mother cells with alpha factor, labeling them with biotin, and then allowing them to divide: the mothers can be pulled away from the daughters by their biotin labels.

As you can see from the image on the right, Keyes and coworkers let a population of biotin-labeled mother cells divide once in the presence of BrdU. Next they separated mothers (M) from daughters (D) and biotinylated the daughters.

Next they let the two populations divide separately one more time in unlabeled thymidine (TdR) instead of BrdU, and separated mothers from daughters again. As shown in the figure, this allowed them to isolate four different cell populations:

1) MM: the original mother cells
2) MD: daughters of the original mothers
3) DM: mother cells derived from the first daughter
4) DD: daughters of the first daughter

The image shows the prediction if the Watson strand (W) is preferentially kept by the mother. As you follow along, remember that the red strand represents the labeled DNA.

If mother cells inherit the Watson strand specifically (W) and the daughter cell always gets the Crick strand (C), then we would predict that only MD and DM cells should have BrdU DNA. The other two cell types should only have unlabeled DNA.

Let’s follow the mother side to see why. The mother cell would inherit the unlabeled Watson strand and a labeled Crick strand because she would keep the original from the first division. The labeled and so copied Crick strand would then be passed preferentially to the daughter.

The same sort of logic applies to the daughter cell. The daughter inherited the labeled Crick strand. Since this daughter now becomes the mother in the next division, the labeled strand now becomes the Watson strand. She would keep this labeled strand and pass the unlabeled Crick strand to her daughter.

These are not the results they got. Instead, all the cell types had pretty close to the same amounts of BrdU. The moms did not preferentially hang on to either the original Watson or Crick strand.

They then followed this up with a separate, similar experiment that looked at each individual chromosome on a microarray. The results were that there was no bias for either strand for any of the chromosomes.

It seems that mom doesn’t hang onto the original DNA even at a single chromosome. In the cases of strand-specific selection that are now being studied, there are most likely other ways to pick a strand that have nothing to do with whether it is an original or a copy. Another great idea foiled by data…

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

Categories: Research Spotlight

Tags: Saccharomyces cerevisiae , chromosome segregation

SGD Server Move Successfully Completed

December 05, 2012

Thank you for your patience during our move to the new data center last week.

SGD servers

Systems Administrator Stuart Miyasato with the SGD servers in their new location

This move is very significant for us, as we are now in a professionally managed computing facility with all the necessary backup cooling and power contingencies available. This move also allows us to continue expanding our services.

The computer room is critical for the stability of SGD and other resources. Our previous location was created when SGD was young, and did not accommodate the amount of growth we have enjoyed. We are very happy to have the opportunity to be located in this brand new facility designed for research computing.

-Mike Cherry

Data Center

The new Stanford Data Center housing SGD’s and other servers

Categories: Website changes

SGD Servers Moving to new Data Center

November 21, 2012

Many SGD tools and resources will be unavailable from Tuesday afternoon, November 27th, until Thursday, November 29th because we will be moving all of our servers to a new data center. This new data center will provide a more secure facility, a faster network, and a diesel generator to provide electricity in the event of a power failure.

You will be able to search the main SGD database during this time. However, many tools and pages, including the following, will be unavailable: Pathway Tools, YeastMine, Downloads/FTP, SPELL, GBrowse, Textpresso, and the SGD Wiki.

We apologize for the inconvenience and thank you for your patience.
Please contact us at sgd-helpdesk@lists.stanford.edu if you have any questions or concerns.

Categories: Maintenance

I See Dead Yeast (in My Beer Foam)

November 19, 2012

A study by Blasco and coworkers confirms that beer foam is littered with corpses of dead yeast. Or at least with bits of their cell walls.

glass of beer

This one has a lot of Cfg1 protein.

This has been known for awhile. But what these researchers did was to identify one of the key proteins in the cell wall important for maintaining a good head on beer.

The authors knew from previous studies that certain mannose binding proteins play an important role in beer foam. So they used primers that lined up with the 5’ and 3’ ends of one of the known foam-related genes from S. cerevisiae, AWA1, to look for similar genes in the brewing yeast S. pastorianus. This allowed them to PCR out the CFG1 gene.

To show that this protein was involved in the foaminess of beer, they next knocked the gene out of S. pastorianus and used this deletion strain to do some brewing. What they found was that while beer made with this strain had about the same amount of foam, it didn’t last as long. This strongly suggested that CFG1 was involved in maintaining a good head on a mug of beer, earning the gene its name: Carlsbergensis Foaming Gene.

As a final experiment, they added the gene back to a strain of S. cerevisiae, M12B, that makes beer without foam. When this strain expressed CFG1 and was used to brew up some beer, that beer was foamless no more. This suggests that CFG1 may be important for foam formation as well as stabilization.

What is probably happening is that during fermentation, yeast cells are autolysing, releasing their cell wall proteins into the beer. Since Cfg1p is hydrophilic on one end and hydrophobic on the other, it forms very stable bubbles. And foam is simply a bunch of stable bubbles.

Hopefully scientists can use this information to tweak the amount of foam a given beer yields. Then a drinker can choose lots or little foam, long lasting or short lived foam, or any combination he or she wants.

Root beer foams for a different reason

NPR’s take on this study

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

Categories: Research Spotlight

Tags: Saccharomyces pastorianus , beer , brewing , Saccharomyces cerevisiae

Identifying the Unstable

November 08, 2012

One of the many stumbling blocks in finding better treatments for genetic diseases is figuring out the cause of the disease.  These days, this doesn’t necessarily mean simply identifying the gene with the mutation.  No, nowadays it can mean figuring out what each specific mutation does to the gene it damages.

See, many genetic diseases are not caused by single mutations.  Instead, lots of different mutations can all damage the same gene in different ways.  And each class of mutation may require different treatments.

It’s easy to tell this bridge is unstable. It is a lot harder to tell with proteins.

Cystic fibrosis (CF) is a great example of this.  While most cases of this ultimately fatal disease are caused by mutations in the CFTR gene, not every mutation does the same thing to the CFTR protein.  Because of this, scientists have found different drugs to treat people with different classes of CFTR mutations.

So one drug, Ivacaftor, targets CFTR proteins that can’t open up as well as they should, while another investigative drug, PTC124, targets prematurely stopped CFTR proteins.  Each only treats a specific subset of CF patients who have the correct CFTR mutation.

All of this screams out for a quick and easy assay to figure out how a mutation actually disables a certain protein.  And this is where a new study by Pittman and coworkers just published in the journal GENETICS can help.

The authors have come up with a sensitive in vivo assay in S. cerevisiae that allows scientists to quickly identify mutations that lead to unstable proteins.  This kind of instability isn’t rare in human disease either.  Some of the more famous examples include a kidney disease called primary hyperoxaluria type 1 (PH1), Lou Gehrig’s disease (ALS), Parkinson’s disease, spinal muscular atrophy (SMA), and even some forms of cancer.

The assay basically inserts wild type and mutant versions of the gene of interest into the middle of the mouse dihydrofolate reductase (DHFR) gene, individually adds these chimeric genes to yeast lacking DHFR, and then measures growth rates.  The idea is that if the mutation leads to instability, the DHFR chimeric protein will be unstable too and the yeast will show growth defects under certain conditions.  This is just what they found.

Initially they focused on a gene involved in PH1, the AGT gene encoding alanine: glyoxylate aminotransferase.  They were able to show that disease causing mutations known to affect protein stability affected growth in this assay.  Not only that, but there was a strong correlation between growth and level of protein stability.  In other words, the more unstable the protein, the more severe the growth defect.

They then expanded their assay beyond known AGT mutations.  First they were able to identify a subset of disease-causing AGT mutations as affecting the stability of the AGT protein.  But the assay ran into trouble when they switched to the more stable SOD1 protein.  This protein, which is involved in most cases of ALS, is so stable that mutations that destabilized it were invisible in the assay.  The authors solved this problem by introducing a mutation into DHFR that destabilized it.  Now they could identify mutants that destabilized SOD1.

As a final step, they used their assay to screen a library of stabilizing compounds to identify those that specifically stabilized their mutant proteins.  Unfortunately, in this first attempt they only found compounds that stabilize DHFR, but the assay has the potential to find drugs that stabilize disease-related proteins as well.

Whether or not that potential is realized, this technique should still be a very useful way to determine whether a mutation affects protein stability.  Then, when drugs that stabilize the protein have been found, using this or other screens, doctors will know which patients can be helped by these compounds.  And this will be a boon for scientists and patients alike.

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

Categories: Research Spotlight Yeast and Human Disease

Tags: yeast model for human disease , protein stability , Saccharomyces cerevisiae

Using Yeast to Discover Treatments for ALS

November 01, 2012

What do Lou Gehrig, Stephen Hawking, David Niven and Mao Zedong have in common?  They all suffered (or in Hawking’s case, continue to suffer) terribly from a disease called amyotrophic lateral sclerosis or ALS.  And now the humble yeast S. cerevisiae may help scientists find new treatments so that others do not need to suffer similarly.

Patients with ALS gradually lose use of their motor neurons and generally die within 3-5 years of diagnosis.  While there are some rare forms that run in families, most are sporadic.  There is no history of the disease in the family and then suddenly, it just appears.

cowboy with lariat

Lariats can also rustle up some TDP-43!
(Image: Rodeo Star sculpture by Clay Hoffman, clayhoffman@frontier.com)

The causes of ALS have remained a mystery for many years but recent work has suggested that RNA binding proteins and RNA processing pathways are somehow involved.  In particular, an RNA-binding protein called TDP-43 appears to be a key player.  Mutations in its gene are associated with ALS, and aggregates of the protein are found in damaged neurons of ALS patients. Unfortunately, since this protein is needed for cell survival it is not an easy target for therapies.  This is where yeast can help.

Scientists have managed to mimic the effects of TDP-43 in yeast.  When this protein is overexpressed, the yeast cells die just like the motor cell neurons do. In a recent Nature Genetics paper, Armakola and coworkers use this model system for finding better therapeutic targets.  And it looks like they may have succeeded.

These authors used two different screens to systematically look for proteins that when deleted or expressed at lower levels rescued yeast overexpressing TDP-43.  They found plenty.  One screen yielded eight suppressors while the other yielded 2,056 potential suppressors.  They decided to focus on one of the stronger suppressors, DBR1.

The first thing they wanted to do was to make sure this wasn’t a yeast specific effect.  If lowering the amount of DBR1 has no effect in mammalian models, it is obviously not worth pursuing!

To answer this question, they created a mammalian neuroblastoma cell line with an inducible system for making a mutant version of TDP-43, TDP-43 Gln331Lys, found commonly in ALS patients.  As expected, these cells quickly died in the presence of inducer.  They could be rescued, though, when DBR1 activity was inhibited with siRNA.  The authors confirmed that decreasing the activity of DBR1 in primary neurons decreased TDP-43 toxicity as well.

So decreasing the amount of DBR1 appears to rescue cells that die from the effects of mutant TDP-43.  This suggests that targeting DBR1 may be useful as a therapy for ALS.  But this study doesn’t stop there.  It also tells us a bit about how lowering DBR1 levels might be rescuing the cells.

DBR1 is an RNA processing enzyme involved in cleaning up the mess left behind by splicing.  It cleaves the 2’-5’ phosphodiester bond of the spliced-out intron (called a lariat).  Previous studies in yeast have shown that when Dbr1p levels are reduced or its catalytic activity is disrupted by a mutation, there is a build up of these lariats. This study showed directly that the accumulated lariats interact with TDP-43 in the cytoplasm to suppress its toxicity. So in ALS, the accumulated lariats may serve as a decoy for the mutant TDP-43 protein, preventing it from binding to and interfering with more essential RNAs.

This last result may also suggest another potential therapy.  If scientists can find other ways to increase the amount of decoy RNA, then they may not need to depend on reducing levels of DBR1.  There may be many possible approaches to soaking up rogue TDP-43.

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

Categories: Research Spotlight Yeast and Human Disease

Tags: RNA binding , Lou Gehrig's Disease , DBR1 , yeast model for human disease , Saccharomyces cerevisiae , ALS

SGD Downloads Site Temporarily Unavailable

October 29, 2012

The server that hosts SGD’s download files was affected by a power outage over the weekend and is currently unavailable. We are working to resolve the problem as quickly as possible. We apologize for the inconvenience, and thank you for your patience.

Categories: Maintenance

When Polymerases Collide

October 25, 2012

Lots of recent studies are showing that transcription happens over way more DNA than anyone previously thought. For example, the ENCODE project has shown that most of a genome gets transcribed into RNA in humans, fruit flies and nematodes. This transcriptional exuberance was recently confirmed in the yeast S. cerevisiae as well.

There is also a whole lot of antisense transcription going on. Taken together, these two observations suggest that there are lots of opportunities for two polymerases to run headlong into each other. And this could be a big problem if polymerases can’t easily get past one another.

Goats butting heads

What happens when RNA polymerases meet head-on?

Imagine that the two polymerases clash in the middle of some essential gene. If they can’t somehow resolve this situation, the gene would effectively be shut off. Bye bye cell!

Of course this is all theoretical at this point. After all, smaller polymerases like those from T3 and T4 bacteriophages manage to sneak past one another. It looks like this isn’t the case for RNA polymerase II (RNAPII), though.

As a new study by Hobson and coworkers in Molecular Cell shows, when two yeast RNAPII molecules meet in a head on collision on the same piece of DNA, they have real trouble getting past each other. This is true both in vitro and in vivo.

For the in vivo experiments, the authors created a situation where they could easily monitor the amount of transcription close in and far away from a promoter in yeast. Basically they pointed two inducible promoters, from the GAL10 and GAL7 genes, at one another and eliminated any transcription terminators between them. They also included G-less cassettes (regions encoding guanine-free RNA) at different positions relative to the GAL10 promoter, so that they could use RNAse T1 (which cleaves RNA at G residues) to look at how much transcription starts out and how much makes it to the end.

When they just turned on the GAL10 promoter, they saw equal amounts of transcription from both the beginning and the end of the GAL10 transcript. But when they turned on both GAL10 and GAL7, they saw only 21% of the more distant G-less cassette compared to the one closer to the GAL10 promoter.

They interpret this result as meaning the two polymerases have run into each other and stalled between the two promoters. And their in vitro data backs this up.

Using purified elongation complexes, they showed that when two polymerases charge at each other on the same template, transcripts of intermediate length are generated. They again interpret this as the polymerases stopping dead in their tracks once they run into one another. Consistent with this, they showed that these stalled polymerases are rock stable using agarose gel electrophoresis.

Left unchecked, polymerases that can’t figure out how to get past one another would obviously be bad for a cell. Even if it were a relatively rare occurrence, eventually two polymerases would clash somewhere important, with the end result being a dead cell. So how do cells get around this thorny problem?

King Arthur and the Black Knight

To get past the Black Knight, Arthur had to destroy him. Hopefully the cell has more tricks up its sleeve than that!

One way is to get rid of the polymerases. The lab previously showed that if a polymerase is permanently stalled because of some irreparable DNA lesion, the cell ubiquitinates the polymerase and targets it for destruction. In this study they used ubiquitin mutants to show that the same system can work at these paused polymerases too. Ubiquitylation-compromised yeast took longer to clear the polymerases than did their wild type brethren.

The authors think that this isn’t the only mechanism by which polymerases break free though. They are actively seeking factors that can help resolve these crashed polymerases. It will be interesting to see what cool way the cell has devised to resolve this dilemma.

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

Categories: Research Spotlight

Tags: transcription , RNA polymerase II , ubiquitin-mediated degradation , Saccharomyces cerevisiae

Better Beer Through Beards

October 16, 2012

Small time craft brewers are always looking for ways to push the envelope of beer taste.  They are trying to find variations in beer’s fundamental ingredients — hops, barley, and yeast — that will make their beer distinctive.  Of these three, the most important is probably yeast (of course, we’re biased here at SGD!).

Think of the tasty beers that could come out of those beards.

Something like 40-70% of beer taste comes from the yeast used to make it alcoholic.  This is why brewers search high and low for new strains of yeast that will give their beer that special something which will make it stand out.  They have looked on Delaware peaches, ancient twigs trapped in amber, Egyptian date palms, and in lots and lots of other places. 

But brewers don’t always have to go far away because sometimes the best yeast is right under their noses.  Literally.

A brewery in Oregon found the yeast they were looking for in one of their master brewers’ beards.  They are now using this yeast to brew a new beer!  This seems uniquely revolting but the beer supposedly is quite tasty.  Perhaps if they don’t advertise the source of their yeast, this beer could become popular.

They aren’t sure where the yeast in his beard came from, but they think it may have come from some dessert he ate in the last 25 years or so (he hasn’t shaved his beard since 1978).  What would be fun is if his beard wasn’t just an incubator, but a breeding ground for new yeast.  Maybe yeast from a dessert from 1982 hooked up with a beer yeast blown into his beard while he was working at the brewery.  The end result is a new improved hybrid yeast! 

Of course we won’t have any real idea about this yeast until we get some sequence data from it.  And all kidding aside, the more yeast that are found that are good for making beer, the better the chances that scientists can home in on what attributes make them beer worthy.  So this beard borne yeast may help many beers in the years to come despite its troubling beginning.

Perhaps brewers also need to start searching through more beards to look for likely beer yeast candidates.  Beard microbiome project anyone?

More information

About beard yeast

What’s in a beer, anyway?

Original lager yeast found in Patagonia

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

Categories: Research Spotlight

Tags: beer , Saccharomyces cerevisiae , fermentation

SGD Hardware Update

October 09, 2012

We are in the process of migrating SGD servers to new faster hardware! You may have already noticed an increase in performance. There could be some teething issues in the next couple days – so please bear with us!

Categories: Website changes

How To Remember Without a Brain

October 02, 2012

Single celled beasts like the yeast S. cerevisiae can “remember” previous insults and so respond better to environmental changes in the future. For example, a yeast cell treated with 0.7M NaCl will respond better in the future to hydrogen peroxide. Not only that but so will its daughters, granddaughters and even its great, great granddaughters.

Who needs a brain to remember things?

In a new study out in GENETICS, Guan and coworkers show at least a couple of ways that this can happen. One is what anyone in biology might expect these days (although with an interesting twist). The NaCl treatment causes a rewiring of the regulatory network at an epigenetic level and this affects future responses to environmental insults.

But fancy epigenetic changes aren’t the only way that yeast remembers things.  No, it also uses a simple, elegant solution—protein stability.
 
Long Live The Protein!
 

The researchers did a set of experiments that showed that a yeast’s memory of a salt treatment did not rely on new protein synthesis and that it slowly faded with each generation.  One possible explanation was that the salt induced a stable factor that was divvied up and diluted with each passing generation. Guan and coworkers found that this was the case and that at least one of these factors was the cytosolic catalase 1 protein, Ctt1p.

The cytosolic catalase 1 or CTT1 gene is induced by salt but quickly returns to normal levels when the salt is removed.  However, Ctt1p is so long lived that it hangs around for at least six hours.  In that time the yeast has budded off multiple daughters, all of which are still better at dealing with hydrogen peroxide than their untreated sisters.

What a marvelously simple way to adapt!  Just make something that hangs around a long time and you and your kids will do better when the next insult comes.  The elegance of evolution.

This explains in part how yeast cells can remember the salt treatment of their ancestors, but a single long-lived protein isn’t the whole story.  No, there is something a bit more complicated going on at the nuclear pore too.

 
Attached for Quick Access
 

Guan and coworkers looked at the gene expression pattern of salt stressed and naïve yeast when exposed to hydrogen peroxide.  They found that 449 genes responded more quickly to hydrogen peroxide treatment if the cell had been pretreated with salt.  Importantly, 51 of these hadn’t reacted previously to the salt treatment, meaning that previous activation wasn’t required.

One idea is that these genes are more accessible to transcription because they are associated with the nuclear pore.  The idea is that faster response happens because the gene is closer to the nuclear envelope and/or because it has been looped near some sort of activator.

This is what has been proposed with inositol starvation and it looks like it may be true here too.  In both cases, eliminating Nup42p, a nuclear pore protein, eliminates the more rapid response to hydrogen peroxide.

So in this case it looks like cells can remember a previous insult with just a long-lived protein and a bit of genetic rewiring.  It will be interesting to see how universal these sorts of mechanisms are for cell memory.

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

Categories: Research Spotlight

Tags: cellular memory , Saccharomyces cerevisiae , catalase , stress response

Wintering in a Wasp’s Gut

September 24, 2012

Anyone reading this blog probably knows how important the yeast S. cerevisiae is.  It makes our bread better, our beer and wine more spirited, and our genetics more understandable. 

Social wasps are a natural reservoir for yeast

Because it is such an important beast, this yeast is also incredibly well characterized.  It was the first non-bacterial organism whose genome was sequenced and is a key model organism for teasing apart how eukaryotes like us work. We may know more about the molecular biology and genetics of S. cerevisiae than about any other organism on the planet.

And yet we know surprisingly little about S. cerevisiae in the wild.  We know that it isn’t on unripe fruits but suddenly appears once they ripen. We also know it doesn’t tolerate winter particularly well.  So where does yeast hang out when there isn’t ripe fruit around and/or it gets chilly?  A group of researchers in Italy thinks a key place is inside a hibernating wasp.

When Stefanini and coworkers looked, they found lots of yeast (including S. cerevisiae) in wasp intestines. They were also able to show that the S. cerevisiae remained viable in a hibernating queen over the winter and that that the queen transferred the yeast to new wasps in the spring by regurgitation.  With this one study, these scientists managed to find at least one way that yeast can survive the winter and get to ripe fruit.

To figure this out, Stefanini and coworkers did experiments both in the field and in the lab.  They first collected wasps and bees from around the Italian countryside and showed that wasps, but not bees, harbored yeast in their gut.  In all they found 393 yeast strains in the 61 wasps they dissected, 17 of which turned out to be S. cerevisiae.   By sequencing and comparing the genes URN1, EXO5, and IRC8, they were able to conclude that these yeast were related to wine, beer, bread, and laboratory strains of S. cerevisiae.

The researchers figured out that the yeast could survive for three months and be passed on to the next generation of wasps with a couple of controlled experiments they did in the lab.  They fed queens GFP labeled yeast and then let them hibernate.  After three months they dissected some of them and found lots of viable yeast in their intestines.

The rest of the queens were allowed to wake up and find new nests.  Larvae were removed from the nests and were found to contain GFP yeast as well.  The yeast not only lived through the winter but passed on to the next generations!

Of course this doesn’t mean that this is the only way that it can happen.  But it is the first time anyone has managed to get such a detailed look at feral yeast.  And this kind of work is important if we want to use S. cerevisiae as a way to study evolution. 

To understand its evolution, we have to understand the natural forces that shaped S. cerevisiae into the organism it now is.  Only then can we piece together why S. cerevisiae has evolved the way that it has and so learn fundamental lessons about the mechanisms of evolution. 

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

Categories: Research Spotlight

Tags: evolution , Saccharomyces cerevisiae , wild yeast

Few Genetic Paths From Here to There

September 12, 2012

Everyone knows that when the environment changes, those individuals with certain DNA differences useful in this new environment thrive while others wither.  But there hasn’t been a lot of work done to investigate how many DNA differences are available to a population for adapting to a particular environmental change.

How many paths lead to adaptation?

This may sound esoteric but the answer has real implications for speciation.  If there are few mutations possible and these mutations are very similar in terms of phenotype, then different populations will travel similar routes in their adaptations to the same environmental change.  This will definitely slow down speciation.  If on the other hand there are many genetic ways to adapt to the same change, then isolated populations will head down different paths leading to faster speciation.

In a new study out in GENETICS, Gerstein and coworkers found that at least for the environmental insult they used (low levels of the fungicide nystatin), there were very few paths to resistance. In fact, just four genes in the ergosterol biosynthesis pathway turned up in the 35 resistant lines they surveyed using whole genome sequencing.

Now that isn’t to say that there were just a few mutations.  There weren’t.  They found eleven unique mutations in the ERG3 gene, seven in ERG6, and one each in ERG5 and ERG7.  There were duplications, deletions, premature stop codons and missense mutations.  So there are lots of ways to mutate these few genes.

The small range of genes affected might suggest that adaptation favors populations evolving along similar paths since the same environmental effects result in the same adaptative mutations.  And yet, not all of these mutations in these few genes are created equally.  Different lines responded differently to other stressors.

For example, lines with mutations in the ERG3 gene responded poorly to ethanol while the other lines did very well.  And the lines with mutations in ERG5 and ERG7 responded less well to salt than the other lines.  So if one population was subjected to salt and nystatin and the other to ethanol and nystatin, the strains would almost certainly adapt with mutations in different genes.  Even within this narrow set of genes, there is room for adaptation by different routes.

While a useful first step, we don’t want to infer too much from this single study.  The researchers used a very specific environmental insult known to work through a specific pathway and found only mutations in that pathway.  The next study might want to focus on something like salt tolerance, a trait predicted to be achieved through multiple pathways.  Then we can get an even better feel for how many options a population has for adaptation.

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

Categories: Research Spotlight

Tags: nystatin , evolution , Saccharomyces cerevisiae , ergosterol biosynthesis

What Happens in Genes, Stays in Genes

September 06, 2012

Chromatin proteins, primarily histones, are a great way to control what parts of a cell’s DNA are accessible to its machinery.  These proteins coat the DNA and are marked up in certain ways to indicate how available a piece of DNA should be.  A methyl group here, an acetyl group there and a cell “knows” where the genes are that it is supposed to read!

Why doesn't RNA polymerase get a strike every time?

Of course this structure needs to be maintained or a cell might start to misread parts of its DNA as starting points of genes.  Then RNA polymerase II (RNAPII), the enzyme responsible for reading most protein-coding genes, would start making RNA from the wrong parts of the DNA, wreaking havoc in a cell.

One place where maintaining chromatin structure might be especially tricky is within the coding parts of genes.  It is easy to imagine RNAPII barreling down the DNA, knocking the proteins aside like pins in a bowling alley.  But it doesn’t.  For the most part the chromatin structure stays the same and survives the onslaught of an elongating RNAPII.

Two key marks for keeping histones in place are the trimethylation of lysine 36 of histone H3 (H3K36me3) that is mediated by Set2p, and a general deacetylation of histone H4 that is mediated by the Rpd3S histone deacetylase complex.  We know this because loss of either complex causes an increase in H4 acetylation and transcription starts from within genes.

In a recent study in Nature Structural & Molecular Biology, Smolle and coworkers identified two key components that help chromatin resist an elongating RNAPII in the yeast S. cerevisiae.  The first, called the Isw1b complex, binds H3K36me3 and the second, the Chd1 protein, binds RNAPII itself.  That these two were involved wasn’t surprising since previous work had suggested they helped prevent histone exchange at certain genes.

What makes this work unique is that the researchers showed the global importance of these proteins in the process and were able to tease out some of the fine details of what is going on at the molecular level. They used electrophoretic mobility shift assays to show that Isw1b bound the trimethylated form of H3 via its Ioc4p subunit and used chromosome immunoprecipitation coupled to microarrays (ChIP-chip) to show that Isw1b localized to the middle of genes in vivo. They also showed that when Set2p was removed, the localization disappeared (presumably because of the loss of the trimethylation of lysine 36).  They clearly demonstrated that Isw1b is found primarily in the middle of genes.

While these results indicate that the Ioc4p-containing Isw1b complex is moored to the middle of genes via its interaction with H3K36me3, it does not establish what it is doing there.  For this the researchers knocked out Isw1b and Chd1 and showed via genome tiling arrays a global increase in cryptic transcription starts.  The DNA in the middle of genes was now being used inappropriately by RNAPII as starting points for transcription.  Further investigation with Isw1b and Chd1 knockouts showed an increase in chromosome exchange and an increase in acetylated H4 in the middle of genes.

Whew.  So it appears that Isw1b and Chd1 inhibit inappropriate starts of transcription by keeping hypoacetylated histones in place over the parts of a gene that are read.   They are two of the key players in maintaining the right chromatin structure over genes.  They help keep RNAPII from railroading histones aside as it elongates, thus protecting the cell from inappropriate transcription starts.

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

Categories: Research Spotlight

Tags: Chd1 , chromatin , Isw1b , RNA polymerase II , Saccharomyces cerevisiae

Prions Let Yeast Take Traits for a Test Drive

August 27, 2012

For the most part, prions have a bad rep. They are the proverbial bad apple that spoils the whole bunch.

One bad apple can spoil the whole bunch...

A prion is a protein that misfolds in a certain way that creates a chain reaction to misfold many additional copies of that particular protein in a cell. This misfolding en masse can cause severe problems like mad cow disease or Alzheimer’s.

As if that weren’t bad enough, this misfoldedness can spread from one organism to another. Once a prion gets into a cell and/or a part of the body, it will cause many of its properly folded brethren to misfold too. This is true even though the prion gene in the new host is happily churning out properly folded protein.

These things look like a nightmare. Why on Earth are prions still around? Because in addition to their bad side, they can sometimes be an advantage too (at least in yeast).

In a study published in Nature in February 2012, Halfmann and coworkers provide compelling evidence that prions can help both laboratory and wild yeast strains to adapt rapidly to a changing environment, by unlocking survival traits hidden in yeast DNA. In other words, prions are a way for a yeast population to hedge its bets against a world of changing environments.

The authors focused on the most famous prion in yeast, the translation termination protein Sup35p. When Sup35p switches to prion mode ([PSI+]), it becomes bound up in insoluble fibers, causing translation termination to become leaky. Now normally untranslated parts of mRNAs become part of their respective proteins. And this can change these proteins’ functions.

Sure, most of this newfound variation will have no effect or maybe even be harmful, but occasionally the prion will reveal a beneficial trait. This yeast can then go on to survive and even thrive in this new environment.

This mechanism may apply to other prions in addition to Sup35p. Prions tend to come from proteins that are global regulators of transcription or translation. In the non-prion form, these proteins do their usual job making sure transcription and translation are following the rules. But when these proteins become misfolded into a prion, they can no longer perform their usual function. This uncovers previously silent bits of DNA or RNA for transcription or translation.

These authors also convincingly showed that prions are not some weird phenomenon found only in laboratory strains of yeast. They found evidence for prions in 255 out of the 690 wild strains they surveyed (although only ten had Sup35p based prions). Not only that, but many of these prions also conferred new traits on the yeast that could be beneficial in certain circumstances. It looks like prions may serve an important function in yeast.

A more surprising result from the study is that these prion-derived traits carry on in later generations even after the prion has been removed. For example, the authors looked at the wine yeast UCD978. They found that when Sup35p was in its prion form in this strain, UCD978 could effectively penetrate agar surfaces and that this trait was lost when the prion was cured, reverting Sup35p to its functional form.

They then took the study further and showed that after meiosis and sporulation, 5/30 haploid progeny of UCD978 retained the trait even after the prion was removed. These five had fixed the new trait and no longer required the prion to maintain it. They got all the benefits with none of the costs.

It isn’t obvious how this trait became independent of the original inducing prion. But that is for another study (or two or ten).

If the results of this study pan out, they show that prions are not just part of a disease but are really just another way to adapt to environmental changes and to pass them down to future generations. Maybe these apples aren’t so bad after all!

Prions allow yeast cells to take various traits out for a test drive.

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

Categories: Research Spotlight

Tags: evolution , S. cerevisiae , prions , variation , SUP35

SGD Summer 2012 Newsletter

August 21, 2012

SGD sends out its quarterly newsletter to colleagues designated as contacts in SGD. This Summer 2012 newsletter is also available online. If you would like to receive this letter in the future please use the Colleague Submission/Update form to let us know.

Categories: Newsletter

GWAS Shows Potential in Yeast

August 09, 2012

Maybe GWAS will prove to be more useful in model organisms like yeast.

The idea behind a genome wide association study (GWAS) makes perfect sense.  Compare the DNA of one group of people with a disease to another group that doesn’t have the disease, identify the DNA region specific to the disease group, and then find the specific gene and mutations that lead to the disease.

In theory, this sort of study should have become routine once we had the human genome sequenced.  In practice, it has turned out to be less useful than everyone hoped.

Now, this doesn’t appear to be any fault with the technique itself.  Instead, it has more to do with the fact that many human diseases are simply too complex for GWAS to handle.

Most common human diseases appear to result from multiple genetic pathways and/or multiple genes.  Throw in environmental effects and GWAS quickly becomes overwhelmed.  At least for now, too many patients and controls would be needed for this powerful technique to have a real chance at deciphering most common human diseases.

But that doesn’t mean the technique isn’t useful.  It is very good at finding single genes involved in strongly expressed traits.  And this might be ideal for certain model organisms.

In a study just out in the latest issue of GENETICS, Connelly and Akey set out to investigate how well GWAS would work in the yeast, Saccharomyces cerevisiae.  In many respects, this yeast appears to be made for GWAS.

It has a small, easily sequenced genome, there is on average a polymorphism every 168 base pairs or so, and its linkage disequilibrium is low.  There are genome sequences from 36 wild and laboratory strains publicly available, all as diverse as can be. 

But this yeast isn’t perfect.  The chromosomal structure between strains tends to be much more varied than between two humans.  This is predicted to introduce a high error rate.  And this is just what Connelly and Akey saw when they ran some simulations.   

They found that the error rate was too high in the simulations to draw any meaningful conclusions.  But they also found that by using a more sophisticated analytical technique called EMMA, they were able to partly correct for some of these errors. 

Simulations are one thing, but how about real life?  Connelly and Akey next tested the method by applying it to a practical problem: identifying the genetic reasons for differences in mitochondrial DNA (mtDNA) copy number in yeast.  What they found mimicked the simulation data. 

Using more traditional analytical approaches on the data obtained from GWAS, they found 73 potential causative SNPs.  But when they switched to analyzing the data with EMMA, they found a single SNP that was significant.  It took a bit of hand waving, but the gene associated with this SNP could possibly be implicated in mtDNA copy number.  And then again, it might not.

This “significant” SNP was found amidst lots of errors and in a background of high p values.  In other words, this finding may not be a real one after all.  This experiment does not give confidence that GWAS can be used when all known strains of yeast are compared.

But if the strains to be included are selected more carefully, it may still prove to be a useful tool.  When Connelly and Akey focused on strains that were structurally similar, they found that the error rate was much lower.  Low enough that in the near term, scientists may be using GWAS to figure out how things work in model organisms.  

Hopefully the findings from GWAS applied to model organisms will illuminate disease mechanisms in humans. Then maybe GWAS can realize its full potential, although not in the way it was originally envisioned.

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

Categories: Research Spotlight

Tags: GWAS , genome wide association study , yeast

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