New & Noteworthy
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.
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
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight
Tags: beer, brewing, Saccharomyces cerevisiae, Saccharomyces pastorianus
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: protein stability, Saccharomyces cerevisiae, yeast model for human disease
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.
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: ALS, DBR1, Lou Gehrig's Disease, RNA binding, Saccharomyces cerevisiae, yeast model for human disease
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.
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?
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: RNA polymerase II, Saccharomyces cerevisiae, transcription, ubiquitin-mediated degradation
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
Original lager yeast found in Patagonia
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight
Tags: beer, fermentation, Saccharomyces cerevisiae
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: catalase, cellular memory, Saccharomyces cerevisiae, 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