New & Noteworthy
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.
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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 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
November 8, 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.
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.
November 1, 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.
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.