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

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