New & Noteworthy

Yeast on Their Best Behavior

December 10, 2015

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As the holidays approach, many of us are getting ready to crowd around the table for a big family dinner. Some of us may behave differently around family than we might with friends or coworkers.

For example, with your relatives, you might bite your tongue if your political views vary greatly from theirs. Where we are and with whom we interact can sometimes affect what we do.

Turns out that yeast growing in a colony can be the same way. Though of course they aren’t keeping their opinions to themselves. (Well, we don’t think they are…)

A yeast cell can end up acting differently depending on where it is in a colony. For example, only a narrow band of cells gets to sporulate while all the others are left to plod through mitosis.

A new study out in GENETICS by Piccirillo and coworkers shows that these cells sporulate because nearby cells “encourage” them to. They are being influenced to sporulate because of the cells around them. Just like your relatives might influence you to change your behavior at the dinner table.

The first step in showing that one set of cells signals a second set to sporulate was to find the genes involved in setting up this pattern. Since the authors were looking at Saccharomyces cerevisiae, it was pretty easy to get mutants to study. They just had to open their freezer and pull out their yeast homozygous diploid deletion library.

Initially, they looked for strains where the usual pattern of sporulating cells was disrupted. They then took these candidates and looked for those that could still sporulate normally in suspension. They wanted mutants that could sporulate but couldn’t do it in the right place.

They found seven strains that fit the bill. Three of the deleted genes, MPK1/SLT2, BCK1, and SMI1, were in the cell-wall integrity pathway (CWI). They also showed that mutation of three other genes in the pathway, SLG1/WSC1,TUS1 and RLM1, all impacted colony sporulation as well.

Further work showed that the transcription factor RLM1 was induced 1-2 days before the master regulator IME1 was turned on. IME1 is a key player in getting meiosis started so that yeast cells can sporulate.

So the story seemed to be that RLM1 is turned up which then turns on IME1, which kick starts meiosis. Makes sense except it is unlikely that Rlm1p is directly activating IME1. There is no obvious Rlm1p site in the IME1 promoter.

A close look at the colonies showed that RLM1 is upregulated in a layer of cells just under the ones where IME1 is upregulated. Deletions in the CWI pathway seemed to have disrupted a group of “feeder” cells whose job it is to get nearby cells to sporulate.

To show this, the authors used a chimeric colony assay that consisted of two strains. The first strain, which had functional Rlm1p, had a reporter, either RFP or lacZ, under the control of the IME1 promoter. The second strain was either wild type or deleted for the transcription factor RLM1.

They created colonies with equal amounts of each strain and looked at IME activation. The idea is that if RLM1 is important in the cells that sporulate, then the second strain shouldn’t matter. You should get the same number of cells in which the IME1 promoter is activated whether or not adjacent cells express RLM1.

But if it is important for RLM1 to be expressed in nearby cells, then there should be a falloff in activation if adjacent cells are deleted for RLM1. This is just what the authors found.

And it wasn’t just the artificial reporter system that was affected either. There was also a drop off in the number of cells that sporulated in the case where some of the cells lacked RLM1.

In a further set of experiments, Piccirillo and coworkers showed that these feeder cells became more osmosensitive compared to the ones that go on to sporulate. While they did not find the signal that prompted the meiosis of nearby cells, this change in osmosensitivity is consistent with the cells preparing to release something into the environment.

So it looks like activating the CWI pathway in one set of cells causes a second set to start down the road of sporulation. And if the CWI pathway is disabled in these cells, then the second set of cells no longer changes their behavior and begin to go through meiosis.

This all seems weird at first until you realize that the cells in a colony usually all share the same DNA. What is good for one set of cells is good for the survival of the DNA even if it is at the expense of other cells in the colony.

Yeast cells tend to sporulate when food grows scarce. But sporulating takes a lot of energy. Colonies may get around this paradox by having some of the cells in the colony give up nutrients or energy to a few cells that go on to sporulate. The feeder cells deprive themselves so that other cells have a better shot at survival.

Now the DNA, shared by all the cells, can live on for the next round of holiday dinners….

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

Categories: Research Spotlight

Tags: cell wall integrity pathway, IME1, RLM1, sporulation

Keeping Gen(i)e Drives in Their Lamps

December 02, 2015

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Everyone knows about genies. They have almost infinite power, can grant you three wishes, and are kept under control by the owner of their lamp.

And as we saw in Disney’s Aladdin, it is a good thing that the lamp is around! When the evil sorcerer Jafar was given the powers of a genie, he began to take over the world. Until, that is, Aladdin forced him back into his lamp where he could be kept under control.

In the last few years, scientists have come up with their own genies. While not as powerful as the “real” ones, these gene drives can still pack quite a punch. And maybe even grant us a few wishes.

Gene drives can force genes to spread quickly through a population whether those genes are good for a species or not. This means we might be able, for example, to force a “bad” gene to spread through the mosquitoes that transmit malaria. By causing the mosquito population to crash, our wish to save hundreds of thousands of lives each year would be granted!

But just like a genie, we need to keep gene drives under control. We do not want something that overrides natural selection to escape and wreak havoc with ecosystems.

Which is where, as usual, our friend yeast can help! In a new study out in Nature Biotechnology, DiCarlo and colleagues use yeast to test two different strategies to make gene drives safe enough to use. And, they argue, safe enough to research.

Gene drives are based on the idea of homing endonucleases. Basically, if a gene associated with a gene drive is on just one of the two chromosomes in a pair, the gene drive will copy and insert the gene into the other chromosome through a precise DNA cut.

Now both chromosomes end up with a copy of the gene. Which of course means all of the offspring will get the altered gene too. This copying will happen generation after generation until the new gene has swept through the population.

The idea for gene drives has been around since 2003 but really only became practical with the discovery of the CRISPR/Cas9 system. This genome editing tool, which is ludicrously simple to program to target most any DNA sequence, allows scientists to create most any gene drive they want.

The CRISPR/Cas9 system has two parts. One part is the guide RNA which leads the second part, the endonuclease Cas9, to the right spot in the genome to cut. What makes the system so powerful is that you just need to make a different guide RNA to target different sequences in the genome.

One easy way to help control a gene drive is to keep these two parts separate. Do not have the guide RNA and the Cas9 on the same piece of DNA. Then, if one part were to escape, it couldn’t do anything on its own.

This is of course easy to do in yeast. Just integrate one part into a chromosome and keep the second part on a plasmid.

This is just what DiCarlo and coworkers did. And they showed that this separation can be very effective.

They integrated a guide RNA into the ADE2 gene of a haploid yeast to create a gene drive designed to disrupt ADE2. As expected, this strain produced red colonies on adenine limiting media.

They next mated this strain to a wild type haploid. All of the resulting diploids were cream colored. This is what would be expected as both copies of ADE2 need to be disrupted to see red colonies in a diploid.

When these diploids were sporulated, the researchers got the expected 2:2 ratio of red to cream colored haploids. This all changed when they introduced a Cas9 containing plasmid into the experiment.

In the presence of Cas9, more than 99% of the resulting diploids were red. And when sporulated, these diploids produced all red haploid colonies.

The two parts of CRISPR/Cas9 together drove the disrupted ADE2 through the population. But importantly, just having the guide RNA integrated into ADE2 had no effect on how the two alleles were passed down. Once one part is removed, the gene drive stalls out.

The same system also worked when the ADE2 gene drive included the URA3 gene so that URA3 spread through the population as well. It also worked when the essential gene ABD1 was targeted.

And genetic background did not significantly affect how well this ADE2 gene drive worked. When they mated their haploid to six different strains of yeast they saw no loss in efficiency.

So separating the two parts of the gene drive is a pretty good failsafe. But of course nothing is perfect.

Ideally we need some way to shut the system down if all of our safety features fail. We want to be able to get rid of Jafar and the lamp entirely if possible.

DiCarlo and coworkers showed that they could create a gene drive that could overwrite and correct the ADE2 they had disrupted with the guide RNA. This new gene drive targeted a synthetic sequence in the original gene which means that it would only affect altered yeast. So even if things go awry, we may be able to erase the changes we made.

These two strategies should help keep gene drives in check both in the wild and the lab. But of course, again, it is important to keep in mind that nothing is foolproof.

At the end of Aladdin, they buried Jafar and his lamp deep in the desert to keep him from causing any more trouble. But his lamp was found and Jafar reemerged to wreak havoc in the second Aladdin movie, reminding us that we must be very careful when unleashing powerful forces.

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

Categories: Research Spotlight

Tags: CRISPR/Cas9, gene drives

Pulsating Yeast

November 19, 2015

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Sometimes when you get a minor injury, doctors will recommend alternating heat and cold as a therapy. The heat opens things up and the cold shuts them back down again.

Now obviously it would be pretty useless to apply both at the same time. Adding a bit of lukewarm water to an injury is not going to be very helpful at all.

The same thing holds true for many genes. If activators and repressors all turned on at the same time, there wouldn’t be much of an effect on the expression of a gene regulated by both. It is no way to respond to something in the environment!

Instead, if you want a gene to go up and then go back down again, you’d have the activator turn on first, followed by the repressor. Another way to put this is you’d have a pulse where all of the activators activate their genes at once and then stop working followed by a pulse where all of the repressors work at once.

This is exactly what Lin and colleagues found in their recent study in Nature. There they looked at the effect of certain external stimuli on the timing of when the activator Msn2p activated genes and when the repressor Mig1p repressed genes in our favorite yeast S. cerevisiae. These transcription factors coregulate many of the same genes.

The authors found that in the presence of either lowered glucose concentrations or 100 mM NaCl, most of the Msn2p in the cell turned on first followed closely by the Mig1p repressors. In the absence of either stimulus, there was no coordination.

So there does seem to be a carefully choreographed dance between these two transcriptional regulators with these signals. But of course gene regulation is a bit more complex than a sprained ankle.

There may be situations where a cell wants both regulators to do their jobs at the same time. Sometimes lukewarm water may be just what the doctor ordered.

And this is what Lin and colleagues found with 2.5% ethanol. Under this condition, the pulses of the two regulators overlapped—both were on at the same time. Apparently different stimuli call for different responses which means different timing of transcription factor pulses.

The authors next wanted to get at why Mig1p repression lagged behind Msn2p activation. Since both transcription factors can only enter the nucleus and do their job after they lose a few key phosphate groups, the authors reasoned that perhaps Mig1p dephosphorylation lagged behind that of Msn2p.

They decided to look at the PP1 phosphatase, Glc7p, as previous work had shown that it can indirectly regulate both Msn2p and Mig1p. And indeed, when the authors lowered the expression of GLC7, Msn2p and Mig1p no longer pulsed one after the other at lower glucose concentrations. It looks like Glc7p is a key player in controlling the pulsing of these two regulators.

Even though much of this work was done with synthetic promoters with Mig1p and Msn2p binding sites, the results were not restricted to these artificial constructs. Lin and colleagues found that around 30 endogenous targets also responded to lowered glucose concentrations in a coordinated way just like their synthetic construct. Yeast regulates genes by controlling when activators and repressors pulse.

Finally, all of these studies were done using fluorescent proteins and filming single cells in real time. (Is biology cool or what?) This makes sense because subtle signs of synchronization can be lost when averaged over a large population.

This also allowed the authors to investigate what happens in unstimulated cells. In other words, what happens when both regulators enter the nucleus at the same time? Or if a repressor gets in first?

The first thing they found was that even in the absence of stimulation, there were still pulses. So at seemingly random times, suddenly all of the Msn2p would swoop into the nucleus at the same time and then all leave a short time later. Or the same thing would happen with Mig1p.

If by chance the two entered the nucleus at the same time, both the synthetic reporter and an endogenous gene, GSY1, were not activated. But if Msn2p happens to get in there first, both were activated.

And if the repressor Mig1p managed to get into the nucleus at least 4-5 minutes before Msn2p, activation by Msn2p was muted. The presence of Mig1p beforehand seemed to keep Msn2p from activating coregulated genes to as high a level.

Taken together these results confirm that just like a synchronized swim team, yeast regulates genes by controlling when activators and repressors can work. First there is a pulse where the all of the molecules of a certain activator are primed to do their job and then, after a short time, they all stop doing their job. This can then be followed later by a pulse of repressors shutting it all down.

And this isn’t just in yeast either. For example, these kinds of pulses are important in neuroscience as well.

This work suggests that in dissecting regulatory pathways, researchers may need to pay more attention to the timing of pulses. Then they can see that hot followed by cold makes much more sense than both together.

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

Categories: Research Spotlight

Tags: Saccharomyces cerevisiae, transcription regulation

Yeast, the Spam Filter

November 11, 2015

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Imagine what our email inboxes would look like if we didn’t have spam filters! To find the meaningful emails, we’d have to wade through hundreds of messages about winning lottery tickets, discount medications, and other things that don’t interest us.

When it comes to sorting out meaningful mutations from meaningless variation in human genes, it turns out that our friend S. cerevisiae makes a pretty good spam filter. And as more and more human genomic sequence data are becoming available every day, this is becoming more and more important.

For example, when you look at the sequence of a gene from, say, a cancer cell, you may see many differences from the wild-type gene. How can you tell which changes are significant and which are not?

SuperBud to the rescue! Because many human proteins can work in yeast, simple phenotypes like viability or growth rate can be assayed to test whether variations in human genes affect the function of their gene products. This may be one answer to the increasingly thorny problem of variants of uncertain significance—those dreaded VUS’s.

In a new paper in GENETICS, Hamza and colleagues systematically screened for human genes that can replace their yeast equivalents, and went on to test the function of tumor-specific variants in several selected genes that maintain chromosome stability in S. cerevisiae. This work extends the growing catalog of human genes that can replace yeast genes.

More importantly, it also provides compelling evidence that yeast can help us tell which mutations in a cancer cell are driver mutations, the ones that are involved in tumorigenesis, and which are the passenger mutations, those that are just the consequence of a seriously messed up cell. Talk about a useful filter!

The researchers started by testing systematically for human genes that could complement yeast mutations. Other groups have done similar large-scale screens, but this study had a couple of different twists.

Previous work from the Hieter lab had identified genes in yeast that, when mutated, made chromosomes unstable: the CIN (Chromosome INstability) phenotype. Reduction-of-function alleles of a significant fraction (29%) of essential genes confer a CIN phenotype. The human orthologs of these genes could be important in cancer, since tumor cells often show chromosome rearrangements or loss. 

So in one experiment, Hamza and colleagues focused specifically on the set of CIN genes, starting with a set of 322 pairs of yeast CIN genes and their human homologs. They tested functional complementation by transforming plasmids expressing the human cDNAs into diploid yeast strains that were heterozygous null mutant for the corresponding CIN genes. Since all of the CIN genes were essential, sporulating those diploids would generate inviable spores—unless the human gene could step in and provide the missing function.

In addition to this one-to-one test, the researchers cast a wider net by doing a pool-to-pool transformation. They mixed cultures of diploid heterozygous null mutants in 621 essential yeast genes, and transformed the pooled strains with a mixture of 1010 human cDNAs. This unbiased strategy could identify unrecognized orthologs, or demonstrate complementation between non-orthologous genes.

In combination, these two screens found 65 human cDNAs that complemented null mutations in 58 essential yeast genes. Twenty of these yeast-human gene pairs were previously undiscovered.

The investigators looked at this group of “replaceable” yeast genes as a whole to see whether they shared any characteristics. Most of their gene products localized to the cytoplasm or cytoplasmic organelles rather than to the nucleus. They also tended to have enzymatic activity rather than, for example, regulatory roles. And they had relatively few physical interactions.

So yeast could “receive messages” from human genes, allowing us to see their function in yeast. But could it filter out the meaningful messages—variations that actually affect function—from the spam? 

The authors chose three CIN genes that were functionally complemented by their human orthologs and screened 35 missense mutations that are found in those orthologs in colorectal cancer cells. Four of the human missense variants failed to support the life of the corresponding yeast null mutant, pointing to these mutations as potentially the most significant of the set.

Despite the fact that these mutations block the function of the human proteins, a mutation in one of the yeast orthologs that is analogous to one of these mutations, changing the same conserved residue, doesn’t destroy the yeast protein’s function. This underscores that whenever possible, testing mutations in the context of the entire human protein is preferable to creating disease-analogous mutations in the yeast ortholog.

Another 19 of the missense mutations allowed the yeast mutants to grow, but at a different rate from the wild-type human gene. (Eighteen conferred slower growth, but one actually made the yeast grow faster!)

For those 19 human variants that did support life for the yeast mutants, Hamza and colleagues tested the sensitivity of the complemented strains to MMS and HU, two agents that cause DNA damage. Most of the alleles altered resistance to these chemicals, making the yeast either more or less resistant than did the wild-type human gene. This is consistent with the idea that the cancer-associated mutations in these human CIN gene orthologs affect chromosome dynamics.

As researchers are inundated by a tsunami of genomic data, they may be able to turn to yeast to help discover the mutations that matter for human disease. They can help us separate those emails touting the virtues of Viagra from those not-to-be-missed kitten videos. And when we know which mutations are likely to be important for disease, we’re one step closer to finding ways to alleviate their effects. 

by Maria Costanzo, Ph.D., Senior Biocuration Scientist, SGD

Categories: Research Spotlight, Yeast and Human Disease

Tags: chromosome instability, functional complementation, Saccharomyces cerevisiae, yeast model for human disease

New SGD Help Video: Variant Viewer

November 05, 2015

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Using SGD’s Variant Viewer, you can compare the nucleotide and protein sequences of your favorite genes in twelve widely-used S. cerevisiae genomes. This tool shows alignments, similarity scores, and sequence variants for open reading frames (ORFs) from the different strains relative to the S288C reference genome. Sequence data are derived from Song et al., 2015.

Take a look at our new video tutorial to get started with the Variant Viewer, and let us know if you have questions or suggestions.

Categories: Sequence, Tutorial

SGD Help Video: Mutant Phenotypes

November 04, 2015

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SGD’s Phenotype pages present detailed information about single mutant phenotypes for a particular gene, along with references for each observation. Phenotype pages are accessible from the ‘Phenotype’ tab of the Locus Summary and is also linked from the Mutant Phenotypes section of the Locus Summary, where the phenotype data are presented in summary form. Data are presented in tabular form on the Phenotype page.

This brief video will give you an overview of the contents and organization of SGD’s Phenotype pages.

Categories: Tutorial

SGD Help Video: Literature Page

October 29, 2015

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If you’re interested in finding all the published literature about a gene or protein, there’s no need to wade through long lists of PubMed results. SGD curators have already done that for you! We review PubMed weekly for new papers about S. cerevisiae. You can find papers about a specific gene or protein on its Literature tab page (see an example).

Articles on the Literature page are categorized by several topics. The Primary Literature section lists papers in which the gene of interest is a primary focus of the study, while the Additional Literature section lists papers in which the gene is mentioned but is more peripheral to the research. There are other categories of references, and also a cool interactive graphic that shows the relationships between papers that are about the same set, or overlapping sets, of genes. You can get to the Literature page for a gene or protein via the Literature tab, located at the top of its Locus Summary page and all of its other tab pages.

Categories: Tutorial

Tags: video

New SGD Help Video: GO Term Finder

October 26, 2015

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Our GO Term Finder tool lets you start with a list of genes—perhaps a set of genes that are co-regulated, or a group of genes that can all mutate to the same phenotype—and analyze their Gene Ontology (GO) annotations to find out what else they might have in common.  GO Term Finder searches for significantly shared terms within the GO annotations associated with the genes in your list. It takes advantage of the tree structure of GO to find terms that are related to each other within the ontology.

Finding shared terms within a gene set can bring meaning to experimental results and suggest new avenues to explore. For example, if the GO Term Finder results show that most of the genes in your co-regulated set mediate steps in a pathway, this might be a hint that the uncharacterized genes in the set also participate in that pathway. Or perhaps GO Term Finder will show that a group of genes that can mutate to confer resistance to a certain drug are all annotated to a certain cellular location, suggesting a mechanism for the effects of that drug. Give it a try and see what interesting results your gene list has in store!

Our new SGD Help video gives you a quick overview of how to use the GO Term Finder. You can find all the details on our GO Term Finder help page.

Categories: Tutorial

Tags: Gene Ontology, GO Term Finder, video

Life Needs to be More Like a 1950’s Chevy

October 21, 2015

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In the old days, a car came with the bare minimum of features to get from point A to point B. The windows rolled down with a crank and it usually had a radio. That was about it.

As the car has evolved, it has gained a huge number of bells and whistles. There are power windows and power brakes, a baffling number of computer-based bonus features, personal wifi hotspots, and so on. All of these have undoubtedly made cars more fun and comfortable to drive. But they have come at a cost. Many cars simply do not last as long as their predecessors because these extras break easily.

Turns out life may be like a modern car. It has lots of nice features that help it to do better in the world. But a lot of these features may shorten its life span.

This point was reinforced in a recent study by McCormick and coworkers. They painstakingly searched through a library of 4,698 single gene deletion strains in S. cerevisiae and found that 238 of these strains were able to produce significantly more buds over their lifetime. Many nonessential genes seem to shorten a yeast’s life.

And boy was it painstaking! Believe it or not, they manually dissected over 2.2 million individual yeast daughter cells to generate these results. Luckily it was worth it, as they found so many interesting things.

First off, many of the genes they found fall into a set of five pathways that includes cytosolic and mitochondrial translation, the SAGA complex, protein mannosylation, the TCA cycle, and proteasomal activity. So there are certain pathways we can target to extend the lifespan of our friend yeast. And even better, yeast may not be the only beneficiary of these studies.

Two of the pathways, cytosolic and mitochondrial translation and the TCA cycle, have also been found to be significant in extending the life of the roundworm C. elegans. These pathways are also shared with humans.

And just because the authors found no overlap with the other three pathways in other beasts doesn’t mean they may not be targets for life extension in them too. It could be that previous screens in C. elegans simply missed genes from these pathways.

It could also be that what is found in yeast may turn out to be important in people but not in C. elegans. For example, the authors failed to find any equivalent to the SAGA complex in C. elegans. Either the roundworm lost this complex during evolution, or the homologs between yeast and C. elegans are so different that they’re unrecognizable. In any event, humans at least do have an equivalent to SAGA, called STAGA.

All of this suggests that there may be common ways to make organisms, including people, live longer, healthier lives. Here’s hoping!

And these five pathways are certainly not the whole story. The majority of the genes McCormick and coworkers identified were not in these five, which means there are probably lots of other ways to get at living longer.

One fascinating example that the authors decided to look at in depth was LOS1. Deleting it had one of the biggest effects on a yeast’s reproductive life span.

At first this seems a little weird, as Los1p exports tRNAs out of the nucleus. As expected, deleting LOS1 led to a buildup in tRNAs in the nucleus. The authors confirmed that this buildup is important by showing that overexpressing MTR10, a gene involved in transporting tRNAs from the cytoplasm to the nucleus, led to a longer lived yeast with a buildup of tRNAs in its nucleus.

The next step was to figure out why having a lot of tRNA in the nucleus makes yeast live longer. It was known previously that Los1p is kept out of the nucleus under glucose starvation conditions. The authors confirmed this result.

Most everyone knows that restricting calorie intake (also called dietary restriction or DR) can extend the lives of most every beast tested so far, including yeast. The authors found that growing a los1 deletion strain at low glucose did not increase the lifespan of this strain any further. It thus appears that an important consequence of DR is keeping Los1p out of the nucleus and thereby increasing the amount of tRNA in the nucleus.

While we don’t know yet exactly why keeping tRNAs in the nucleus helps yeast live longer, it is interesting that the increased lifespan associated with the loss of LOS1 is linked to caloric restriction. Finding a way to inhibit Los1p has to be better than starving yourself!

This study has identified 238 genes to follow up on for future studies. And of course there is a whole class of genes that haven’t yet been investigated—the essential genes! Many of these may be important for extending life too. 

Stripping life down to its bare essentials may help individuals live longer at the expense of being the most fit in terms of survival in the hurly burly world of nature. After all, those “nonessential” genes undoubtedly have a function in helping yeast outcompete their less well-endowed yeast neighbors. Just like those power sliding doors are way better than the manual ones on a minivan.

But if you want a long-lived minivan, get the one with the manual doors. And if you want a long-lived yeast (or person), get rid of some of those nonessential genes that cause you to break down.

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

Categories: Research Spotlight

Tags: aging, lifespan, Saccharomyces cerevisiae

You Can Take Yeast Off of the Grapevine, But…

October 14, 2015

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All over the world and through the ages, people have moved from the country into the big city to look for a better life. These folks often find that even though they can adapt to city life and city ways, they still hang on to their core country values. As the old saying goes, “You can take the boy out of the country, but you can’t take the country out of the boy.”

Our friend Saccharomyces cerevisiae didn’t migrate voluntarily into the lab. But it ended up there, and has been as lonely as a new migrant in a big city. 

Which is of course how we need it to be. One of the basic tenets of classical microbiology is that you can’t begin to study an organism until you’ve isolated it in a pure culture.

And studying pure S. cerevisiae has yielded a huge body of knowledge about molecular biology, cell biology, and genetics. But by not studying yeast in the context of its old country home, we may have missed a few things.

In a new article in PLOS ONE, Rossouw and colleagues uncover one of them. S. cerevisiae has a family of FLO genes that promote flocculation, the adherence of yeast cells to each other. It has always been a bit puzzling why a whole family of genes that are pretty much redundant with each other would be maintained through evolution.

When the researchers took S. cerevisiae out of its lab isolation by mixing it with other yeast species, they found that the different flocculation genes actually determine which species it can co-flocculate with. Different Flo proteins prefer different partners. 

This discovery helps us understand the evolution of this gene family and also opens the door to further study of inter-species interactions in the vineyard. And since flocculation is an important property in winemaking and brewing, there could even be tasty practical applications of this knowledge.

The researchers started by surveying 18 non-Saccharomyces yeast strains that are found in vineyards. They looked at the ability of the yeasts to flocculate both as pure strains and when mixed with either of two S. cerevisiae wine strains.

Intriguingly, certain species showed a synergistic effect when mixed with S. cerevisiae, flocculating more than either species on its own. Rossouw and colleagues used microscopy to confirm that the “flocs” did indeed contain both yeast species—a simple observation, since the cells of different species have slightly different shapes.

To test the effects of different FLO genes on co-flocculation, the authors assayed the co-flocculation ability of flo1, flo10, and flo11 deletion mutants as well as Flo1, Flo5, and Flo11 overproducers in individual combinations with six of the non-Saccharomyces yeasts. 

The results showed that Flo1 has general effects on flocculation. Overproduction increased co-flocculation across the board with all the species tested, while deletion of FLO1 consistently decreased it. In contrast, deletion of FLO10 didn’t have much effect on co-flocculation.

It was a different story for Flo5 and Flo11, though. Overproduction of each of these not only affected co-flocculation, but had species-specific or even strain-specific effects. Flo5 overproduction caused a relative increase in co-flocculation with Metchnikowia fructicola and a substantial decrease in co-flocculation with two different strains of Hanseniaspora opuntiae. Flo11 overproduction reduced co-flocculation with one of the Hanseniaspora opuntiae strains but not with the other. 

All of these experiments were done on mixtures of two species at a time. To get S. cerevisiae even further out of the lab, Rossouw and colleagues created a “consortium” of wine yeasts, a mixture of six species that are found in wine must (freshly pressed grapes) at the start of fermentation. They then added the FLO overproducer strains individually to the consortium, to see their effects in a more natural situation.

They let the yeast consortium flocculate, extracted total DNA from the flocculated or supernatant parts of the culture, and then used automated ribosomal intergenic spacer analysis (ARISA) to see which strains had co-flocculated. This technique can determine the relative abundance of different yeast species in a sample by sequencing a particular region of ribosomal DNA.

In this experiment, overexpression of each of the three FLO genes had significant effects on at least one of the species in the consortium. The species composition of the flocculated yeasts was uniquely different, depending on which gene was overexpressed.

The discovery that the flocculation genes have individual effects on association with other species goes a long way towards explaining why S. cerevisiae has maintained this gene family with so many members that apparently have the same function—at least, when you study a pure culture. Differential regulation of the FLO genes could affect the spectrum of other species that our favorite yeast interacts with. 

So, our friend S. cerevisiae didn’t actually get out of the lab in these experiments, but at least it got to rub shoulders with some of its old friends (buds?) from the vineyard. These experiments are a good reminder for researchers to think outside the lab.

And when S. cerevisiae and its friends get together outside the lab, beautiful things can happen. We’ll drink a toast to that!

by Maria Costanzo, Ph.D., Senior Biocuration Scientist, SGD

If yeast could sing about its forced migration to the lab, it might sound like this.

Categories: Research Spotlight

Tags: flocculation, Saccharomyces cerevisiae, vineyard

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