New & Noteworthy

Making the Best of a Sticky Situation

June 05, 2017


Lemonade stand

Like turning lemons into lemonade, Hope and coworkers turned gummy yeast into a useful strain. from Pixabay.

 Back in 1915, writer Elbert Hubbard coined the phrase, “When life gives you lemons make lemonade.” (His actual quote was “He picked up the lemons that Fate had sent him and started a lemonade-stand.”)

The idea of course is to take something bad and make it into something good. Like, if your research gives you terribly weak glue, invent Post-It notes.

Or as Hope and coworkers show in a new study in GENETICS, when your yeast experiment gets gummed up because the yeast evolves a sticky trait, do additional experiments to learn about the evolution of that complex trait. And “invent” a way to make the yeast less likely to flocculate, or stick together.

This new “invention” will be very useful for anyone trying to evolve yeast to create new products or to study how evolution works. It also showed that for this trait, under these conditions with this strain, there was one major way to get to flocculence—up-regulating the FLO1 gene. This meant they could greatly reduce the risk of this trait popping up by simply deleting FLO1.

Studying evolution in yeast often involves using a chemostat, an automated way to keep the yeast growing through repeated dilutions with fresh media. Scientists use this method to study how yeast evolves under varying conditions over hundreds of generations.

An unfortunate side effect of this method is that it also tends to select for yeast that stick together. These yeast are diluted away less, often meaning they become more common over the generations.

In this study, Hope and coworkers ran 96 chemostats under three different conditions for 300 generations and found that in 34.7% of the cultures, the yeast ended up aggregated. This was even though they used a strain of S288c in which the FLO8 gene was mutated. This strain flocculates less often than wild type!

These authors picked the 23 most aggregated cultures to study in more detail. They found that 2 out of 23 strains aggregated because of a mother/daughter separation defect. The rest were more run-of-the-mill flocculent strains. 

They next used whole genome sequencing to try to identify which genes when mutated caused flocculence in the strains. They saw no FLO8 revertants.

The two strains with a mother/daughter separation defect both had mutations in the ACE2 gene, which encodes an important transcription factor for septation as well as other processes.

Flos

By CBS Television (eBay item photo front photo back) [Public domain], via Wikimedia Commons. Progressive insurance facebook flo advertising failsBy woodleywonderworks, via Flickr
No, these Flos don’t cause flocculence, FLO1 does.

FLO1-related mutations dominated the other 21. They found a couple of different Ty insertions in the promoter region of FLO1 in 12 of the strains and mutations in TUP1 in 5 more. Tup1p is a general repressor known to repress FLO1, so it looks like up-regulating FLO1 leads to flocculent cultures. Other candidate mutations were found in FLO9 and ROX3.

They wanted to try to identify the responsible gene(s) in strains where there was no obvious candidate gene and also to confirm that the genes they identified really caused the trait, so they next did backcrosses between each mutant strain and a wild type strain.

The backcrosses identified two other ways to get flocculence— by mutating either CSE2 or MIT1. The researchers also confirmed that the mutations they found, including these two new ones, were probably the main cause of the flocculence in each individual strain.  This trait co-segregated with the appropriate mutation in a 2:2 pattern for 20/21 strains as is predicted for a single causal mutation.

The results are even more FLO1-heavy than they appear. Further studies showed that ROX3, CSE2, and MIT1 all require a functional FLO1 to see their effects.

So FLO1 appears to be the main route to flocculence in this strain. And the next set of experiments confirmed this.

Hope and coworkers ran 32 wild type and 32 FLO1 knockout strains in separate chemostats for 250 generations and found that 8 wild-type strains flocculated while only 1 FLO1 knockout strain did. Knocking out FLO1 seems to make for a more well-behaved yeast (at least in terms of evolving a flocculent trait in a chemostat).

And the strain can probably be improved upon even more. For example, researchers may want to limit Ty mobility as this was a major way that FLO1 was up-regulated, increase the copy number of key repressors or link those repressors to essential genes. Another possibility is to also mutate FLO9 as its up-regulation was the cause of that one aggregated strain in the FLO1 knockout experiment.

Researchers no longer need to “settle” (subtle, huh?) for big parts of their experiments being hampered by gummy yeast. Hope and coworkers have created a strain that is less likely to flocculate by simply knocking out FLO1. Not as ubiquitously useful as a Post-It note but a potential Godsend for scientists using yeast to understand evolution.

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

Categories: Research Spotlight

Tags: FLO9 , FLO8 , flocculation , FLO1 , evolution , ROX3 , MIT1 , TUP1 , CSE2 , ace2

Yeast Tackles Climate Change

November 29, 2016


With its new superpower of turning xylose into ethanol, yeast is just about ready to provide the low carbon fuel we need to help us stave off climate change. Image from pixabay.

One of the best parts about doing outreach with a museum is creating a successful hands on activity for the visitors. This is not an easy thing to do.

You first create something that you think will appeal to and educate visitors. When that falls flat on its face, you then do a series of tweaks until it is working smoothly. In the end you have smiling kids who understand DNA better (or at all)!

Genetic engineering can be similar. You can import the genes for a complex pathway into your beast of choice but it may not work first try. A bit of tinkering, evolution style, is often needed to get the engineering working well enough to be useful.

This is exactly what happened when a group of researchers tried to get our favorite beast, the yeast Saccharomyces cerevisiae, to turn the sugar xylose into ethanol. They added the right genes from either fungi or bacteria, but S. cerevisiae couldn’t convert enough xylose into ethanol to be useful.

And it is important for all of us that some beast be able to do this well. A yeast that can turn xylose into ethanol means a yeast that can turn a higher percent of agricultural waste into a biofuel. Which, of course, means lots of low carbon fuel to run our cars so that we have a better shot of limiting the Earth’s heating up by 1.5-2 degrees Celsius.

To get their engineered yeast to better utilize xylose, Sato and coworkers forced it to grow with xylose as its only carbon source. Ten months and hundreds of generations later, this yeast had evolved into two new strains that were much better at turning xylose into ethanol. One strain did its magic with oxygen, the other without it.

In a new study in PLOS Genetics, Sato and coworkers set out to figure out which of the mutations that came up in their evolution experiments mattered and why.

Two genes were common in both the aerobic and anaerobic strains – HOG1 and ISU1. Both needed to be nonfunctional in order to maximize ethanol yields from xylose. They confirmed this by deleting each individually and together from the parental strain.

HOG1 encodes a MAP kinase, and ISU1 encodes a mitochondrial iron-sulfur cluster chaperone. These probably would not have been the first genes to go after with a more biased approach. The benefits of evolution and natural selection!

Further experiments showed deleting each gene individually was not as good as deleting both at once when oxygen was around. In fact, while deleting only ISU1 had a small effect on the ability of this yeast to convert xylose into ethanol, deleting HOG1 alone had no effect at all. Its deletion can only help a strain already deleted for ISU1.

In the absence of oxygen, yeast needs a couple of additional genes mutated – GRE3 and IRA2. GRE3 is an aldose reductase and IRA2 is an inhibitor of RAS. Again, not very obvious genes!

Still, once you find the genes you can come up with reasonable hypotheses for why they are important.

Some are easier than others. Hog1p, for example, is known to enhance a cell’s ability to turn xylose into xylitol, which shunts the xylose away from the ethanol conversion pathway. GRE3 is involved in this as well. Deleting either should make more xylose available to the yeast.

This doesn’t mean this is Hog1p’s only role in boosting this yeast’s ability to turn xylose into ethanol of course. It also probably “…relieves growth inhibition and restores glycolytic activity in response to non-glucose carbon sources.” Consistent with this, the authors found that the xylose-utilizing strain deleted for HOG1 was also better at using glycerol and acetate as carbon sources.

activity

The tweaks needed to make a successful museum activity are often nonintuitive. Much like the tweaks needed to maximize the performance of genetically engineered yeast. Image courtesy of The Tech Museum.

Other genes were less obvious. For example, perhaps mutating ISU1 frees up some iron so extra heme can be made. Or alternatively, it may have increased the mass of mitochondria available. Again, probably would not have been the first gene to go after to improve yeast’s ability to convert xylose to ethanol.

Which again underlines the importance of letting natural selection improve an engineered organism as opposed to only trying to pick and tweak the genes you think are important. Biology is simply too complicated and our understanding too limited to be able to know which are the best genes to go after. This is reminiscent of prototyping museum activities.

Some tweaks are obvious but others you would never have guessed would be needed. For example, we had visitors spreading bacteria on a plate and found that if they labeled their plate first, they almost always put their transformation mixture on the lid instead of on the LB agar. This problem was solved by having them add their mixture first and then labeling their plate.

It would be very hard to predict something like this from the get-go. The activity needed to evolve on the museum floor to work optimally. Much like the yeast engineered to utilize xylose needed to evolve in the presence of xylose to work optimally. And to perhaps take a big step towards saving the Earth from warming up too much.

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

Categories: Research Spotlight

Tags: evolution , biofuel , xylose , ethanol , glucose metabolism , fermentation

Ancient Hybridization Causes Revision of Yeast’s Calendar

August 19, 2015


Just as this monk had trouble pinpointing when Jesus was born, so too did yeast biologists have trouble figuring out when the genome of S. cerevisiae doubled in size. But they had trouble for very different reasons… Image via Wikimedia Commons

Most people assume that Jesus Christ was born around 1 A.D. or 1 B.C. or something like that. After all, that dating system is based on when he was born. 1 A.D. is by definition “the first year of the Lord.”

Now of course no one was actually marking years this way from the get go. In fact, this dating system wasn’t really invented until 525 A.D. by the monk Dionysius Exiguus.

And as can sometimes happen when you look that far back in time, he didn’t quite get the date of Jesus’ birth right. In point of fact, Jesus was most likely born between 4 and 6 B.C.

While not nearly as momentous, yeast biologists may have done something similar with the genome of our friend Saccharomyces cerevisiae. For many years scientists have believed that this yeast underwent a whole genome duplication (WGD) event around 100 million years ago

But if the conclusions reached by Marcet-Houben and Gabaldón in a new PLOS Biology article are correct, then what looks like the WGD event actually happened before there was a S. cerevisiae around to duplicate its genome.

Which would mean that there couldn’t have been a WGD. There is no way for a species to double its genome if it doesn’t yet exist! Instead the authors propose that what looks like a WGD was actually a hybridization of two related yeasts from longer ago than 100 million years.

Once you get past the vocabulary, the idea behind this study is actually pretty easy. Basically, they took pairs of genes that most likely came from a duplication event (ohnologs) and figured out when they diverged away from one another. This is the same idea behind figuring out when chimps and humans shared a common ancestor by comparing homologous genes.

When Marcet-Houben and Gabaldón compared every potential ohnolog in S. cerevisiae, they found that a WGD event could explain the origin of only 15% of these genes. These 15% could be traced back to a time after S. cerevisiae was already around.

The other 85% all looked to have been duplicated before S. cerevisiae yet existed. Which of course means these could not have come from a WGD. A genome that does not yet exist cannot be duplicated. This set of genes must have arisen in a different way.

An origin story that makes more sense than a WGD for these genes is one in which two related species hybridize to form one new species. This kind of thing definitely happens, especially with yeast (and if you like lagers, you can be glad it does!).

Here the idea is that the related species share a subset of their genes from when they had a common ancestor. When these species fuse, the new beast has both sets of genes. A cursory look might suggest that these genes were from a duplication event, especially if there are many large tracts of them. After all, many genes share a lot of homology between species.

It is only with a closer look that you might trace these genes back to a common ancestor that came before the species you are studying even existed. This is, in essence, what Marcet-Houben and Gabaldón found.

From the phylogeny of reference species that they created, the authors were able to get a general idea about which clades these prehybridization species may have come from. The largest peak of duplication from their analysis came from before Saccharomyces split from a clade containing Kluyveromyces, Lachancea, and Eremothecium (KLE). The other major peak came before Saccharomyces separated from a clade that contains Zygosaccharomyces rouxii and Torulaspora delbrueckii (ZT). So the simplest interpretation is that at some point long ago, an ancestor of modern S. cerevisiae was formed from the hybridization of a pre-KLE and a pre-ZT species.

Showing that something like this happened so long ago is fraught with peril. All sorts of things can happen to a genome in more than a hundred million years. And duplicated genes are even trickier because they can mutate at different rates when one copy is gaining a new function. (After all, that is one way that new genes are born.)

So Marcet-Houben and Gabaldón threw everything but the kitchen sink at the genome sequences. They tried at least three different methods for comparing the various sequences, using alternative reference phylogenies and a variety of techniques to show what might happen to their results if genes were mutating at different rates.

With each method they got similar results. Many or even most of the “duplication” events happened before S. cerevisiae was even a species. And on top of all of this they were able to show that they got a similar result when they used a known hybrid, S. pastorianus, and its two founding species, S. cerevisiae and S. eubayanus.

All of this taken together argues that S. cerevisiae did not undergo a WGD in the deep, dark past. Instead, it is the result of two closely related species getting together and creating a new species.

Now this does not necessarily mean there was no WGD in our favorite yeast’s past. There are at least two ways that a hybrid species might have formed, and as you can see in this image from Marcet-Houben and Gabaldón’s article, one of them involves a duplicated genome:

Fig 6. Hybridization scenarios. From Marcet-Houben and Gabaldón (2015), PLOS Biol. 13(8):e1002220.

In the first scenario, shown on the top left, two diploids of different species fuse together to create a yeast with two sets of chromosomes. Eventually, through mutation, translocation, gene loss and whatever other genome sculpting mechanisms are handy, the yeast ends up with double the number of chromosomes of its predecessors.

In the second possibility, shown on the bottom left, two haploids fuse. This fused yeast then undergoes a whole genome duplication and then goes through similar processes as the first model to get to the current genome.

So, although there may have been a WGD, it looks unlikely that it happened to S. cerevisiae. Just as the placement of historic events in our calendar changes when more information is available, the generation of genome sequences for more and more yeast species and new methods for analyzing them are giving us deeper insight into the history of our friend S. cerevisiae.

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

Categories: Research Spotlight

Tags: whole-genome duplication , evolution , Saccharomyces cerevisiae , hybridization

Yeast are People Too

June 03, 2015


Cars on the road today all look pretty similar from the outside, whether they’re gasoline-fueled or electric. On the inside, they’re fairly similar too. Even between the two kinds of car, you can probably get away with swapping parts like the air conditioner, the tires, or the seat belts. Although cars have changed over the years, these things haven’t changed all that much.

Just like these cars, yeast and human cells have some big differences under the hood but still share plenty of parts that are interchangeable. Nissan Leaf image via Wikimedia Commons; Ford Mustang image copyright Bill Nicholls via Creative Commons

The engine, though, is a different story. All the working parts of that Nissan Leaf engine have “evolved” together into a very different engine from the one in that Ford Mustang. They both have engines, but the parts aren’t really interchangeable any more.

We can think of yeast and human cells like this too. We’ve known for a while that we humans have quite a bit in common with our favorite little workhorse S. cerevisiae. But until now, no one had any idea how common it was for yeast-human pairs of similar-looking proteins to function so similarly that they are interchangeable between organisms.

In a study published last week in Science, Kachroo and colleagues looked at this question by systematically replacing a large set of essential yeast genes with their human orthologs. Amazingly, they found that almost half of the human proteins could keep the yeast mutants alive.

Also surprising was that the degree of similarity between the yeast and human proteins wasn’t always the most important factor in whether the proteins could be interchanged. Instead, membership in a gene module—a set of genes encoding proteins that act in a group, such as a complex or pathway—was an important predictor. 

The authors found that genes within a given module tended to be either mostly interchangeable or mostly not interchangeable, suggesting that if one protein changes during evolution, then the proteins with which it interacts may need to evolve as well. So we can trade air conditioner parts between the Leaf and the Mustang, but the Mustang’s spark plugs won’t do a thing in that newly evolved electric engine!

To begin their systematic survey, Kachroo and colleagues chose a set of 414 yeast genes that are essential for life and have a single human ortholog. They cloned the human cDNAs in plasmids for yeast expression, and transformed them into yeast that were mutant in the orthologous gene to see if the human gene would supply the missing yeast function.

They tested complementation using three different assays. In one, the human ortholog was transformed into a strain where expression of the yeast gene was under control of a tetracycline-repressible promoter. So if the human gene complemented the yeast mutation, it would be able to keep the yeast alive in the presence of tetracycline.

Another assay used temperature-sensitive mutants in the yeast genes and looked to see if the human orthologs could support yeast growth at the restrictive temperature. And the third assay tested whether a yeast haploid null mutant strain carrying the human gene could be recovered after sporulation of the heterozygous null diploid.

Remarkably, 176 human genes could keep the corresponding yeast mutant alive in at least one of these assays. A survey of the literature for additional examples brought the total to 199, or 47% of the tested set. After a billion years of separate evolution, yeast and humans still have hundreds of interchangeable parts!

That was the first big surprise. But the researchers didn’t stop there. They wondered what distinguished the genes that were interchangeable from those that weren’t. The simplest explanation would seem to be that the more similar the two proteins, the more likely they would work the same way. 

But biology is never so simple, is it? While it was true that human proteins with greater than 50% amino acid identity to yeast proteins were more likely to be able to replace their yeast equivalents, and that those with less than 20% amino acid identity were least likely to function in yeast, those in between did not follow the same rules. There was no correlation between similarity and interchangeability in ortholog pairs with 20-50% identity.  

After comparing 104 different types of quantitative data on each ortholog pair, including codon usage, gene expression levels, and so on, the authors found only one good predictor. If one yeast protein in a protein complex or pathway could be exchanged with its human ortholog, then usually most of the rest of the proteins in that complex or pathway could too.

This budding yeast-human drives home the point that humans and yeast share a lot in common: so much, that yeast continues (and will continue) to be the pre-eminent tool for understanding the fundamental biology of being human. Image courtesy of Stacia Engel

All of the genes that that make the proteins in these systems are said to be part of a gene module. Kachroo and colleagues found that most or all of the genes in a particular module were likely to be in the same class, either interchangeable or not. We can trade pretty much all of the parts between the radios of a Leaf and a Mustang, but none of the engine parts.

For example, none of the tested subunits of three different, conserved protein complexes (the TriC chaperone complex, origin recognition complex, and MCM complex) could complement the equivalent yeast mutations. But in contrast, 17 out of 19 tested genes in the sterol biosynthesis pathway were interchangeable.

Even within a single large complex, the proteasome, the subunits of one sub-complex, the alpha ring, were largely interchangeable while those of another sub-complex, the beta ring, were not. The researchers tested whether this trend was conserved across other species by testing complementation by proteasome subunit genes from Saccharomyces kluyveri, the nematode Caenorhabditis elegans, and the African clawed frog Xenopus laevis. Sure enough, alpha ring subunits from these organisms complemented the S. cerevisiae mutations, while beta ring subunits did not.

These results suggest that selection pressures operate similarly on all the genes in a module. And if proteins continue to interact across evolution, they can diverge widely in some regions while their interaction interfaces stay more conserved, so that orthologs from different species are more likely to be interchangeable.

The finding that interchangeability is so common has huge implications for research on human proteins. It’s now conceivable to “humanize” an entire pathway or complex, replacing the yeast genes with their human equivalents. And that means that all of the versatile tools of yeast genetics and molecular biology can be brought to bear on the human genes and proteins.

At SGD we’ve always known that yeast has a lot to say about human health and disease. With the growing body of work in these areas, we’re expanding our coverage of yeast-human orthology, cross-species functional complementation, and studies of human disease-associated genes in yeast. Watch this space as we announce new data in YeastMine, in download files, and on SGD web pages.

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

Categories: Research Spotlight Yeast and Human Disease

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

The Failed Hook Up: A Sitcom Starring S. cerevisiae

May 27, 2015


As anyone who watches a situation comedy knows, long range relationships are tricky. The longer the couple is separated, the more they drift apart. Eventually they are just too different, and they break up.

Jerry Seinfeld finds girlfriends incompatible for seemingly minor reasons like eating peas one at a time. Different yeast strains become incompatible over small differences in certain genes as well. Image by Dano Nicholson via Flickr

Of course if this were the end of the story, it would be the plot of the worst comedy ever. What usually happens in the sitcom is that one or both of them find someone more compatible and live happily ever after (with lots of silliness and high jinks).

Turns out that according to a new study by Hou and coworkers, our friend Saccharomyces cerevisiae could star in this sitcom. When different populations live in different environments, they drift apart. Eventually, because they accumulate chromosomal translocations and other serious mutations, they have trouble mating and having healthy offspring.

Now researchers already knew that big changes in yeast, like chromosomal translocations, affect hybrid offspring. But what was controversial before and what this study shows is that, as is known for plants and animals, smaller changes like point mutations can affect the ability of distinct populations of yeast to have healthy progeny. It is like Jerry Seinfeld being incompatible with a girl because she eats her peas one at a time (click here for other silly reasons Jerry breaks up with girlfriends).  

The key to finding that yeast can be Seinfeldesque was to grow hybrid offspring in different environments. Hybrids that did great on rich media like YPD sometimes suffered under certain, specific growth conditions. Relying on the standard medium YPD masked mutations that could have heralded the beginnings of a new species of yeast.

See, genetic isolation is a powerful way for speciation to happen. One population generates a mutation in a gene and the second population has a mutation in a second gene. In combination, these two mutations cause a growth defect or even death. Now each population must evolve on its own, eventually separating into two species.

To show that this is a route that yeast can take to new species, Hou and coworkers mated 27 different Saccharomyces cerevisiae isolates with the reference laboratory strain S288C and grew their progeny under 20 different conditions. These strains were chosen because they were all able to produce spores with S288C that were viable on rich medium (YPD).

Once they eliminated the 59 pairings that involved parental strains that could not grow under certain conditions, they found that 117 out of 481 or 24.3% of crosses showed at least some negative effect on the growth of the progeny under at least some environmental conditions. And some of these were pretty bad. In 32 cases, at least 20% of the spores could not survive.

The authors decided to focus on crosses between S288C and a clinical isolate, YJM241, where around 25% of spores were inviable under growth conditions that required good respiration, such as the nonfermentable carbon source glycerol. They found that rather than each strain having a variant that affected respiration, the growth defect happened because of two complementary mutations in the clinical isolate.

The first mutation was a nonsense mutation in COX15, a protein involved in maturation of the mitochondrial cytochrome c oxidase complex, which is essential for respiration. The second was a nonsense suppressor mutation in a tyrosine tRNA, SUP7. So YJM241 was fine because it had both the mutation and the mechanism for suppressing the mutation. Its offspring with S288C were not so lucky.

Around 1 in 4 progeny got the mutated COX15 gene without SUP7 and so could not survive under conditions that required respiration. Which of course is why this was missed when the two strains were mated on YPD, where respiration isn’t required for growth.

So this is a case where the separated population, the clinical isolate YJM241, changed on its own such that it would have difficulty producing viable progeny with any other yeast strains. Like the narrator in that old Simon and Garfunkel song, it had become an island unto itself.

The researchers wondered whether this kind of change—a nonsense mutation combined with a suppressor—occurs frequently in natural yeast populations. They surveyed 100 different S. cerevisiae genome sequences and found that nonsense mutations are actually pretty common. Nonsense suppressor mutations were another story, though: they found exactly zero.

Apparently nonsense suppressor mutations are really rare in the yeast world, and Hou and colleagues wondered whether this was because they had a negative effect on growth. They added the SUP7 suppressor mutant gene to 23 natural isolates. It had negative effects on most of the isolates during growth on rich media, but it was more of a mixed bag under various stress conditions. Sometimes the mutation had negative effects and sometimes it had positive effects.

The fact that a suppressor mutation can provide a growth advantage under the right circumstances, combined with the fact that they are very rare, suggests that a new suppressor arising might help a yeast population out of a jam, but once the environment improves the yeast are free to jettison it. Suppressor mutations may be a transitory phenomenon, a momentary dalliance.

So, separate populations of yeast can change over time in subtle ways that prevent them from mating with one another. This can eventually lead to the formation of new species as the changes cause the two to drift too far apart genetically. It is satisfying to know that yeast drift apart like any other plant, animal, or sitcom character.

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

Categories: Research Spotlight

Tags: evolution , Saccharomyces cerevisiae

A Factory Without Doors

April 29, 2015


Every factory needs raw materials. Without steel, this is just a pretty factory. And without incoming xylose, a yeast cell set up to make ethanol from biomass is just a pretty cell. Image by Steve Jurvetson via Flickr

Imagine you have built a state-of-the-art factory to make a revolutionary product. The place is filled with gleaming assembly lines and you have hired the best talent in the world to run the place.

Unfortunately there was a glitch in the factory design—the builders forgot to put doors in! Now you can’t get the raw materials in to make that killer product that will change everything.

This may sound contrived or even silly, but it is sort of what is happening in attempts to use yeast to make biofuels from agricultural waste. Scientists have tweaked yeast cells to be able to turn xylose, a major sugar found in agricultural waste, into ethanol. But yeast has no transporter system for this sugar. A bit can get in through the windows, so to speak, but we need to put in a door so enough can get inside to make yeast a viable source for xylose-derived ethanol.

An important step was taken in this direction in a new study by Reznicek and coworkers. They used directed evolution to transform the Gal2 transporter of Saccharomyces cerevisiae into a better xylose transporter. And they succeeded.

After three successive rounds of mutagenesis, they transformed Gal2 from a transporter that prefers glucose into one that prefers xylose. When put in the right background, this mutant protein opens the door for getting yeast to turn agricultural waste into ethanol. Perhaps yeast can help us stave off cataclysmic climate change for just a bit longer. 

The first step was to find the right strain for assaying xylose utilization. They needed a strain lacking 8 hexose transporters, Hxt1-7 and Gal2, because these transporters can take up xylose (albeit at a very low efficiency). Deleting these genes “shuts the windows” and completely prevents the strain from utilizing xylose as a substrate (as well as impairing its ability to use glucose).

This strain was also engineered to be able to utilize xylose. It contained a xylose isomerase gene from an anaerobic fungus and also either overexpressed or lacked several S. cerevisiae genes involved in carbohydrate metabolism. With this strain in hand, the researchers were now ready to add a door to their closed off factory.  

The authors targeted amino acids 292 to 477 in Gal2. This region is thought to be critical for recognizing sugars, based on homology with other hexose transporters. They used mutagenic PCR conditions that generated an average of 4 point mutations in this region, and screened for mutants that grew better than others on plates containing 0.1% xylose.

In their initial screen they selected and replated the 80 colonies that grew best. They then chose the best 9 to analyze further. Of these 9, one mutant which they dubbed variant 1.1 grew better on xylose than a strain carrying wild-type GAL2. Variant 1.1 had a single amino acid change, L311R.

They repeated their assay using variant 1.1 as their starting source. Out of the 14,400 mutants assayed, they found four that did better than variant 1.1. These variants, dubbed 2.1-2.4, all shared the same M435T mutation.  Variant 2.1 had three additional mutations—L301R, K310R, and N314D.

These four new mutants showed better growth on 0.45% xylose, and after 62 hours, all the strains had pretty much used up the xylose in their media. Of the four, variant 2.1 appeared to be the best xylose utilizer: after 62 hours the authors could detect no xylose in the media at all. This variant also grew faster than the others in 0.1% xylose.

Reznicek and coworkers had definitely made Gal2 a better xylose transporter, but they weren’t done yet. They wanted to try to make a door that only let in the raw supplies (xylose) they wanted and not other sugars (glucose).

Up until now, the screens had been done with xylose as the sole carbon source. When they grew variant 2.1 in the presence of both 2% glucose and 2% xylose, they found that it preferentially used the glucose first. Their evolved transporter still preferred glucose over xylose!

Now in some ways this wasn’t surprising, as the mutations had not really affected the part of the protein thought to be involved in recognizing sugars. They next set out to evolve Gal2 so that it would transport xylose preferentially over glucose.

This time they used a slightly different background strain for their screen. This strain, which was deleted for hxk1, hxk2, glk1, and gal1, was unable to use glucose although it could transport it.

They repeated their mutagenesis and looked for mutants that grew best in 10% glucose and 2% xylose. We would predict that any growing mutants would have to transport xylose better than glucose. And this is just what they found.

When they analyzed the mutants, they found that the key mutation in making Gal2 prefer xylose over glucose in the variant 2.1 background was T386A. Based on homology with Hxt7, this mutation happens smack dab in the middle of the sugar recognition part of the protein. Most likely this mutation compromised the ability of Gal2 to recognize glucose, as opposed to improving recognition for xylose.

These experiments represent an important but by no means final step in engineering yeast to make fuel from biomass. We are on our way to a smaller carbon footprint and perhaps a world made somewhat safer from climate change.

First, beer, wine, and bread; next, keeping coral alive and saving countless species from extinction. Nice work, yeast.

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

Categories: Research Spotlight

Tags: evolution , Saccharomyces cerevisiae , biofuel

Once You Start Looking, Shu2 Homologs Are Everywhere

April 09, 2015


Once you notice the first robin of the spring, you see them everywhere. And once you notice an important protein, you might well find it all over the evolutionary tree. Image by Jerry Friedman via Wikimedia Commons

Have you ever heard of the Baader-Meinhof phenomenon? (Don’t worry, we hadn’t either!) But you’ve surely experienced it.

The phenomenon describes the experience of suddenly seeing something everywhere, after you’ve noticed it for the first time. For those of us in Northern climates, it’s like like robins coming back in the springtime: one day, you see a single robin hopping on the grass; the next day, you look around and realize they’re all over.

Something similar happened to Godin and colleagues as they investigated the S. cerevisiae Shu2 protein. People knew about the protein and its SWIM domain but no one had looked to see just how conserved it was.

When the researchers looked for homologous proteins that contained the characteristic SWIM domain, they found homologs in everything from Archaea through primitive eukaryotes, fungi, plants, and animals. They were practically everywhere (in the evolutionary sense).

In a new paper in GENETICS, Godin and coworkers described their studies on this relatively little-studied, unsung protein. They found that it is an important player in the essential process of DNA double-strand break repair via homologous recombination, and its SWIM domain is critical to this function. Not only that, but being able to compare the SWIM domains from so many different homologs allowed them to refine its consensus sequence, identifying a previously unrecognized alanine within the domain was both highly conserved and very important.

Double-strand DNA breaks (DSBs) can happen because of exposure to DNA damaging agents, but they are also formed normally during meiotic recombination, in which a nuclease actually cuts chromosomes to start the process. Homologous recombination to repair DSBs is a key part of both mitosis and meiosis.

During homologous recombination, one strand of each broken DNA end is nibbled back to form a single-stranded region. This region is then coated with a DNA-binding protein or proteins, forming filaments that are necessary for those ends to find homologous regions and for the DSB to be repaired.

Shu2 is one of the proteins that participates in the formation of these filaments in S. cerevisiae. It was known that it was part of a complex called the Shu complex, and a human homolog, SWS1, had been identified. But the exact role of Shu2 and the significance of the SWIM (SWI2/SNF2 and MuDR) zinc finger-like domain that it contains were open questions.

One of the first questions the authors asked was whether Shu2 was widely conserved across the tree of life. Genes with similar sequences had been seen in fission yeast (Schizosaccharomyces pombe) and humans, but no one had searched systematically for orthologs. They used PSI-BLAST, a variation of the Basic Local Alignment Search Tool (BLAST) algorithm that that is very good at finding distantly related proteins, to search all available sequences.

Querying with both yeast Shu2 and human SWS1, the researchers found hits all across the tree of life—both in “lower” organisms such as Archaea, protozoa, algae, oomycetes, slime molds, and fungi, and in more complex organisms like fruit flies, nematode worms, and plants. The homologous proteins that they found across all these species also had the SWIM domain, suggesting that it might be important.

The sequence similarity was all well and good, but did these putative Shu2 orthologs actually do the same job in other organisms that Shu2 does in yeast? One way to test this is to do co-evolutionary analysis. Proteins that work together are subject to the same evolutionary pressures, so they tend to evolve at similar rates. Godin and colleagues found that evolutionary rates of the members of the Shu complex in fungi and fruit fly did generally correlate with those of other proteins involved in mitosis and meiosis.

The awesome power of yeast genetics offered Godin and coworkers a way to look at the function of Shu2. They first tested the phenotype of the shu2 null mutation, and found that it decreased the efficiency of forming filaments of the Rad51 DNA-binding protein on the single-stranded DNA ends that are created at DSBs. Formation of these filaments is a necessary step in repairing the DSBs by homologous recombination.

The comparison of SWIM domains from so many different proteins highlighted one particular alanine residue. This alanine hadn’t previously been considered part of the domain’s consensus sequence, but it was conserved in all the domains.

When the researchers changed the invariant alanine residue in yeast Shu2, the mutant protein bound less strongly to its interaction partner in the Shu complex, Psy3. When they mutated the analogous residue in the human Shu2 ortholog SWS1, this also decreased its binding to its partner, SWSAP1.

Other mutations within the SWIM domain of Shu2 also affected its interactions with other members of the Shu complex, and made the mutant cells especially sensitive to the DNA-damaging agent MMS. Diploid cells with a homozygous mutation in the Shu2 SWIM domain had very poor spore viability, suggesting that the SWIM domain is important for normal meiosis.

As one more indication of the SWIM domain’s importance, Godin and colleagues took a look in the COSMIC database, which collects the sequences of mutations found in cancer cells. Sure enough, a human cancer patient carried a mutation in that invariant alanine residue of the SWIM domain in the Shu2 ortholog, SWS1.

There’s still much more to be done to figure out exactly what Shu2 and the Shu complex are doing during homologous recombination. Yeast obviously provides a wonderful experimental system, and the discovery of Shu2 orthologs in two other model organisms that also have awesomely powerful genetics and happen to be multicellular, Drosophila melanogaster and Caenorhabditis elegans, expands the experimental possibilities even further.

There’s also a lot to be learned about the SWIM domain in particular. The discoveries that it affects the binding behavior of these proteins and that it is mutated in a cancer patient show that it’s very important, but just what does it do in Shu2? It will be fascinating to find out exactly how this domain works to help cells recover from the lethal danger of broken chromosomes. And it is amazing what you can see, once you start looking. 

by Maria Costanzo, Ph.D., Senior Biocurator, SGD

Categories: Research Spotlight

Tags: Saccharomyces cerevisiae , evolution , model organism , homologous recombination

Conformity Preferred in Yeast

March 25, 2015


In a classic Apple ad, the world is a gray, dreary place where everyone is the same. In charges a woman dressed in brightly colored clothing who hurls a hammer into a big black and white screen, smashing the old conformist world order. Individuality can now blossom.

Mother Nature frowns on mutations that add to cell to cell variation in gene expression. Image by SUNandMooN363 via Creative Commons

This is a powerful story for people, but it turns out that if Mother Nature had her druthers, she would like that woman to either stay at home or dress and act like everyone else. At least this is true if we are talking about individuals that are genetically identical. In this case, alleles that cut down on cell to cell variation tend to be the ones that prosper.

This is confirmed in a new study in Nature in which Metzger and colleagues in the Wittkopp lab showed that there is a selection against mutations that cause increased variation between individuals in the S. cerevisiae TDH3 gene. In other words, mutations that cause more “noise” are selected against. The squeaky wheel is eliminated.

The TDH3 gene encodes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an important metabolic enzyme. Yeast cells can survive a deletion of this gene, but their fitness is greatly reduced. Overexpression of the gene also has noticeable effects. This suggested to the researchers that the level of TDH3 expression would be under selection pressure during evolution.

Metzger and coworkers compared the promoters of the TDH3 gene from 85 different strains of S. cerevisiae and found that the promoter had undergone selection out in the wild. The authors were interested in why certain polymorphisms were selected for and why others were selected against. To try to tease this out, they compared the activities of evolved changes to randomly selected ones.

The authors first used the sequences of the 27 haplotypes they saw in the 85 strains to predict what the original, ancestral promoter probably looked like. They then re-created this sequence and also sequences that represented the most likely intermediates on the way to the current promoters. They linked each of these promoter sequences to a yellow fluorescent protein (YFP) reporter and looked at mean activity and expression noise. In other words, they looked at how much promoter activity there was in aggregate and how much it varied between individual cells for the 10,000 cells in each culture.

They next set out to generate a pool of polymorphisms that didn’t make the cut during evolution, so they could compare these to the successful ones. To do this, they individually mutated 236 G:C to A:T transitions throughout the promoter region. They chose this transition because these are the most common spontaneous mutations seen in yeast and the most common SNP seen in this promoter out in the wild.

Now they were ready to do their experiment! Comparing the randomly created mutations to the evolved changes, they looked at both the overall level of expression and how much variation there was between each of the 10,000 individual cells in the tested culture.

What they found was that the effects on the mean level of activity were pretty comparable between the “selected” mutations and the random ones. But the same was not true for individual variation. The random mutations were much more likely to increase expression noise compared to the “selected” mutations.

From these results the authors conclude that there is a selection against mutations that increase the level of noise. In fact, they go a step further and conclude that at least for the TDH3 promoter, there was more of a selection against noise than there was a selection for a particular level of activity. It was more important that individuals had consistent activity than it was to have some mean level of activity.

This makes some sense, as a cell is a finely tuned machine where all the parts need to work in harmony together to succeed. If one part is erratic and shows different levels of activity in different individuals, then some of those individuals won’t do as well and so won’t survive.

This also means that certain paths to a more fit organism will be selected over others. And it could be that organisms miss out on some potential fitter states because they can’t survive the dangerous evolutionary journey that would be needed to get there.

So the cell prefers that all the parts work together in a predictable way. When you’re a population of individuals that are more or less genetically identical, nonconformists are dangerous.  The gray sameness of the Nineteen Eighty-Four world is preferable to a more bohemian atmosphere where diversity is celebrated.

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

Categories: Research Spotlight

Tags: evolution , Saccharomyces cerevisiae , promoter

Those Yeast Got Talent

March 11, 2015


Thriving in a yeast culture is a lot like becoming a finalist on American Idol—you need some minor advantage to hang around and then a big finish to dominate. Image by Michael Tanne via Wikimedia Commons

The winners of American Idol go through quite a selection process. They start out as one of tens of thousands of people who audition, and survive each cut until they are finally crowned.

At the first cuts, those with any sort of advantage are kept in the pool and the others dropped. As the cuts continue, contestants not only need to have had that stronger initial advantage (or a bit of luck), but they also need to have picked up some new skills from all of those off- and on-air performances.

Some of these contestants start with a lot of raw talent but then progress only a little, while others are able to hone their weaker initial talent with lots of practice. Once their numbers are winnowed down to a handful, it gets close to being anyone’s game because the remaining contestants are so talented.

A new study by Levy and coworkers paints a similar sort of picture for evolving populations of yeast. Very early on a whole lot of yeast stumble upon weak, beneficial mutations that keep them going in the population. These are the yeast that make the initial cut in the hurly-burly world of the Erlenmeyer flask.

At later times a few yeast end up with strongly beneficial mutations that allow them to start to dominate. These are the pool of yeast that are the finalists of the flask.

Of course a big difference (among many) between American Idol and the yeast in this experiment is that the pool of contestants in the flask hangs around—they are not thrown off the show. This means that some cell that didn’t do too well early on can suddenly gain a strongly beneficial mutation and begin to dominate. Until, of course, that cell is usurped by another more talented yeast, in which case that finalist will fade away unless it can adapt.

And this study isn’t just a fascinating dissection of evolutionary population dynamics either. It might also have implications for treating bacterial infections and even cancer.

Bacteria and cancer cells live in large populations with each cell trying to outcompete the others. By understanding the set of mutations that allow some cells to succeed against the others and become more harmful, researchers may be able to come up with new ways to treat these devastating diseases.

One of the trickiest parts of this experiment was figuring out how to follow lots of yeast lineages all at once in a growing culture. Levy and coworkers accomplished this by adding 500,000 unique DNA barcodes to a yeast population and using high-throughput DNA sequencing to follow the lineages in real time.

They set up two replicate cultures and followed them for around 168 generations. In both cultures the researchers saw that while most of the lineages became much less common, around 5% happened upon a beneficial mutation that allowed them to increase in number by generation 112.

In other words, around 25,000 lineages ended up with beneficial mutations that let them make the first cut in both cultures. This translates to a beneficial mutation rate of around 1 X 10-6 per cell per generation and means that around 0.04% of the yeast genome (around 5000 base pairs) can change in a way that confers a growth advantage.

But of course not all mutations are the same. Weakly beneficial mutations are very common, which means both cultures have plenty of these early on. This is why the replicate cultures behave so similarly up to around generation 80.

Eventually, though, a few yeast stumble upon stronger, more beneficial mutations. Since these are rarer and harder to get, each replicate culture gets them at different generations. This is why the cultures begin to diverge as the 100 or so of the strongest beneficial mutations begin to dominate.

The experiment did not go on for long enough to see many double mutations. In other words, it was very rare in this experiment to see a yeast lineage succeed because it had developed additive beneficial mutations. This is because there simply wasn’t enough time for a yeast cell to get a beneficial mutation and establish itself and then have one of its lineage gain and establish a second beneficial mutation. There was no Jennifer Hudson who came in 7th but then went on to win a Grammy and an Oscar.

When a cancerous tumor is developing, however, there is plenty of time for multiple “beneficial” mutations to be established. These mutations are only beneficial for the tumor; they are devastating for the person with cancer. This is why it is so critically important to understand not only which mutations are implicated in cancer, but also the dynamics of how they accumulate in the cancer cell population during progression of the disease. Talented yeast in the hands of talented researchers are helping us figure this out.

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

Categories: Research Spotlight

Tags: evolution , Saccharomyces cerevisiae , cancer

Mannan From Heaven

February 25, 2015


Once we started eating the yeast in breads and beer, our microbiota adapted to feast on these mannans raining down on them from heaven. Manna reigning from heaven on the Israelites (Exodus 16), from the Maciejowski Bible, c. 1250 C.E.

Thousands of years ago, humans encountered very little yeast in their diet. This all changed with the invention of beer and bread.

Now of course we didn’t include the yeast as a carbon source…we just liked fluffy bread and getting hammered. But it was a different story for the bacteria in our gut. They now found the mannans in yeast cell walls raining down on them like manna from heaven.

Unlike the Israelites, who could eat manna, most of our microbiota probably couldn’t use this newfound carbon source. But at some point, the polysaccharide utilization loci (PULs) of a few species changed so that they could now digest the mannans found in yeast cell walls. And, as a new study in Nature by Cuskin and coworkers shows, it was almost certainly a great boon for them.

They found that at least one gram negative bacterium, Bacteroides thetaiotamicron, can live on just the mannans found in the yeast cell walls. This isn’t just a cool story of coevolution either. It might actually help sick people get better one day.

Glycans, like the mannans found in yeast cell walls, have been implicated in autoimmune diseases like Crohn’s disease. One day doctors may be able to treat and/or prevent diseases like this by providing bacteria that can eliminate these potentially harmful mannans: a probiotic treatment for a terrible disease.

Cuskin and coworkers identified three loci in B. thetaiotamicron, MAN-PUL1, MAN-PUL2, and MAN-PUL3, that were activated in the presence of α-mannan. Deletion experiments showed that MAN-PUL2 was absolutely required for the bacteria to utilize these mannans.

The authors next wanted to determine whether the ability to use yeast cell walls as a food source provided an advantage to these bacteria. For this work they turned to a strain of mice that lacked any of their own gut bacteria.

The authors colonized these gnotobiotic mice with 50:50 mixtures of two different strains of B. thetaiotamicron—wild type and a mutant deleted for all three PULs. They found that in the presence of mannans, the wild type strain won out over the mutant strain and that in the absence of mannans, the opposite was true.

So as we might expect, if you feed mice mannans, bacteria that can break them down do best in the mouse gut. The second result, that mutating the PULs might be an advantage in the absence of glycans, wasn’t expected. This result suggests some energetic cost of having active PULs in the absence of usable mannans.

To better mimic the real world, Cuskin and coworkers also looked at the gut bacteria of these mice when they were fed a high bread diet. In this case, the mutant still won out over wild type but not by as much. Instead of being reduced to 10% as was true in the glycan-free diet, the final proportion of wild type bacteria in guts of mice on the high bread diet was 20%. Modest but potentially significant.

We do not have the space to discuss it here, but the authors next dissected the mannan degradation pathway in fine detail. If you are interested please read it over. It is fascinating.

The authors also analyzed 250 human metagenomic samples and found that 62% of them had PULs similar to the ones found in B. thetaiotamicron. So the majority of the people sampled, but not all, had gut microbiota that could deal with the mannans in yeast cell walls.

Human microbiota have adapted to use the energy from the bits of yeast left in bread and alcoholic beverages. Given how little there is, it might be better to think of it as the filth the peasants collected in Monty Python and the Holy Grail instead of manna. Even though it isn’t much, it has given these bacteria a niche that no one else has (or probably wants).

Still, it has allowed them to at least survive. And if these bacteria can one be repurposed as a treatment for disease like Crohn’s disease, thank goodness they adapted to using these mannans.  

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

Categories: Research Spotlight

Tags: glycans , evolution , gut microbiome

Species Can’t Risk the New Coke

January 29, 2015


Genome organization may protect key genes from the ravages of increased mutation rate during meiosis:

Back in 1985, Coca Cola decided to completely rejigger the flavor of their flagship soft drink, calling it the New Coke. This radical change to the product was a colossal failure. Toying with such an essential part of a key product was simply too risky a move. If only they had learned from our favorite beast, Saccharomyces cerevisiae.

If only Coke had protected its essential recipe as well as yeast protects its essential genes! Image via Wikimedia Commons

In a new study in PLOS Genetics, Rattray and coworkers show that the mutation rate is higher during meiosis in yeast because of the double-strand breaks associated with recombination. This makes sense, because any new mutations need to be passed on to the next generation for evolution to happen, and germ cells are made by meiosis. But their results also bring up the possibility that key genes might be protected from too many mutations by being in recombination cold spots. Unlike the Coca Cola company, yeast (and everything else) may protect essential genes from radical change.

Previous work in the Strathern lab had suggested that when double strand breaks (DSBs) in the DNA are repaired, one result is an increased mutation rate in the vicinity. The major culprit responsible for the mutations appeared to be DNA polymerase zeta (Rev3p and Rev7p).

To test whether the same is true for the DSBs that happen during the first meiotic prophase, Rattray and coworkers created a strain that contained the CAN1 gene linked to the HIS3 gene. The idea is that mutants in the CAN1 gene can be identified as they will be resistant to canavanine. The HIS3 gene is included as a way to rule out yeast that have become canavanine resistant through a loss of the CAN1 gene. So the authors were looking for strains that were both resistant to canavanine and could grow in the absence of histidine.

The first things the authors found was that the mutation rate during meiosis was indeed increased as compared to mitosis in diploids. For example, when the reporter cassette was inserted into the BUD5 gene, the mitotic mutation rate was 5.7 X 10-8 while the meiotic mutation rate was 3.7 X 10-7, a difference of around 6.5 fold.

This effect was dependent on the DSBs associated with recombination, since the increased mutation rate wasn’t seen in a spo11 mutant; the SPO11 gene is required for these breaks. Using a rev3 mutant, the authors could also conclude that at least half of the increased mutation rate is due to DNA polymerase zeta. This all strongly suggests that the act of recombination increases the local mutation rate.

If recombination is associated with the mutation rate, then areas on the genome that recombine more frequently should have a higher rate of mutation during meiosis. And they do. The authors inserted their cassette into a known recombination hotspot between the BUD23 and the ARE1 genes and saw a meiotic mutation rate of 1.77 X 10-6  as compared to a rate of 4.9 X 10-7 when inserted into a recombination coldspot. This 3.6 fold increase provides additional evidence that recombination is an important factor in meiotic recombination.

This may be more than just an unavoidable side effect of recombination. It could be that yeast and perhaps other beasts end up with their genes arrayed in such a way as to protect important genes by placing them in recombination dead zones.

And perhaps genes where lots of variation is tolerated or even helpful are placed in active recombination areas. In keeping with this, recent studies have shown that essential S. cerevisiae genes tend to be located in recombination cold spots, and that this arrangement is conserved in other yeasts.

It is too early to tell yet how pervasive this sort of gene placement is.  But if this turns out to be a good way to protect essential genes, Coca Cola should definitely have left the Coke formula in a part of its genome with little or no recombination. Mutating that set of instructions was as disastrous as mutating an essential gene!

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

Categories: Research Spotlight

Tags: recombination , evolution , Saccharomyces cerevisiae , meiosis

Lots of Ways to Get to the Same Place

November 13, 2014

 

An advantage of taking one route to the post office might be that you get to see the elephant topiary in front of the zoo. For a yeast cell, taking a roundabout route to a wild-type phenotype might confer a big advantage in a different environment. Image from Wikimedia Commons

How Bad Mutations Can Help Yeast Thrive in New Environments:

If you’ve ever asked for directions from more than one person, you know there are many ways to get to the same place. But not all routes are created equal. You might be trying to get to the post office, but on some routes you’ll pass by the zoo, while on others you might pass the museum or the bus station.

Turns out that something like this may be happening with individuals in a population too. Each may be well adapted for its environment, but each may have arrived there in different ways. And although they may seem similar, they might actually be more different than they look on the surface.

In a new study in PLOS biology, Szamecz and coworkers show that a lot of these different routes to the same place happen in yeast because of bad mutations. They found that when a yeast gets a deleterious mutation, it is sometimes able to mutate its way back to being competitive with wild type again. But the evolved strain is genetically distinct from the original wild type strain. Similar phenotype, distinct genotype.

And this isn’t just an interesting academic exercise either. As any biologist knows, bad mutations are much more common than are good ones. This means that populations may often be evolving to overcome the effects of these bad mutations. This process may help to explain the wide range of diverse genotypes seen in any wild population. 

The authors started out by focusing on 187 yeast strains in which a single gene had been deleted. Each strain grew more poorly than wild type under the tested conditions.

They then took 4 replicates of each mutant along with the wild type strain and grew them for 400 or so generations. They looked for strains that had evolved to overcome the growth defect caused by the mutation. 

To take into account the fact that every strain would probably evolve a bit to grow better in the environment, they only looked for those that had gained more growth advantage than the wild type had. Around 68% of the strains showed at least one replicate that met this criterion.

So as we might expect, it is possible for a strain that grows poorly to mutate its way closer to a wild type growth rate. The next question was whether these mutant strains had mutated back to something close to wild type or to something new.

The authors decided to answer this question by doing a gene expression analysis of the wild type, the eight mutant strains, and a corresponding evolved line from each of these eight. After doing transcriptome analysis, they found that for the most part the evolved lines did not simply revert back to the original gene expression pattern of the wild type strain. Instead, they generated a novel gene expression pattern to deal with the consequences of having lost the original gene. And in the next set of experiments, the authors showed that this matters when the evolved strains are put in a new environment.

The researchers took 237 evolved lines that grew nearly as well as the original wild type strain and tested how well they each did in 14 different environments. In other words, they tested genetically distinct, phenotypically similar strains in new environments.

They found that even though the original mutant strains grew poorly in all the environments tested, the evolved ones sometimes did better. Fitness improved in 52% of the strains and declined in 8%. What is even more interesting is that a few stumbled upon genotypes that were significantly better than the evolved wild type in a particular environment.

A couple of great examples are the rpl6b or atp11 deletion mutant strains. Strains evolved from either mutant did around 25% better than the evolved wild type strain in high salt, even though both of the original mutants did significantly worse than the wild type strain. By suffering a bad mutation, the evolved strain had been rerouted so that it now grew better than wild type. 

So it looks like getting a bad mutation may not be all bad after all. It might just give you that competitive edge you need when things change. Sometimes the best way to get from point A to point B is not a straight line. 

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

Categories: Research Spotlight

Tags: evolution , Saccharomyces cerevisiae

If Yeast Can’t Stand the Heat, Our World May Be in Trouble

October 23, 2014

In case there were still any doubters, the world is definitely heating up. April to September 2014 were the warmest these months have ever been since we started keeping records in 1880. And about 35,000 walruses were forced ashore this summer in Alaska because there wasn’t enough room left for them on the sea ice where they normally hang out. Unfortunately, the trend looks to be more record breaking heat for as long as we can see in the future.

Like the X-men, mutated yeast might one day be the superheroes that save us from the worst effects of global warming. Image by Gage Skidmore via Wikimedia Commons

This is bad news for us, and even worse news for nature.  But all is not lost! Our hero yeast may be able to swoop in and make us biofuels to replace gasoline.  This won’t stop global warming, but it might at the very least slow it down.

But yeast in its current form probably couldn’t make enough biofuels to make a difference without using too much precious food in the process. Like the X-men, it will need a helpful mutation or two to increase its efficiency to the point where it can make a real dent in slowing down the relentless rise in global temperatures. And ironically, one new trait yeast needs to pull this off is to be able to survive better in the heat.

To efficiently turn the parts of the plants we can’t use (mostly anything to do with cellulose) into biofuels, we need for yeast to grow in cultures where enzymes are chopping cellulose into bite sized pieces at the same time.  These enzymes work best at temperatures that are a real struggle for wild type yeast.

In a new study in Science, Caspeta and coworkers isolated mutants better able to tolerate high temperatures by simply growing Saccharomyces cerevisiae at around 39.5° C for over 300 generations.  They did this in triplicate and ended up with strains that grew on average about 1.9 times faster than wild type yeast at these temperatures.

The next step was to use genome sequencing and whole-genome transcription profiling to figure out why these mutant strains were heat tolerant. This is where things got really interesting.

They found many genes that were affected, but identified ERG3 as the most important player. All of the thermotolerant strains that they selected contained a nonsense mutation in ERG3. And in fact, when they introduced an erg3 nonsense mutation into wild type yeast, they found that this engineered strain grew 86% as well as the original mutant strain at high temperatures. In other words, most of the heat tolerance of these strains came from mutations in the ERG3 gene.

This makes sense, as membrane fluidity is a key factor in dealing with higher temperatures, and ERG3 codes for a C-5 sterol desaturase important for membrane composition.  When Caspeta and coworkers looked at the membranes of the mutant strains, they found a buildup of the “bended” sterol fecosterol.  Since “bended” sterols have been shown to protect the membranes of plants and Archaea from temperature swings, this could be the reason that the mutant strains dealt with the heat so well.

So as we might predict, changing the composition of the cell membrane affects how the yeast respond to temperature. What we couldn’t predict is that in order to get to this point via artificial selection, the yeast had to have a second mutation in either the ATP2 or the ATP3 genes that actually make yeast less able to grow at higher temperatures.

Because ATP2 and ATP3 are needed for yeast to grow on nonfermentable carbon sources, the authors hypothesized that perhaps thermotolerance could not evolve while oxidative respiration worked at full speed. Consistent with this, Caspeta and coworkers found that their evolved thermotolerant strains were more susceptible to oxidative stress and could not grow on nonfermentable carbon sources. This was not true of the strain they engineered to only have the mutation in the ERG3 gene—it grew well at 40° C and could use nonfermentable carbon sources.

So not only have these authors found a mutant yeast with the superpower of growing at higher temperatures, but they also showed that sometimes engineering works better than natural selection at creating the mutant they want. Evolving sometimes requires passing through an intermediate that makes the final product less useful than it could have been. In this case it worked best to use a combination of artificial selection and engineering to build a better mutant.   

Even this super engineered yeast mutant won’t stop global warming in its tracks, but it might help us to slow it down enough so that natural systems have a chance to adapt. A superhero indeed… 

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

Categories: Research Spotlight

Tags: evolution , Saccharomyces cerevisiae , biofuel , thermotolerance

RNase P, Unmasked

August 28, 2014

No matter how fancy, all masks hide the identity of a wearer. And no matter how fancy an RNase P is, all it likely does is trim tRNA precursors. Images from Wikimedia Commons

Masks for a masquerade party come in a dazzling array of shapes and sizes.  And yet they all pretty much serve the same purpose — they hide the identity of the wearer.

Biology sometimes has its own dazzling array of cellular machines all doing the same thing.  One of the best examples of this is RNase P.   This enzyme trims tRNA precursors into mature tRNAs and has pretty much been around in one form or another since there were cells.  And yet, despite this common heritage and its one apparent job, it seems that no two are exactly alike.

In bacteria, RNase P is a piece of RNA that serves as the enzymatic component, complexed with a single protein.  Most Archaea and eukaryotes kept the RNA and added a varying number of protein subunits to make some wildly complex enzymes.  But in a few eukaryotes, the RNA has been dropped completely and a single protein substituted to provide the enzymatic activity. 

A new study out in PLOS Biology by Weber and coworkers shows that, despite this structural diversity, all the different forms of RNase P pretty much do the same thing.  Just like someone can hide who they are with any old mask, a cell can trim its tRNA precursors with any old RNase P.  Well, at least the simple RNase P of Arabidopsis thaliana, comprised of a single enzymatic protein subunit, can replace the enzymatic RNA and at least one protein subunit from the much more complex RNase P of our friend Saccharomyces cerevisiae!

This suggests that evolution has done something weird here.  It took what most likely started out as an RNA enzyme and made various changes to it over time.  Despite these changes, the enzyme kept doing the same thing: trimming tRNA precursors.  It is as if the enzyme went through a bewildering set of evolutionary changes and ended up at nearly the same place doing the same thing.   

How did Weber and coworkers arrive at this startling finding? Yeast RNase P consists of nine protein subunits and an RNA component that comes from the RPR1 gene.  The first thing Weber and coworkers did was to show that the lethal phenotype of a rpr1 knockout could be rescued by the single-subunit RNase P from either the plant Arabidopsis thaliana or the trypanosome Trypanosoma brucei.  The RNase P in these beasts consists of only a single polypeptide.

The authors next integrated the RNase P gene of A. thaliana into the genome of a yeast cell lacking both RPR1 and one of the protein subunits of RNase P, Rpr2p, and put it through a set of rigorous tests.  To their surprise, they found that this strain does a perfectly fine job of processing tRNA precursors.  There was no buildup of intermediates and, if anything, the A. thaliana RNase P proved to be a bit more efficient at trimming these tRNA precursors.

Of course just because the simpler RNase P can substitute for the RNA subunit of the more complex RNase P, that does not mean the two do the exact same thing.  It could be that the more complex form of RNase P has a broader set of functions, but that the only function absolutely required for life is the trimming of tRNA precursors.  But this does not appear to be the case.

Previous research showing that unprocessed forms of other RNAs accumulate at the restrictive temperature in an rpr1-ts mutant had suggested that yeast RNase P also processes a number of other RNAs besides tRNAs.  Since Weber and coworkers didn’t see these unprocessed forms accumulating in their strain, either the simple A. thaliana RNase P was able to process those other RNAs, or they’re actually not RNase P substrates. 

By analyzing the phenotypes of several different RNase P mutants, they showed that the other RNAs aren’t RNase P substrates; apparently their accumulation in the rpr1-ts mutant is an indirect effect.  All in all, these results show that the added complexity of yeast RNase P did not arise so that the enzyme could also process these other RNAs.

The authors next set out to see if there was any subtle difference between the two strains.  In other words, does replacing the RNA component of yeast RNase P with the catalytic protein subunit from A. thaliana have any effect on the yeast whatsoever? 

Weber and coworkers tested this by comparing the growth of the two strains under a wide range of conditions.  They saw no significant effects in any of the over 30 conditions tested.  If the yeast RNase P has any added features over the A. thaliana one, they are very, very subtle.

Pushing to see if they could find any differences, they even set the two up in direct competition to see which was the best suited for survival.  They did this by adding GFP to one or the other strain so that they could follow it, putting the two strains together, and growing them for many generations to see if one routinely outcompeted the other.  Neither did…it was a draw.  There appears to be no advantage to having the yeast RNase P despite its complexity!

This is weird.  It is almost like round trip evolution.  RNase P starts out as a single RNA that processes tRNA precursors.  Then as it moves around the tree of life, it picks up various bells and whistles and occasionally is even replaced by a protein.  And yet in the end, all RNase P’s are strangely equivalent.  As if all of that evolving was for naught!

Obviously there are still plenty of unanswered questions.  Why did yeast build up this complexity if there is seemingly no advantage?  And is the protein subunit superior to the RNA subunit?  If so, this last question would at least explain why a few beasts evolved away from the RNA catalytic subunit to the protein one – but still wouldn’t answer why all those proteins are glomming onto the perfectly adequate RNA that probably predates proteins.  More studies in yeast may help us “unmask” the answer to this fundamental question.

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

Categories: Research Spotlight

Tags: evolution , Saccharomyces cerevisiae , RNase P , tRNA processing

Slipping Through Haldane’s Sieve

May 22, 2014

Just as harsh panning can uncover hidden gold nuggets, so too can loss of heterozygosity reveal beneficial new recessive mutations. Image via Wikimedia Commons

Imagine you are panning for gold in a river and there are two kinds of nuggets.  One type is naked gold while the other is gold hidden inside of normal rock.  Pretty easy to figure out which nuggets you’ll gather first!

Now imagine instead that the process of panning is a rough one that knocks the shell off of the second type of nugget revealing the gold inside.  Now there won’t be any difference between the two.  You will be just as likely to keep both types of nuggets.

The same sort of situation applies to new beneficial mutations in a changing environment.  Back in 1927, J. B. S. Haldane predicted that the more dominant a mutation, the more likely it was to help a diploid beast adapt to a new environment.  The naked gold was more likely to be taken over the covered gold.

Gerstein and coworkers show in a new study that at least in the yeast Saccharomyces cerevisiae, Haldane’s sieve (as it is called) may not always apply.  The process of adapting to a new environment can strip away the dominant older allele, revealing the recessive one.  Loss of heterozygosity (LOH) uncovers the hidden gold of the recessive phenotype.

The authors had previously identified haploid mutants that were able to survive in the presence of the fungicide nystatin.  They mated these mutants to create either heterozygotes or homozygous recessive mutants and compared these to wild-type diploids growing either in the presence or absence of nystatin. 

Gerstein and coworkers found a wide range of effects of these mutations in the absence of nystatin.  Sometimes heterozygotes grew better than either homozygote, sometimes homozygous recessive strains did best, and sometimes wild type grew best.   Phenotypes were all over the map.

The story was very different in the presence of nystatin where only the homozygous recessives managed to grow.  This appears to contradict Haldane’s sieve.  Here there were no dominant mutations that allowed for survival.

Gerstein and coworkers found that some heterozygote replicates started to grow after a prolonged lag period.  A closer look at the heterozygotes that grew showed that they had lost the dominant allele so that they could now show the recessive phenotype and survive.  LOH had broken Haldane’s sieve. 

The authors found that the lower the nystatin levels, the more likely a population was to break through Haldane’s sieve.  They postulate that the populations survive longer at lower levels of nystatin, which increases the chances that a LOH will happen.  It is a race between survival and eliminating the dominant allele that keeps them from growing. 

The next step was to determine if LOH was common enough that populations with a small percentage of heterozygotes could survive.  They found that even in populations where only 2% were heterozygotes, around 5% of the 96 replicate populations managed to lose an allele and grow.  So even at low levels, a recessive mutation can give a population the advantage it needs to adapt and survive.

Combining the awesome power of yeast genetics with cheap sequencing is allowing scientists to test fundamental models of genetics that will unearth how populations adapt and survive in new environments.  We are finding those nuggets of scientific knowledge that have remained hidden.

Now of course, not every diploid is as numerous or as genetically flexible as yeast.  Cows, chickens, lizards, and people may all still be slaves to Haldane’s sieve.  We will need more studies to see if our recessive treasures can be uncovered in time to save us. 

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

Categories: Research Spotlight

Tags: evolution , Saccharomyces cerevisiae , antifungal resistance

How Yeast Populations Make the Cut

April 15, 2014

Kids like these can overcome some physical limitations with lots of hard work and practice. But yeast needs to stumble upon the right mutations to win out over its peers. Image from the U.S. Navy via Wikimedia Commons

Imagine that your dream is to be a professional basketball player.  Unfortunately for you, you are only five feet six inches tall and you can’t jump very high.  No matter how much you practice and work out, it is exceedingly unlikely you will be a starter for the Miami Heat.

Now imagine instead that you are six feet tall with a reasonable vertical jump.  Here, with enough effort you have a shot at beating out the guy with the genetic advantage of being six foot six inches high who doesn’t work as hard as you do.  Keep practicing and you might be passing the ball to LeBron James instead of him! 

In a new study in GENETICS, Frenkel and coworkers show that something similar can happen in yeast too.  If a population of yeast has some overwhelming advantage over a second population, the first will quickly outcompete the second every time.  But if the first population is just a bit better than the second, then the second can sometimes end up with a mutation that gives it an even better advantage than the first.  Now the first population is outcompeted and the second takes over.

Of course, when presented in a general way this is sort of obvious.  But Frenkel and coworkers set up their experiments in such a way that they got some hard numbers for just how much of an advantage one population needs to overcome to have a chance at winning.  If six feet is tall enough, what about five feet eleven inches?

The first step was to generate a number of mutants with different measured fitness advantages.  They selected mutant populations with advantages of 3, 4, 5, or 7%.  These populations were all tagged with a fluorescent marker.

They then seeded these mutants individually into 658 replicate reference populations that were tagged with a different fluorescent marker.  The mutants were seeded at a high enough level to prevent genetic drift from wiping them out.  The authors then followed each population for hundreds of generations by determining the levels of each population every 50 or so generations.

Their first finding was that mutants with a 7% advantage won out every time.  The reference population had no chance at getting a good enough mutation to beat it out.  No one is going to beat LeBron James out for his starting position with the Miami Heat.

Once the advantage was only 5%, around 16% of the time the second population won out.  As the advantage got smaller and smaller, the second population won out more and more often.   Even a genetically less gifted player has a shot at beating out the 12th guy on the Heat’s roster!

These results can tell us quite a bit about the mutational landscape of haploid Saccharomyces cerevisiae.  For example, from these data Frenkel and coworkers figured out that only populations that get mutations that give at least a 2% advantage have a chance at outcompeting other populations.  By assuming a mutation rate of 4X10-3, around 1 in 1000 mutations fit this bill, which might seem surprisingly high but is consistent with previous studies.  With a bit more hand waving, the authors hypothesize that disruption of something like 1 in 100 yeast genes is actually beneficial!

So yeast have a surprisingly level playing field.  Unless they are up against the equivalent of Kobe Bryant or Michael Jordan, they have a good shot at stumbling on a mutation that gives them an edge over their peers.    

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

Categories: Research Spotlight

Tags: evolution , Saccharomyces cerevisiae , population genetics , mutation

Gene Knockouts May Not Be So Clean After All

November 18, 2013

Imagine you have the instructions for building a car but you don’t know what any of the specific parts do.  In other words, you can build a working car but you don’t understand how it works.

If a cell were a car and you removed its radiator, it might adapt by evolving an air cooled system. If it happened soon enough, you might never figure out what the radiator did. Image by Joe Mazzola obtained from Wikimedia Commons.

One way to figure out how the car works would be to remove a part and see what happens.  You would then know what role that part played in getting a car to run.

So if you remove the steering wheel, you’d see that the thing runs into a wall.  That part must be for steering.  When you take out the radiator, the car overheats so that part must be for cooling the engine.  And so on.

Sounds like a silly way to figure out how the car works, but this is essentially one of the key ways we try to figure out how a cell works.  Instead of parts, we knock out genes and see what happens. A new study by Teng and coworkers is making us rethink this approach.

See, one of the big differences between a machine and a cell is that the cell can react and adapt to the loss of one of its parts.  And in fact, it not only can but it almost certainly will.

Each cell has gone through millions of years of evolution to adapt perfectly to its situation.  If you tweak that, the cell is going to adapt through mutation of other genes.  It is as if we remove the radiator from the car and it evolves an air cooling system like the one in old Volkswagen Bugs.

Teng and coworkers decided to investigate whether or not knocking out a gene causes an organism to adapt in a consistent way.  In other words, does removing a gene cause a selection pressure for the same subset of mutations that allows the organism to deal with the loss of the gene.  The yeast knockout (YKO) collection, which contains S. cerevisiae strains that individually have complete deletions of each nonessential gene, gave them the perfect opportunity to ask this question.

There have long been anecdotal reports of the YKO strains containing additional, secondary mutations, but the authors first needed to assess this systematically. They came up with an assay that could detect whether secondary mutations were occurring, and if so, whether separate isolates of any given YKO strain would adapt to the loss of that gene in a similar way.  The assay they developed had two steps.

The first step was to fish out individual substrains from a culture of yeast that started from a single cell in which a single gene had been knocked out.  This was simply done by plating the culture and picking six different, individual colonies.  Each colony would have started from a single cell in the original culture.

The second step involved coming up with a way to distinguish differently adapted substrains.  The first approach was to see how well each substrain responds to increasing temperatures.  To do this, they looked for differences in growth at gradually increasing temperatures using a thermocycler.

They randomly selected 250 YKO strains and found that 105 of them had at least one substrain that reproducibly responded differently from the other substrains in the assay.  In contrast, when they looked at 26 isolates of several different wild type strains, including the background strain for the YKO collection, there were no differences between them. This tells us that the variation they saw in the knockout substrains was due to the presence of the original knockout.

So this tells us that strains can pretty quickly develop mutations but it doesn’t tell us that they are necessarily adapting to the knocked out gene.  To see if parallel evolution was indeed taking place, the authors chose to look at forty strains in which the same gene was independently knocked out.  They found that 26 of these strains that had at least one substrain with the same phenotype, and fifteen of those had mutations that were in the same complementation group.  So these 15 strains had evolved in similar ways to adapt to the loss of the same gene.

Teng and coworkers designed a second assay independent of the original heat sensitivity assay and tested a variety of single knockout strains.  They obtained similar results that support the idea that knocking out a gene can lead cells to adapt in similar ways.  This is both good and bad news.

The bad news is that it makes interpreting knockout experiments a bit trickier.  Are we seeing the effect of knocking out the gene or the effect of the secondary mutations that resulted from the knockout?  Are we seeing the loss of the radiator in the car or the reshaping that resulted in air cooling?  We may need to revisit some earlier conclusions based on knockout phenotypes.

The good news is that not only does this help us to better understand and interpret the results from yeast and mouse (and any other model organism) knockout experiments, it also gives us an insight into evolution and maybe even into the parallel evolution that happens in cancer cells, where mutations frequently co-occur in specific pairs of genes.  And while we may never be able to predict if that knock you hear in your engine really needs that $1000 repair your mechanic says it does, we may one day be able to use results like these to predict which cells containing certain mutated genes will go on to cause cancer and which ones won’t.   

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

Categories: Research Spotlight

Tags: deletion collection , evolution , Saccharomyces cerevisiae

When One Spark Isn’t Enough

August 07, 2013

A single mutation, just like a single spark, is more likely to fizzle out.

As anyone who has ever tried to start a fire with flint knows, a single spark is rarely enough.  You need to get a bunch of sparks all working at once to end up with that roaring campfire.  And with the wrong kindling or wood, even lots of powerful sparks just can’t get it done.

A new study in the yeast S. cerevisiae by Lang and coworkers suggests that evolution may be similar.  A single helpful DNA change may not be enough to give an individual yeast that leg up it needs to spread through the population.  Turns out that more often than not it needs something like 5-7 mutations.   And again, even that may not be enough if the rest of its DNA isn’t up to par.  A set of powerful sparks on soggy wood still won’t light a fire.

Lang and coworkers followed 40 different yeast populations for a thousand generations as the yeast adapted to a new environment (rich medium).  They sequenced each population to 100-fold depth at 12 different time points.  Not only did this allow the researchers to watch mutations rise and fall over time, it also let them screen out sequencing errors.  Real mutations will correlate over time, sequencing errors won’t.

The key finding of their research was that mutations that increased in the population over time almost always came in bunches (or cohorts) and that not all the mutations were beneficial.  Neutral mutations invariably hitchhiked along with strongly beneficial ones.

A great example of this involves the ELO1 and GAS1 genes.  These two mutations arose together in a yeast population but when the researchers looked at each individually, only GAS1 was beneficial.  ELO1 appeared to go along for the ride.

Another key point of this study is that mutations do not happen in a vacuum…beneficial mutations only “catch” in the context of a good background.  This is clearly shown in one of the populations they followed. 

In this population, yeast with a mutation in the SPC3 gene began to spread through the population.  After about 300 generations, though, a second yeast with mutations in the WHI2 and ROT2 genes began to outcompete the SPC3 mutant.  If things stayed like this, the SPC3 mutation would disappear from the population even though it was obviously helpful.

What happened instead was that a yeast with the SPC3 mutation developed a useful mutation in the YUR1 gene.  This combination was strong enough for this yeast to stay in the game until one of them developed a third mutation in the WHI2 gene.  This triple threat proved too much for the yeast with the mutations in the WHI2 and ROT2 genes – they were driven to extinction.

No wonder people refer to evolution as a dynamic process!  This example shows just how tumultuous it actually is.  Even helpful mutations like the ones in ROT2 and WHI2 can disappear over time if they happen in a weaker background.  And presumably even potentially harmful mutations can spread if they hitchhike along with a cohort of strongly beneficial mutations.

These results not only shed light on how evolution works, but could also spark other discoveries on how cancer progresses, how bacteria become resistant to antibiotics, and how viruses deal with our immune system, just to name three.  And that could help kindle a brighter future.  

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

Categories: Research Spotlight

Tags: evolution , Saccharomyces cerevisiae

Flip-Flopping Yeasts

June 20, 2013

We all have certain things we can’t live without. But what’s essential to one person may be completely trivial to another. For example, a teenager who can’t live without his video games is fine without that antique tea set,  while the opposite may be true for grandma. 

Just like all of the pieces of this set are essential to grandma, so too are all of the proteins in an essential complex usually essential to yeast (and maybe to us too!).

In a new article in Genome Biology and Evolution, Ryan and coworkers find that the same holds true for different yeast species.  One gene that is essential in one yeast is dispensable in another.  And furthermore, they tend to be essential or nonessential in sets. Just like grandma’s tea set and the teenager’s games.

It’s been seen in S. cerevisiae that the genes that encode the proteins in a complex tend to be either mostly essential or mostly nonessential.  It is like the teapot and the cups and saucers all being essential to grandma or all being nonessential to the teenager.  This is called modular essentiality.

Ryan and coworkers found that if some protein complex is essential in one yeast, most or all of those genes will be essential in that species.  On the other hand, if that protein complex is dispensable in a different yeast, then most or all of those genes will be nonessential.  The genes encoding the entire complex flip together from essential to nonessential – again, just like the tea set.  It isn’t as if one of the tea cups happens to stay essential when the teenager gets ahold of the set! 

To do this work, Ryan and coworkers started out by looking at S. cerevisiae.  As all of us here at SGD know, this was an excellent choice!  But not just because we work on it…

Because the S. cerevisiae genome sequence has been available for quite a while, we know which genes are essential to life, which genes interact genetically, and which proteins interact physically with each other. We also have a very good list of the protein complexes that exist in yeast and what their subunits are.

Ryan and coworkers used updated data to confirm that modular essentiality exists in S. cerevisiae.  Most of the proteins in an essential complex tend to come from essential genes and most of the proteins in nonessential complexes come from nonessential genes.  There is very little overlap…the tea set does not often contain a video game!

Next the authors asked whether this modular essentiality is found in other species too. At the moment, Schizosaccharomyces pombe, or fission yeast, is the only other eukaryote with complete data on the essentiality of genes. Although it’s also a single-celled yeast, S. pombe is about as far away from S. cerevisiae as you can get and still be a yeast. The two are thought to have diverged as much as 400 million years ago.

Even though it is so different, S. pombe also shows modular essentiality. And using an incomplete set of data from knockout mice, the authors see a similar pattern! So it looks like modular essentiality is at least conserved across fungi, and may be universal.

Next they asked if complexes that have flipped from essential to nonessential over time still maintain their modular essentiality.  Do all the tea cups become nonessential, or just some of them? 

When grandma tidies up the video games, none of them will seem important to her. The same thing happens when a complex switches from essential to nonessential as a species evolves.

Most (83%) of the genes that are present as one-to-one orthologs in both yeasts are either essential in both or nonessential in both. Ryan and coworkers focused on the other 17%, where a gene was essential in one species but not in the other.

In the cases where essentiality is “flipped” between the species, whole protein complexes tend to flip as a unit. The subunits of a complex that is nonessential in budding yeast are mostly nonessential, while the subunits of the analogous complex in fission yeast are mostly essential.

An example of this is the large subunit of the mitochondrial ribosome. Mitochondrial translation is optional for S. cerevisiae, but obligatory for S. pombe. In keeping with this, almost all the proteins that make up the S. cerevisiae large mitochondrial ribosomal subunit are nonessential. In S. pombe, the situation is flipped.

So the essentiality of a complex mirrors the lifestyle of its owner, just like the teenager and his grandmother.  The two yeasts, with their different lifestyles, place different importance on the mitochondrial ribosome. This wasn’t a big surprise, since this lifestyle difference was already known. But other complexes that are flipped between the species may point to things that we don’t yet know about their physiology.

These results support the idea that modular essentiality is universal, which would mean that in various organisms we can expect that mutants in subunits of a complex will share the same phenotype, disease association, and drug sensitivity. Obviously there are important implications here for antifungal drug design or for disease treatment: if you want to stop a complex from working, any of its subunits (or perhaps several at the same time) might prove to be good targets.

But another bigger point is how much we can learn from a deep understanding of an organism’s genome.  By teasing apart what is essential and what isn’t we can learn a lot about the beast we’re studying.  And someday, maybe a lot about ourselves.

by Maria Costanzo, Ph.D., Senior Biocurator, SGD

Categories: Research Spotlight

Tags: evolution , Saccharomyces cerevisiae , essentiality

Codependent Genes

May 16, 2013

When a gene is duplicated, one copy usually dies. It is battered by harmful mutations until it eventually just fades into background DNA.

Genes can be codependent too. Sometimes this is what keeps a duplicated gene alive.

But this isn’t the fate of all duplicated genes.  Sometimes they can survive by gaining new, useful functions.  The genes responsible for snake venom proteins are a great example of this.

Another way for a duplicated gene to live on is when both copies get different mutations that confer different functions, so that a cell needs both to survive.  Two examples of this type of codependent gene survival are highlighted in a new study by Marshall and coworkers.  They compared various fungal species and identified cases where two functions were carried out by either one gene or by two separate genes.  Surprisingly, these cases involve alternative mRNA splicing, which is a rare process in fungi.

The first gene pair they focused on was SKI7 and HBS1 from Saccharomyces cerevisiae.  In this yeast these two genes exist as separate entities, but in other yeasts like Lachancea kluyveri they exist as a single gene which the authors have called SKI7/HBS1

The SKI7/HBS1 gene makes two differently spliced mRNAs, each of which encodes a protein that matches up with either Ski7p or Hbs1p.  In addition, the SKI7/HBS1 gene can rescue a S. cerevisiae strain missing either or both the SKI7 and HBS1 genes.  Taken together, this is compelling evidence that SKI7 and HBS1 existed as a single gene in the ancestor of these two fungal species. In S. cerevisiae, after this gene was duplicated each copy lost the ability to produce one spliced form.

The second gene Marshall and coworkers looked at experienced the reverse situation during evolution.  PTC7 exists as a single gene that makes two mRNA isoforms in S. cerevisiae: an unspliced form that generates a nuclear-localized protein, and a spliced form that produces a mitochondrial protein. 

But in Tetrapisispara blattae, these two forms exist as separate genes.  The PTC7a gene is similar to the unspliced form in S. cerevisiae and the protein ends up in the nucleus, while the PTC7b gene is similar to the spliced S. cerevisiae version and its product is mitochondrial.  

Because an ancestor of S. cerevisiae had every one of its genes duplicated about 100 million years ago, yeasts have been a great system to study the fate of duplicated genes.  This study shows that even though gene duplication is widespread in fungi and alternative splicing is rare, these mechanisms are actually interrelated and each can increase the diversity of the proteins produced by a species.

Fun fact: 544 genes survived duplication in S. cerevisiae.  That is around 10%.

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

Categories: Research Spotlight

Tags: alternative splicing , Saccharomyces cerevisiae , evolution , gene duplication

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