New & Noteworthy

Insight into Evolution of Paralog Doublets

July 29, 2022

Sequencing of Saccharomyces cerevisiae in the 1990s revealed blocks of duplicated genes, suggesting an ancient whole-genome duplication. The original duplication has been postulated as a means to restore fertility to an interspecies hybrid of ancestral yeast parents; however, the question remains as to why so many paralog doublets have been retained through eons of evolution. Are these paralogs the residual clutter that has yet to be selected away, or has it represented an opportunity that created positive selection? Said another way, if every gene once had a twin, why have some been retained and others lost?

To dig further into understanding doublet evolution, a study by Purkanti and Thattai in a recent issue of Scientific Reports looks at the functional significance of these yeast paralog doublets from an evolutionary perspective. To ask whether retention of doublets across sub-clades of the yeast phylogenic tree might suggest selective advantage, they looked at which Gene Ontology biological processes showed the greatest enrichment of observed over expected for the 887 genes with conserved doublets. The greatest enrichment for over-represented doublets was in the GO process of endocytosis, with its parent term of vesicle-mediated transport also showing significant enrichment.   

The authors then assigned vesicle-traffic genes to function-specific and pathway-specific modules to ask about doublet retention. Among the modules, the greatest enrichment of doublets was observed for coat/adaptor genes and lipid control genes. Other modules, such as the endosomal sorting complex required for transport (ESCRT), had no doublets at all, i.e., the genes in this module have completely reverted to singletons.

Intriguingly, the classes with singletons and the classes with doublets can be broadly grouped, where doublets tend to be retained in secretory and early endocytic pathways, whereas singletons largely act in retrograde Golgi traffic and late endocytic steps.

The authors separated all 360 ancestral vesicle traffic doublets into two groups: those that are retained as doublets in present-day species and those that are not. They looked at the nucleotide sequence identity as a proxy for evolutionary rate and found that retention of doublets is strongly associated with lower evolutionary rates. From this they conclude that doublet retention is under significant selection, as the purifying selection that lowers evolution rates is well correlated with functional significance.

While it may be mere coincidence that budding yeast happened to undergo ancestral genome duplication, it appears that this event has proven fortuitous for making S. cerevisiae even more useful as a model. As revealed in this study, the ease of manipulation and the vast knowledge base in yeast makes it possible to ask these deeply important questions about evolutionary impact on paralog doublets.  

Categories: Research Spotlight

Tags: evolution, paralog doublets, paralogous genes, Saccharomyces cerevisiae, vesicle traffic, whole genome duplication

Out of China: Changing our Views on the Origins of Budding Yeast

April 17, 2018

1,011. That’s the number of different Saccharomyces cerevisiae yeast strains that were whole-genome sequenced and phenotyped by a team of researchers jointly led by Joseph Schacherer and Gianni Liti, published this week in Nature (Peter et al., 2018; data at: http://bit.ly/1011genomes-DataAtSGD).

Scrupulously gathering isolates of S. cerevisiae from as many diverse geographical locations and ecological niches as possible, the authors and their collaborators plucked yeast cells not only from the familiar wine, beer and bread sources, but also from rotting bananas, sea water, human blood, sewage, termite mounds, and more. The authors then surveyed the evolutionary relationships among the strains to describe the worldwide population distribution of this species and deduce its historical spread.

They found that the greatest amount of genome sequence diversity existed among the S. cerevisiae strains collected from Taiwan, mainland China, and other regions of East Asia. This means that in all likelihood the geographic origin of S. cerevisiae lies somewhere in East Asia. According to the authors, our budding yeast friend began spreading around the globe about 15,000 years ago, undergoing several independent domestication events during its worldwide journey. For example, it turns out that wine yeast and sake yeast were domesticated from different ancestors, thousands of years apart from each other. Whereas genomic markers of domestication appeared about 4,000 years ago in sake yeast, such markers appeared in wine yeast only 1,500 years ago.

Additionally — and similar to the situation where human interspecific hybridization with Neanderthals occurred only after humans migrated out of Africa — it appears that S. cerevisiae has inter-bred very frequently with other Saccharomyces species, especially S. paradoxus, but that most of these interspecific hybridization events occurred after the out-of-China dispersal.

There are many more gems to be found among the treasure trove of information in this paper. Some notable conclusions from the authors include: diploids are the most fit ploidy; copy number variation (CNV) is the most prevalent type of variation; most single nucleotide polymorphisms (SNPs) are very rare alleles in the population; extensive loss of heterozygosity is observed among many strains. There are also phenotype results (fitness values) for 971 strains across 36 different growth conditions.

As is often the case for yeast, the ability to sequence and analyze whole genomes at very deep coverage has yielded broad insights on eukaryotic genome evolution. The team’s work highlights this by presenting a comprehensive view of genome evolution on many different levels (e.g., differences in ploidy, aneuploidy, genetic variants, hybridization, and introgressions) that is difficult to obtain at the same scale and accuracy for other eukaryotic organisms.

SGD is happy to announce that in conjunction with the authors and publishers, we are hosting the datasets from the paper at this SGD download site. These datasets include: the actual genome sequences of the 1,011 isolates; the list of 4,940 common “core” ORFs plus 2,856 ORFs that are variable within the population (together these make up the “pangenome”); copy number variation (CNV) data; phenotyping data for 36 conditions; SNPs and indels relative to the S288C genome; and much more. We hope that the easy availability of these large datasets will be useful to many yeast (and non-yeast) researchers, and as the authors say, will help to “guide future population genomics and genotype–phenotype studies in this classic model system.”

Categories: Announcements, New Data

Tags: evolution, genome wide association study, Saccharomyces cerevisiae, strains

Making the Best of a Sticky Situation

June 05, 2017

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 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.

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

Yeast Tackles Climate Change

November 29, 2016

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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.

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

Ancient Hybridization Causes Revision of Yeast’s Calendar

August 19, 2015

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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:

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

Yeast are People Too

June 03, 2015

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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.

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.

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

The Failed Hook Up: A Sitcom Starring S. cerevisiae

May 27, 2015

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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.

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

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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: biofuel, evolution, Saccharomyces cerevisiae

Once You Start Looking, Shu2 Homologs Are Everywhere

April 09, 2015

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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: evolution, homologous recombination, model organism, Saccharomyces cerevisiae

Conformity Preferred in Yeast

March 25, 2015

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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.

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, promoter, Saccharomyces cerevisiae

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