New & Noteworthy

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

Getting the Big Picture from 100 Genomes

May 20, 2015


Like the Peruvian Hairless dog, in some ways the S288C genome looks quite different from other members of its species. Image via Wikimedia Commons

Imagine if aliens visited the earth to learn about dogs, but they stumbled upon a colony of the very rare Peruvian Hairless. Taking a sample for DNA analysis, they would retreat to their home planet, do their studies, and conclude that all dogs had smooth, mottled skin and a stiff mohawk—as well as whatever crazy mutations the Peruvian Hairless happens to carry. 

Until recently, S. cerevisiae researchers have been a bit like those aliens. The genomic sequence of the reference strain S288C was completed in 1996, and for a long time it was the only sequence available. Scientists knew a lot about the S288C genome, but they didn’t have any perspective on the species as a whole.

In the past few years, genomic sequences have become available from a handful of other strains. But now, as described in a new paper in Genome Research, Strope and colleagues have determined the genomic sequences of 93 additional S. cerevisiae strains to make the number an even hundred.

This collection of strains and sequences has already provided new insights into yeast phenotypic and genotypic variation, and represents an incredible resource for future studies. And the comparison with this collection of other strains suggests that in some ways, S288C may be just as unusual as the Peruvian Hairless.

This collection of strains and their sequences gave the researchers a much broader perspective across the whole S. cerevisiae species. It’s as if the aliens discovered Golden Retrievers, Great Danes, Chihuahuas, and more. We only have space here to touch upon a few of the highlights.

First off, they confirmed what many yeast researchers have suspected for a while—S288C is a bit odd.  We already knew that a S288C carries polymorphisms in several genes that affect its phenotype. For example, the MIP1 gene in S288C encodes a mitochondrial DNA polymerase that is less efficient than in other strains, making its mitochondrial genome less stable.

Back when fewer strain sequences were available, it wasn’t clear whether the S288C polymorphisms in other genes like MKT1, SSD1, MIP1, AMN1, FLO8, HAP1, BUL2, and SAL1 were the exception or the rule. Now that Strope and colleagues had 100 genomes in hand, they could see that these differences are indeed peculiar to S288C and its close relative W303.  They might have arisen because of the long genetic isolation of the strains, or because of special selective pressures they faced during growth in the lab.

They also found a lot of variation in how often S. cerevisiae strains have acquired whole chromosomal regions from other Saccharomyces species. This process, known as introgression, happens when related species mate to form hybrids. Stretches of DNA that are transferred in this way are recognizable because gene order is preserved, but all the genes they contain are highly diverged.

The researchers found 141 of these regions containing 401 genes. Many showed similarity to S. paradoxus, which is known to hybridize with S. cerevisiae, but others apparently came from unknown, as yet un-sequenced Saccharomyces species. In a couple of cases that the authors looked at closely, the introgressed genes had slightly different functions from their native S. cerevisiae counterparts.

Another notable finding by Strope and colleagues concerned some genes that exist in multiple copies. The ENA genes, encoding an ATP-dependent sodium pump, are present in 3 copies in S288C (ENA1ENA2, and ENA5), while the CUP1-1 and CUP1-2 genes, encoding metallothionein that binds to copper and mediates copper resistance, are present in 10-15 copies.

To get perspective on a whole species, you need to look at lots of different examples. Image by Sue Clark via Flickr

The sequence coverage in these regions relative to their flanking regions allowed the researchers to see exactly how many repeats are present in each strain. All had between 1-14 copies of ENA genes and 1-18 copies of CUP genes. Interestingly, the strains of clinical origin had significantly higher copy numbers of CUP genes than the non-clinical strains, suggesting that copper resistance is an important trait for virulence.

So, instead of being confined to the S288C genome, S. cerevisiae researchers can now get a much fuller idea of the range of genetic and phenotypic variation within the species. The strains (available at the Fungal Genetic Stock Center), along with their genome sequences (available in GenBank), are an amazing resource for classical and quantitative genetics and comparative genomics.

Unlike those aliens, we won’t end up thinking of yeast as a mostly bald dog with a mohawk. No, we will have a fuller picture of S. cerevisiae strains in all their glory.

A few technical details

In selecting the strains to sequence, Strope and colleagues chose from a wide variety of yeast cultures isolated from the environment and from hospital patients with opportunistic S. cerevisiae infections. But they faced a problem: many of the cultures had irregular numbers of chromosomes or genome rearrangements, which would complicate both interpretation of the sequence data and any future genetic analysis.

To avoid this problem, the researchers selected only strains that were able to sporulate and produce four viable spores—showing that their genomes weren’t messed up. They also wanted strains with no auxotrophies (nutritional requirements), since these can negatively affect growth and complicate the comparison of phenotypes. In some cases, they corrected specific mutations in the strains to increase their fitness.

They ended up with 93 homozygous diploid strains to sequence. Producing paired-end reads of 101 bp, they generated genome assemblies that had 22- to 650-fold coverage per strain.

Because the sequence reads were relatively short, they didn’t provide enough information to assemble the sequence across repetitive regions. So Strope and colleagues used a genetic method to determine gene order. They crossed haploid derivatives of the strains to the reference strain S288C; if their genomes were not colinear with that of S288C, then some of the resulting spores would be inviable.

This analysis showed that 79 of the strains had chromosomes colinear to those of S288C, and allowed assembly of their genomes across multicopy sequences. The remaining strains had chromosomal translocations relative to S288C. Twelve of these carried the same reciprocal translocation between chromosomes 8 and 16.

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

If it Swims Like a Duck…

May 6, 2015


It may swim like a duck, but this beast is obviously not a duck. Just like the glycine patch of Pxr1 looks like an interaction region when it isn’t. Image by Yotujonoo via Creative Commons

Back in the 1980’s some U.S. politicians were proposing to raise money by something they called “revenue enhancements”. Richard Darman, the budget director at the time, correctly pointed out that a revenue enhancement really is just a tax increase by another name. 

To make his point, he used the expression, “If it looks like a duck and quacks like a duck, it’s a duck.” In other words, just because politicians call it something else, if a revenue enhancement does everything a tax increase does then it really is just a tax increase.

This same reasoning is often used in biology. If two regions of a protein look the same (are homologous) and the proteins do similar things, then the two similar regions do the same thing. Except, of course, when they don’t.

This probably isn’t what Conan O’Brien had in mind when he changed the famous expression a bit to say, “If it looks like a duck and quacks like a duck, it’s a little person dressed as a duck,” but as is often the case with Team Coco, he was right in both biology and life. Not everything that looks and quacks like a duck is a duck, and not every homologous region in proteins that do similar things does the same thing.

Conan’s point is borne out in a new study out in GENETICS where Banerjee and coworkers show that even though the yeast Prp43 RNA helicase shares glycine patches with three of the proteins with which it interacts, this doesn’t mean the glycine patches are used the same way in each case. They may all look and act like ducks, but they are not all ducks! 

Glycine patches are short, glycine-rich protein motifs that are thought to help proteins recognize other proteins or RNAs. Two of the proteins that the researchers looked at, Spp382 and Sqs1, have glycine patches that are only subtly different from that of Prp43. In both of these, the glycine patch is important for interacting with Prp43, but that isn’t its only role. The patches really are ducks in this case, just different kinds of ducks—maybe a mallard and a mandarin duck.

In the case of the third protein, Pxr1, the glycine patch seems to have a completely different (albeit important) role. In this case, it really is a little person in a duck costume!

Prp43 is involved in two different kinds of RNA processing in the yeast cell—pre-mRNA splicing and rRNA maturation. It is one of the few proteins shared between the two complexes involved in each process.

Previous work had shown that different factors in each complex are important for bringing Prp43 to each party. For rRNA maturation, Sqs1 and Pxr1 are the critical players, while for pre-mRNA splicing, Spp382 is key. Since all four proteins share little else beyond a shared weakly conserved, 45-50 amino acid glycine-rich patch, one idea was that all of these proteins use the patch to interact with one another. As is true of much in life, the real answer is a bit more complicated than that.

The first set of experiments was to determine how well Prp43 interacts with each of the other glycine patches, using yeast two-hybrid assays. With full length proteins, the authors found that Spp382 interacted most strongly with Prp43, Pxr1 was the weakest, and Sqs1 was intermediate. They got a similar order of interaction when using just the glycine patches of each of these three proteins, with one small difference: the Pxr1 glycine patch did no better than the empty vector control.

This last result suggested that the glycine patch of Pxr1 was insufficient on its own to interact with Prp43. This was confirmed when they found no difference in the interaction of full length Pxr1 and Pxr1 deleted for the glycine patch.

The Pxr1 glycine patch apparently plays no role in interacting with Prp43—it really isn’t a duck at all. But that doesn’t mean it is dispensable! They showed later that it is critical for snoRNA processing, an important step needed for rRNA maturation.

Mandarins and mallards look like ducks and quack like ducks…and they are ducks. Like these ducks, the glycine patches of Spp82 and Sqs1 look and act like interaction regions, and in fact they are interaction regions. Image via Wikimedia Commons

Of course, sometimes if it looks and quacks like a duck, it is indeed a duck. This was the case for Sqs1 and Spp382.

As shown by two-hybrid and glycine patch swap assays, each of these glycine patches do seem to be important for interacting with Prp43. But each patch was more than just a way for two proteins to hook up.

To show this, Banerjee and coworkers looked for chimeras of Spp382, Pxr1, and Sqs1 that could rescue the lethal phenotype of a Spp382 deletion. First off, they showed that deleting the glycine patch from Spp382 was equivalent to deleting the whole protein—it was a lethal event. And as expected, replacing the Spp382 glycine patch with the one from Pxr1 was still lethal. But the Sqs1 glycine patch was able to rescue the deletion strain although it grew more slowly. So the Spp382 and Sqs1 glycine patches could to some extent substitute for one another.

One way to interpret the difference in growth rates is that it has to do with the fact that the glycine patch of Spp382 bound more strongly to Prp43 than did the one from Sqs1. The glycine patch from Sqs1 can’t fully rescue the Spp382 deletion strain because it is a weaker binder. But a set of mutagenesis experiments suggests that this is not the case.

The authors basically took the Spp382/Pxr1 chimera in which the Pxr1 glycine patch replaced the one from Spp382 and made a series of point mutations that slowly converted the glycine patch back to the one from Spp382. What they found was that the strength of interaction in the two-hybrid assay does not correlate with the level of rescue in the complementation assay. One interpretation is that the Spp382 glycine patch is doing more than recruiting Prp43.

Taken together, these results are a bit of a biological cautionary tale. Just because a protein region looks like another one, do not assume they are doing the same thing. Sometimes what looks and acts like a duck is just a man dressed as a duck.

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

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

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