New & Noteworthy

The Dark Yeast Rises

April 04, 2017

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Sometimes in life you need to take risks to survive and prosper. Maybe you need to take a leap of faith like Bruce Wayne does to escape that pit in The Dark Knight Rises. Or you need to try a risky business strategy to put your company out in front.

While these are critical things to do at the time, chronic risk-taking is not usually a good idea. Even Bruce Wayne retires to Florence at the end of The Dark Knight Rises, his risky life choices done now that he has saved his beloved Gotham City. He can now settle down with Selina Kyle (Catwoman) and live happily (and safely) ever after.

Something similar can happen in the budding yeast, Saccharomyces cerevisiae. In the right environment, certain yeasts can build up mutations like mad, hoping to hit on one that lets them make it out of that pit.

But then, over time, yeasts with the successful mutation lose that frenetic mutation rate—they take fewer risks with their DNA. One way they can do this is by ending up with a mutation that suppresses the high mutation rate. This is like Bruce Wayne retiring to a nice villa on the Arno.

Another way they can reestablish their old mutation rate is to mate with a nonmutator, a yeast strain that does not risk its DNA with a high mutation rate. Now, the next generations have the beneficial mutation and a lowered mutation rate as well. It is as if Bruce Wayne settled down with an accountant instead of Catwoman and had risk-averse children to carry on the Wayne name.

Bui and coworkers investigate this phenomenon in yeast in a new study out in GENETICS. They show that natural isolates exist that can sporulate into one of these mutator strains. And that at least one strain predicted to have a high mutation rate has a number of suppressor mutations that tamp down that higher rate.

Previous work had shown that a high mutation rate can happen with mutations in two genes in the mismatch repair (MMR) pathway, MLH1 and PMS1. This was discovered when researchers saw that some of the haploid strains from a mating of two laboratory strains, S288C and SK1, showed this mutator phenotype. A closer look revealed that S288C has a mutation in MLH1, and SK1 has a mutation in PMS1.

Bui and coworkers show that these mutator haploid strains outcompete other strains in high salt media. The strain hits upon mutations that allowed for better survival in high salt faster than its slower mutating brethren. But this advantage does not last.

After 120 generations these mutator strains start to lag behind strains with more typical mutation rates. Their DNA becomes as beat up as Bruce Wayne’s body by the third movie.

The researchers next looked at the MLH1 and PMS1 genes of 1,010 natural isolates of S. cerevisiae to see if there were any that might be able to sporulate into a mutator strain. Or that were already mutator strains themselves.

Since the mutations that lead to the mutator phenotype are recessive, strains that are heterozygotes for mutations in both genes should be able to form haploid spores with the higher mutation rate. They found 18 that fit the bill.

They also found one strain, YJM523, that was homozygous for both mutations and so would be predicted to have a high mutation rate. They further investigated this strain.

The first thing they did was to test the YJM523 mutant genes in an S288C background. They found that these two mutant genes did indeed give S288C a mutator phenotype. In fact, it had a higher mutation rate than the original genes from S288C and SK1 did, suggesting there were other mutations in one or both YJM523 genes that further increased the mutation rate.

These researchers next wanted to determine if YJM523 had a higher mutation rate or not, but needed to first develop a new assay that could be used in natural isolates. They came up with a reporter system that is not dependent on the typical markers used in yeast experiments, but instead relies on two antibiotic markers.

The reporter uses the NatMX antibiotic resistance marker for selection and the KanMX marker to identify revertant mutants. By scoring the number of colonies resistant to both antibiotics, they could determine the mutation rate.

When the researchers did this experiment, they found that YJM523 did not have a particularly high mutation rate. Sporulation experiments showed that this was most likely due to multiple suppressor mutations.

So out in the wild, the potential for mutator strains exists. And a close look at the genome of YJM523 showed that if it did have a mutator phenotype, it was for a short time. This yeast at least quickly lost its high mutation rate (assuming it ever had one).

This study helps to explain how yeast can obtain that mutator phenotype that researchers have seen before. And how yeast can escape that mutation pit to get back to the safety of a reasonable DNA repair pathway. To quote the prisoners in The Dark Knight, “Deshi Basara”, or ‘rise up’ yeast!

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

Categories: Research Spotlight

Tags: adaptation, DNA mismatch repair, experimental evolution, genetic incompatibility, mutator phenotype, natural yeast isolates

Yeast on the Red Carpet

September 28, 2016

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Awards season just kicked off with the Emmys a couple of weeks ago. Julia Louis-Dreyfus won her fifth Emmy in a row for her work on Veep, and Tatiana Maslany finally got an Emmy for her incredible work on Orphan Black. (If you haven’t seen Orphan Black, it’s a fascinating look at the ramifications of human cloning and genetic engineering.)

For a lot of people, the awards themselves are secondary to what everyone is wearing and who shows up with whom. You can learn a lot about the evolution of a performer’s career through these subtle (and not so subtle) cues.

Turns out that PLOS just had their own awards. Not quite as glamorous as the Emmys or the Oscars but almost certainly more significant.

The PLOS Genetics Research Prize for 2016 went to a fascinating paper from December of 2015 that uses yeast to explore evolution and provide ways to get at the underlying mechanisms of polygenic inheritance. And it does this by studying the subtle cues of what happens when two different yeast species show up on a yeast plate together (and mate).

The study uses a technique called a sign test. This is a way to find a set of genes that have been up- or down-regulated in response to some sort of selection pressure.

The first step is to mate two different strains or species. In this case Naranjo, Smith and coworkers used two different yeasts – Saccharomyces cerevisiae and Saccharomyces paradoxus.

The next step is to take the result of this mating, the F1 hybrid, and to compare the expression from each species’ alleles to the other’s. In other words, how does the expression of gene A compare in the two species? And gene B? And so on, through all of the genes.

What they are looking for in this allele specific expression (ASE) is a set of genes in the same pathway that are all affected in the same way. In this case they found a set of eleven genes linked to resistance to the toxin citrinin that was upregulated in S. paradoxus, but not in S. cerevisiae, in the absence of citrinin. This suggested that there was some sort of evolutionary pressure on S. paradoxus to become resistant to citrinin.

An obvious prediction from this is that their strain of S. paradoxus, CBS432, is more resistant to citrinin than is their strain of S. cerevisiae, S288C. They tested this and their S. paradoxus strain did indeed do better than S288C when citrinin was around.

They next did RNA-seq on the F1 hybrid yeast in the presence and absence of citrinin to find the up-regulated genes responsible for the ability of S. paradoxus to better tolerate citrinin. They ultimately settled on five genes that were both more highly expressed in the absence of citrinin, and more strongly induced by citrinin.

To figure out which of these genes is critical for resistance, they next deleted each gene individually and tested each deletion strain for its ability to grow in citrinin. Four of the five genes – GPX2, FRM2, RTA1, and CIS1 – made it through this test.

They next checked to see if making more product from all of these genes at once increased the strain’s resistance to citrinin. To pull this off they turned to everyone’s favorite genetic tool, CRISPR/Cas9.

Unless you’ve been hiding under a rock, you already know that CRISPR/Cas9 uses a guide RNA to get the protein Cas9 to the specific spot in the genome you want to edit. But in this case they aren’t editing a gene. Instead they are activating genes by using a version of Cas9 with two important changes: it can’t cut DNA anymore and it has a transcription activation domain added to it.

The idea is to activate all four genes at once by providing the yeast with guide RNAs that can lead this Cas9 to each of the four genes. What a powerful and simple way to easily activate all four genes at once.

They found that this overexpression strain was able to better tolerate citrinin but that it came at a cost – the strain grew more poorly in the absence of citrinin.

They next set out to see if the mutations that distinguish S. paradoxus from S. cerevisiae were in the promoters of these four genes. First, they replaced the S. cerevisiae promoter with the S. paradoxus promoter for each gene in the citrinin-sensitive S. cerevisiae strain. This created four new strains, each with one of the promoters swapped.

They found that in all cases the S. paradoxus promoter led to increased gene activity. Expression from these genes increased by anywhere from 1.6-6.9 fold.

Their final experiment was a competition between the original S. cerevisiae parent and the four strains in which the native S. cerevisiae promoter had been swapped out with the S. paradoxus one. They found that except for the strain overexpressing RTA1, these strains did better than the original S. cerevisiae strain in the presence of citrinin, but worse in its absence. Each of the three strains alone did not provide as much advantage as all three together did.

This is pretty powerful stuff! They used the sign test and CRISPR/Cas9 to nail down the three differences between S. paradoxus and S. cerevisiae that help to explain the polygenic trait of citrinin resistance.

And this isn’t just some cool yeast experiment either (although it is definitely that – #APOYG!). Sign tests may provide a new way for all those geneticists dutifully doing genome-wide association studies (GWAS) to find the set of genes responsible for polygenic traits that have so far eluded them. This is the kind of work that definitely deserves an award!

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

Categories: Research Spotlight

Tags: adaptation, allele-specific expression, cis-regulation, natural selection

Can’t Get There Like That

May 04, 2016

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As HBO’s Silicon Valley scathingly relates, the mapping app from Apple was truly terrible when it was first launched. There are all kinds of funny (scary?) stories in which people following the directions ended up in the wrong place. (Click here for a few more of the epic fails.)

And sometimes it would show impossible ways to get from one location to the other. For example, to get to a certain place, my iPhone would recommend two different routes. After choosing the seemingly easiest route I quickly realized that it would take me through a building. Sometimes, even though it seems there are a couple of different ways to get somewhere, there is actually only one.

It turns out that this can be true in gene expression too. While you might increase expression by either mutating the promoter or duplicating the whole gene, sometimes only duplication is enough. There is just one route from here to there.

This point is driven home in a new study out in GENETICS by Rich and coworkers. Here they show that, under sulfate-limiting conditions, the only way that yeast can boost the expression of the SUL1 high affinity sulfate transporter enough to thrive is by duplicating it.

In many previous experiments, whenever yeast is starved for sulfate, after 100 generations or so the population almost always ends up selecting for a SUL1 duplication rather than increasing expression with a promoter mutation. This duplication results in a fitness advantage of 35% or more, which makes sense – when there isn’t much sulfate available, those cells that can get more of what’s there will outcompete their neighbors.

There are a couple of possible reasons things go down like this. It could be that the duplication is simply the most likely way to increase expression enough to survive, meaning that promoter mutations are possible but rare. Alternatively there may be no way to mutate the promoter enough to adequately increase the activity in such a short window of time. You simply can’t get to enough increased fitness by this route.

The first step in figuring out how to get somewhere is to map the roads in the area. This is also what Rich and coworkers needed to do – they needed to figure out how SUL1 is regulated. They did this in the standard way by nibbling away bits of the sequences upstream of the gene until there was a significant impact of gene expression. This is how they identified the 493 base pairs upstream of SUL1 as its promoter.

In the meantime, they also managed to develop a broadly applicable methodology for investigating any promoter – saturation mutagenesis, chemostat selection, and DNA sequencing to track variants.

The next step was to generate a library of mostly single point mutations in this promoter using error-prone PCR. As might be expected, most of the mutants had no effect or decreased gene expression but a few did increase activity. However, none of these last set increased expression as much as duplicating the gene.

They found 8 mutants that gave a 5% or better increase in fitness with the best being a 9.4% increase. Even when they combined these point mutations they could not increase the fitness much beyond 11%, not even half of the increase in fitness that an extra SUL1 gene gives. It seems that to get the most bang for its buck, yeast needs to duplicate SUL1. At least in the time frame of the experiment, that is.

Using what is essentially the scanning mutagenesis of this promoter, they were able to identify three sites that were important for SUL1 regulation. One site at -465 to -448 corresponded to a Cbf1-Met28-Met4 regulatory site, the second site at around -407 was most similar to either a Met31 or Met32 site and the third site at around -350 matched a Met32 site.

Mutations that resulted in increased activity tended to bring one of these three sites closer to the consensus transcription factor binding site. For example, the strongest point mutation, -353T>G, did this with the Met32 site at -350.

Even with these stronger consensus sequences for sulfate-regulatory transcription factors, none of these point mutations could get the yeast to where it needed to be in the fitness landscape in order to be able to thrive under sulfur limitation. Point mutations just can’t hold a candle to simply duplicating the SUL1 gene. Sometimes there really is just one way to get from point A to point B.

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

Categories: Research Spotlight

Tags: adaptation, chemostat selection, fitness, gene duplication, saturation mutagenesis

Be Good, For Adaptation’s Sake

December 17, 2012

You may never have herded cows. But in one way or another, you’ve certainly experienced the tragedy of the commons.

This happens when a village shares a pasture that can only feed a certain number of cows. For the system to work, everyone has to cooperate and keep the total number of cows under that limit. But inevitably, one cheater comes along and adds extra cows to his herd. At no immediate cost to himself, he gets all the benefits of the extra cows. But then tragedy kicks in as the pasture is overgrazed until no one can have any cows.

This doesn’t just happen out in the village. We can see it in the overfishing of the oceans, the production of carbon dioxide contributing to global warming, the milking of investors on Wall Street, and many other aspects of modern life. But perhaps surprisingly, we can even see it in cultures of the humble yeast S. cerevisiae.

In a recent issue of the Proceedings of the National Academy of Sciences, Waite and Shou set up a yeast system to look at the factors influencing the tragedy of the commons. In human society, sometimes cheaters cause a collapse, but other times, cooperators get together and exile the cheaters from the village. You might think that since yeast aren’t quite as smart as humans, in yeast “society” the cheaters would always win. However, Waite and Shou found that sometimes the cheaters were marginalized or even driven out, and the cooperators thrived!

To do these experiments, the researchers set up a very clever pasture in miniature. They engineered three strains, each marked with a different-colored fluorescent marker so they could be distinguished from each other.

The two “cooperator” strains needed each other to survive on minimal medium: one required lysine and produced excess adenine, while the other required adenine and produced excess lysine. The “cheater” strain required lysine but it didn’t provide any nutrients. So the cheater needed one of the cooperators to survive, but didn’t contribute anything to the common good.

As we might expect, being cooperative has a cost. The generous production of extra nutrients made the cooperator grow slower than the otherwise identical cheater strain. So you would predict that if you mixed the cooperators and the cheater in equal numbers and grew them together, the cheater would take over and collapse the culture every time.

However, when the researchers mixed all three strains in a 1:1:1 ratio and grew lots of replicate cultures, they found that cheaters didn’t always prosper. Sometimes the nice guys finished first.

Of course some of the cultures did collapse under the influence of the rapacious cheaters. After a while these cultures stopped growing and turned out to be made up of mostly dead or dying cheater cells. The cheaters had taken over the culture, selfishly using up the lysine until eventually there was not enough to continue growing.

But unexpectedly, other replicate cultures were growing much faster, at rates similar to cultures without any cheaters. In these cultures, the two cooperator strains had either dominated the culture or even driven the cheaters extinct!

To explain this, the researchers proposed that the intense selection pressure led to an adaptive race between cooperators and cheaters. In surviving cultures, the rare cooperator with a small advantage had outcompeted the cheaters.

To confirm this, they took a close look at the winning cooperators. They found that the fitness advantage could be inherited, so they used whole-genome resequencing to find out why the cooperators were outcompeting the cheaters. They kept finding mutations in the same five genes.

These genes all made sense, as mutating them would help in an environment with limited amounts of lysine. For example, most of the mutations were found in ECM21 and DOA4. Both of these gene products are important in pathways that break down proteins like permeases. Knocking them out would keep the permeases around longer, making for better lysine uptake. But this newfound advantage did not come without a price.

The researchers tested directly whether the adaptive mutations improved growth in limiting amounts of lysine. Without exception, they did. But almost all these strains grew more slowly in abundant lysine than did their ancestor strains. That explains why these mutant strains only became a significant proportion of the population late in the life of the culture, when lysine levels were very low.

The same mutations can arise in both cooperators and cheaters, of course. But when cheaters become better at growing in low lysine levels, they just become that much better at making themselves extinct. When cooperators get better at growing in low lysine levels, they are better able to keep growing and keep the cheaters at bay.

So the take-home lesson is that cooperation does pay, after all. Especially in a constantly changing environment, cooperators can often win the adaptive race and squelch the cheaters. Maybe we should take a hint from little S. cerevisiae that being kind to each other is not only a nice thing to do, it’s in all of our best interests!

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

Categories: Research Spotlight

Tags: adaptation, evolution, Saccharomyces cerevisiae

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