New & Noteworthy

Saccharomyces cerevisiae, the Party Animal

June 26, 2013

S. cerevisiae would be the life of a fraternity party. With its high alcohol tolerance, it could win every beer drinking contest. And if the party ran out of alcohol, it could make lots more!

Like a barfly before his liver gives out, the yeast S. cerevisiae can tolerate incredibly high levels of ethanol.  But unlike the town drunk, S. cerevisiae uses this skill to its own advantage.

In the wild, this yeast ferments sugars to flood the local environment with alcohol.  The end result is that it does just fine but any nearby microorganisms are killed.

Humans also take advantage of this unique property of S. cerevisiae.  Not only does it allow us to brew beer and ferment grapes into wine without worrying too much about bacterial contamination, but it also helps us generate ethanol as a biofuel.  What a cool and useful little beast!

Given how important this property is for both yeast and us, it is perhaps surprising how little we know about how S. cerevisiae pulls this off.  A new study out in PLOS Genetics by Pais and coworkers sets out to rectify this situation.

The study yielded a number of interesting findings.  It might seem obvious that an organism that can make a lot of ethanol should able to grow in the high-ethanol environment that it created. However, by looking at these characteristics in 68 different S. cerevisiae strains, the authors found that the ability to produce ethanol was at least partially separate at the genetic level from the ability to thrive in it. Pais and coworkers called the first process “high ethanol accumulation capacity” and the second “tolerance of cell proliferation to high ethanol levels.”

Second, they identified DNA differences in three different genes – ADE1, URA3, and KIN3 – that all work together to give certain strains of yeast their high ethanol accumulation capacity.  The most interesting of these three is KIN3.

Kin3p is a protein kinase that has a role in DNA repair.  Since ethanol is a known mutagen, it may be that the DNA differences in the KIN3 gene make its protein better at DNA repair, rescuing the cell from the DNA damage from high ethanol levels.

The authors found these genes as part of a larger study to identify at the genetic level why some strains of S. cerevisiae did better than others in ethanol.  They focused on two strains, CBS1585 and BY710.  CBS1585 is a sake yeast strain that can tolerate and grow in high levels of ethanol while BY710 is a laboratory strain that doesn’t do well with either (although still better than most any other beast out there in nature!).

Yeast holds its liquor way better than this guy.

They created diploids using these two strains, sporulated them into haploids, and then screened these haploids for their ability to deal with high levels of alcohol.  As would be predicted from their survey of the 68 strains, the haploids could be grouped into three distinct but overlapping pools: 

1)   High ethanol accumulation capacity in the absence of cell proliferation

2)   Cell proliferation at high ethanol levels

3)   Poor tolerance and growth in high ethanol

The authors then used pooled-segregant whole-genome sequence analysis to identify the DNA regions critical to the first two functions.  Basically this is just what it sounds like.  They isolated DNA from the three pools and looked for differences in pools 1 and 2 that weren’t in 3.  (OK that is dangerously simplified, but that is the gist of it.)

This is how they identified ADE1URA3, and KIN3 as important in high ethanol accumulation capacity.  We will have to wait for them to pinpoint the important genes in the regions they identified for pool 2 to begin to understand why some strains can proliferate in the presence of high alcohol concentrations.

Once they have identified all of the genes that make certain yeast strains so good at dealing with alcohol, we may be able to engineer a yeast that can make more ethanol for less money.  We can make a great little alcohol producer even better!  

Flip-Flopping Yeasts

June 20, 2013

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

SGD Summer 2013 Newsletter

June 10, 2013

SGD periodically sends out its newsletter to colleagues designated as contacts in SGD. This Summer 2013 newsletter is also available online. If you would like to receive the SGD newsletter in the future please use the Colleague Submission/Update form to let us know.

Alternative Ways to Increase a Cell’s Shelf Life

June 5, 2013

Like milk or eggs, most cells with linear chromosomes have a shelf life. Each time these cells divide, they lose a little off the end of their chromosomes. Eventually, too much is lost and the cells crap out. Or, to use a more scientific term, they become senescent.

expiration date

Cells have lots of ways to keep their telomeres long and extend their “cell-by” dates.

But this is not the fate of every cell. Some cells, like those that go on to become sperm or eggs, use a reverse transcriptase called telomerase to extend their telomeres as part of their normal life cycle. And they aren’t the only ones. Around 85% of cancers hijack the telomerase and use it for their own nefarious ends.

The other 15% of cancers use a variety of different mechanisms to keep their telomeres from getting too short (Cesare and Reddel, 2010). All these different ways are lumped together in a single category called alternative lengthening of telomeres or ALT. The telomeres are lengthened in these cells by recombination with other telomeres, either those on other chromosomes or those that exist as shed, extrachromasomal bits. 

While telomere extension may keep cells alive, it can sometimes be a double-edged sword. A double stranded DNA break is usually recognized as DNA damage. However, if the break happens near a telomere seed (a sequence that looks like a telomere), then the DNA damage response can be suppressed and the end can be extended into a new telomere, in a process called chromosome healing. But now the cell could be in trouble, with new, partial chromosomes being created and getting pulled this way and that.

In a new study out in GENETICS, Lai and Heierhorst decided to investigate whether chromosome healing happens in yeast cells that have stayed alive because of ALT.  What they found was that chromosome healing at telomere seeds was suppressed in these post-senescence survivors.

They created these ALT dependent, post-senescence survivors from an est2 mutant strain that lacked the catalytic subunit of telomerase.  Without telomerase, the only way for these cells to survive is by using ALT. 

In the first experiment, they looked at whether the post-senescence survivors could create a new telomere by chromosome healing.  The authors used a galactose inducible HO endonuclease to create a double stranded break near an 81 base pair sequence known to be a telomere seed sequence in wild type. 

Broken DNA usually signals cells to pause the cell cycle until the damage is repaired. This is known as the DNA damage checkpoint. During chromosome healing in wild type, this checkpoint is suppressed so the chromosome break isn’t recognized as DNA damage.

In the post-senescence survivors, even after 21 hours there was no evidence of a telomere forming.  They didn’t suppress the DNA damage checkpoint either.

Lai and Heierhorst determined that these ALT-dependent cells could still repair a different break that was not near a telomere seed sequence. They just couldn’t repair the break at the telomere seed. And this wasn’t because the DNA damage checkpoint was active. When they prevented the checkpoint by using a rad53 mutant, the telomere still wasn’t repaired.

Instead, the post-senescence survivors eventually repaired the break by some other mechanism, generating lots of differing products in the process. When they repaired breaks at sites that were not telomere seeds, they were able to use homologous recombination. But homologous recombination was suppressed at the telomere seed site.

Since ALT is used in cancer cells, and happens most often in some of the least-curable types of cancer, whatever we can learn about the process in yeast is valuable. It may give us clues on how to change the expiration date of those cancer cells to “ASAP”.