New & Noteworthy

Getting Into Yeast’s Genes

September 26, 2013

Yeast has been responsible for a lot of hook ups in its day (think beer goggles and margaritas on the beach).  Now it is payback time.  In a new study, Giraldo-Perez and Goddard have figured out how to make yeast more promiscuous.

No, they don’t get the yeast drunk.  Instead, they found that strains containing VDE, a homing endonuclease gene (HEG), entered meiosis more often than genetically identical strains that lacked VDE.  The yeast that contained this “selfish” gene (well, actually intein) were ready to go haploid more often than those that didn’t.

VDE and its ilk are said to be selfish because they end up getting passed down to more offspring than a certain Austrian monk might have predicted.  When a diploid is heterozygous for an HEG, the homing endonuclease cuts the sister chromosome at the equivalent spot. Then, when the diploid undergoes meiosis, the sister is repaired through recombination causing both chromosomes to contain the VDE gene.  Now instead of two spores containing VDE, all four will.

Giraldo-Perez and Goddard monitored the percentage of sporulating cells over a 30 day period and found that after five days, a higher percentage of diploids homozygous for VDE sporulated compared to diploids heterozygous for or lacking VDE.  The authors contend that under the right conditions, this increased sporulation would allow VDE to spread through a population 20 times faster than it might otherwise.  And the authors found that VDE needs something like this or it might disappear.

Like alcohol, VDE isn’t all lowered inhibitions and good times.  For example, yeast homozygous for VDE grow significantly more slowly than do yeast lacking VDE in YPD, grape juice, vineyard soil, vine bark (heterozygotes fall in between).  This obviously puts yeast carrying VDE at a disadvantage, meaning that if it didn’t have another trick up its sleeve, it would dwindle away to nothing.  That trick is speeding up sporulation. 

The authors weren’t able to determine why this little bit of DNA can have such a profound effect on the growth rate of yeast.  It is almost certainly too little DNA to affect the time it takes the yeast to copy its DNA.  And the endonuclease itself is probably not randomly nicking the chromosomal DNA in the mitotic state, since it is kept out of the nucleus by host encoded karyopherins.

So VDE is a truly a parasitic selfish gene.  It is parasitic because it sucks a little of the life out of a yeast cell.  And it is selfish because way more daughters end up with it than might be predicted.  Sounds like a nice description for many drunk people…

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

Categories: Research Spotlight

Tags: homing endonuclease, intein, Saccharomyces cerevisiae, selfish gene, VDE

Have Your Fuel and Eat It Too

September 19, 2013

Back in 2008 and 2011 there were huge spikes in the cost of food that caused riots in various parts of the world.  These things were pretty bad and one of our favorite beast’s best products, ethanol, may have been at least partly to blame.  In an attempt to deal with global warming, governments had created incentives that made it more lucrative to turn food into ethanol to power cars rather than keeping it as food to feed people.  The law of unintended consequences reared its ugly head and caused food prices to rise high enough to be unaffordable by the very poor.   

This situation arose because right now, pretty much the only commercially viable way to make ethanol is to use sugars like those found in sugar cane or starches like those found in corn.  Ultimately this won’t be a problem once scientists learn to coax yeast or other microorganisms to make ethanol out of agricultural waste.  Until then, though, one way to lessen the impact of ethanol production on food supplies might be to engineer a yeast strain that can more efficiently turn sugars into ethanol. 

One of the most inefficient parts of yeast fermentation is that the silly thing converts anywhere from 4-10% of the sugars it gets into glycerol instead of ethanol.   In a new study, Guadalupe-Medina and coworkers have engineered a strain of yeast that produces 60% less glycerol and 8% more ethanol than other commercial strains.  If they can scale this up, it might help us feed both the world’s population and our cars.

It has been known for some time that yeast end up making glycerol during fermentation because of redox-cofactor balancing issues.  In essence, the excess NADH that is made in fermentation reactions is reoxidized by converting part of the sugar into glycerol.  One obvious way to get less glycerol would be to give the yeast some other way to reoxidize its NADH. 

Guadalupe-Medina and coworkers decided to persuade yeast to use carbon dioxide instead of sugars.  Not only would this make sugar use more efficient, but their particular plan would also convert that carbon dioxide into a precursor that could be shunted into the ethanol producing pathway.  Theoretically the yeast should now increase its ethanol production both by wasting less sugar on glycerol and by turning carbon dioxide into ethanol.  And it turns out that this idea actually worked in practice.

The first step was to introduce the Rubisco enzyme into the yeast.  Rubisco (ribulose-1,5-bisphosphate carboxylase oxygenase) is really one of the key enzymes in life…it provides the foundation for almost all life on the planet by fixing carbon dioxide from the air into ribulose-1,5 phosphate.  But that isn’t the important point here.  No, the key point for this work is that in the process of doing this, the enzyme oxidizes NADH.  By putting Rubisco in yeast, the yeast should now be able to reoxidize its NADH without making useless glycerol.

Of course this is easier said than done!  Rubisco is multi-subunit in most beasts and persnickety to boot.  But with a bit of work, they managed to get Saccharomyces cerevisiae to express a working copy of Rubisco.

So they would only have to introduce a single gene, the authors used the single subunit enzyme from T. dentrificans. As expected, this gene alone was not enough.  They knew from previous work that Rubisco would not work in yeast without the help of a couple of E. coli chaperones, groEL and groES.  When they expressed all three genes at the same time, they got Rubisco to fix carbon dioxide in Saccharomyces cerevisiae.  

The next step was to introduce the enzyme phosphoribulokinase (PRK) so that the ribulose-1,5 phosphate could be converted into 3-phosphoglycerate, a precursor in the ethanol pathway.  Luckily this was much easier than Rubisco and worked on the first try.  They had now engineered a Frankenyeast that should be able to make more ethanol and less glycerol.

When they tested the new strain, Guadalupe-Medina and coworkers found they had indeed engineered a more efficient yeast.  In anaerobic chemostat conditions, this yeast made 68% less glycerol and 11% more ethanol than the usual commercial strain.  They obtained similar results, 60% less glycerol and 8% more ethanol, in batch fermentations.  They had succeeded in improving an already awesome beast.

If this strain works on an industrial scale and if commercial producers all used this strain instead of the ones they currently use, the authors calculate we could get an extra 5 billion liters of ethanol added to the 110 billion we are already making.  That might just be enough to tide us over until scientists come up with a way to make ethanol commercially from non-food sources.

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

Categories: Research Spotlight

Tags: biofuel, Saccharomyces cerevisiae

Parthenogenesis, Saccharomyces Style

September 10, 2013

Parthenogenesis is one of the cooler things in biology. When a female Komodo dragon can’t find a mate, her eggs simply double their DNA and voila, a whole litter of female Komodo dragons is born. (Interestingly, they aren’t clones of mom…)

Now, this doesn’t work in mammals like us (curse you imprinting!), but something similar can happen in yeast. Given the right conditions and the right mutations, yeast can go from haploid to diploid without all that messy mating.

In a new study out in GENETICS, Schladebeck and Mösch uncover the newest mutation to be shown to cause whole genome duplication (WGD) in haploid Saccharomyces cerevisiae: the whi3 deletion. And this mutant is no slouch…the haploid will go diploid in no time flat if given the right conditions.

Schladebeck and Mösch looked at the stability of the haploid state of the whi3 mutant in both minimal and rich media, either in liquid culture or on solid agar. They generated fresh whi3 deletion strains and then followed them in each of these growth conditions for 72 days, passaging them every two days. 

What they found was that the haploid state was actually pretty stable in liquid culture using minimal media. They found very few diploid cells after 72 days. The same was not true for the other growth conditions.

On solid minimal media and liquid rich medium, there was a complete switch after 72 days. And on solid rich medium, the cells were all diploid after only 14 days. Genome duplication appeared to stop at the diploid level though. Even after 72 days on solid rich media there was no sign of tetraploids.

The authors next set out to figure out why deleting WHI3 had such a profound impact on haploid stability. They have not yet figured out everything that is going on, but they did uncover some interesting clues.

First they looked at the protein Nip100p. They already knew that NIP100 interacted genetically with WHI3, and that a nip100 deletion mutation affected chromatid separation. They found that Nip100p levels were significantly reduced when WHI3 was deleted, and even more so when the whi3 mutant strain was grown on solid rich medium. These are the conditions that most favored the transition from haploid to diploid. This suggests that NIP100 might be a key player in maintaining the haploid state.

The authors also compared transcriptional profiles of the wild type haploid strain, the whi3 deletion in a haploid background, and the whi3 homozygous mutant diploid. One of the findings from these experiments was that most of the genes involved in the yeast cohesion complex were upregulated in the absence of WHI3. Since this complex is required for sister chromatid cohesion, the idea would be that inefficient separation of chromatids in the whi3 mutant would increase the rate of whole genome duplication.

One of the as yet unexplained aspects of all of this is why the diploid state remains stable. There was no difference between the haploid and diploid deletion strains with regard to either Nip100p levels or transcription of cohesion-relate4d genes – the cohesins were upregulated in both and Nip100p was reduced in both.

One idea Schladebeck and Mösch put forth is that the diploid state isn’t inherently stable in this mutant. Instead, they do not see tetraploids simply because tetraploids have decreased viability. They appear but are quickly outcompeted by their diploid sisters.

The discovery about WHI3’s role in controlling ploidy is just one aspect of this new study. The authors also found important new information about the central regulatory role of WHI3 in cell division and biofilm formation.

The finding about ploidy control is important because maintaining haploid and diploid status is obviously a big deal: you don’t want to switch willy nilly from one to the other. And many pathogenic fungi, such as Candida albicans, change the organization of their genomes to adapt to changing growth conditions in their human hosts. They have WHI3 homologs, so these results could lead to better ways to cure fungal infections. Just one more example of how basic research can lead scientists to stumble on unexpected but ultimately important results…

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

Categories: Research Spotlight

Tags: ploidy, Saccharomyces cerevisiae

Using Yeast to Find New Treatments for Huntington’s Disease

September 05, 2013

Huntington’s disease (HD) is a truly awful, inherited and ultimately fatal genetic disease.  People with this neurodegenerative disorder typically start having trouble with their coordination and displaying mild cognitive and psychiatric problems in mid-adulthood.  Their symptoms continue to worsen, with most of these folks passing away within 20 years of their diagnosis.  This disease strikes down adults in their prime.

Scientists have known for decades what causes HD—too many CAG repeats in the huntingtin (htt) gene.  What they haven’t been able to figure out is what to do about this misfolded protein.  To date, the treatment options are very limited.

A new study out by Mason and coworkers has a chance to change all of that.  Using an unbiased screen in Saccharomyces cerevisiae, these authors were able to identify a class of proteins, the glutathione peroxidases, that when overexpressed protected yeast from the harmful effects of the mutant htt protein.  They then followed up and showed that these proteins had a similar effect in fruit fly and mouse HD cell models as well as in a whole fruit fly model.  And this isn’t even the exciting part.

There are druggable small molecules that when added to cells (or whole animals) can upregulate the activity of glutathione peroxidases.  The authors used one of these molecules, ebselen, and showed that it mimicked the effects of overexpressing various glutathione peroxidases in cells and, more importantly, in whole fruit flies.  When these flies were fed ebselen, their neurons degenerated at a much slower rate.  Mason and coworkers have identified a small molecule that can mitigate the effects of the mutant htt protein in model systems.

While we shouldn’t get ahead of ourselves here, this is all pretty exciting news.  How cool would it be if one day people with HD lived longer, happier lives because of a drug identified using our favorite model organism?  (Pay attention NIH!)

Mason and coworkers looked in S. cerevisiae for open reading frames that, when overexpressed, would lower the toxicity of the mutant htt protein.  They identified 317 of these, and used a variety of bioinformatics tools to group them into different pathways and gene networks.

In the end, they decided to focus on two powerful suppressors, the glutathione peroxidases Gpx1p and Hyr1p (also known as Gpx3p), for a variety of different reasons. These proteins are powerful antioxidants, and oxidative stress is known to contribute to HD symptoms.  Also, these proteins aren’t already upregulated in patients with Huntington’s disease, suggesting that it might be possible to increase their activity using drug therapy.

Now of course yeast aren’t mammals, so Mason and coworkers needed to show that having extra glutathione peroxidase activity would help in mammalian cells too. And this is just what they did: adding a mouse version of glutathione peroxidase, mGPx1, suppressed cellular toxicity in mouse cells that overexpressed the mutant form of htt.

Next they tested whether activating glutathione peroxidases would have the same effect.  They focused specifically on a selenocysteine-containing molecule called ebselen because it is highly bioavailable, can cross the blood-brain barrier (critical for HD) and has been used in treating stroke and noise induced hearing loss. When added to the mouse HD model cell system, ebselen had very similar effects to overexpressing mGPx1.

So upregulating glutathione peroxidase activity by either overexpressing mGPx1 or adding the small molecule ebselen appears to help in a couple of different model cell systems.  But what about a whole animal?  Looks like it can help there too.

Mason and coworkers looked at HD in a fruit fly.  When they added mGPx1 to this model fly, various neurons in these flies were protected from the effects of HD.  And they got similar results when they fed these flies the molecule ebselen.

As a final experiment, the authors wanted to figure out whether glutathione peroxidases were really having their effect because of their antioxidant activity.  In one way it makes sense that this activity is why they are so effective at mitigating the effects of the mutant HD—scientists have known for a while that oxidative stress is a major contributor to symptoms of HD. But on the other hand no antioxidant therapies have worked to date for HD.  In fact, if anything they made matters worse.  So one thought was that there was something special about the antioxidant activity of these proteins.  For these experiments, they needed to go back to yeast.

The authors looked at a variety of antioxidant proteins, including superoxide dismutase, catalases, and glutathione reductases, and none protected the yeast from the effects of the mutant htt protein.  They then checked the effects of catalase and superoxide dismutase in the HD mouse cells, and again saw no effect.

It is well known that antioxidants negatively affect autophagy and that disrupting this process can make HD symptoms worse.  From this the authors reasoned that glutathione peroxidases were special because they were antioxidants that did not affect autophagy.  They provided support for their idea by showing that ebselen did not affect autophagy in yeast while a control antioxidant, N-acetylcysteine, did.

Once again, yeast shows why it is such an important tool in finding potential new treatments for human disease.  Without the unbiased screen, it’s difficult to imagine how scientists would have found this target. You can really only do this easily in a beast like yeast. 

 

Symptoms like these may one day be delayed because of the awesome power of yeast genetics.

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

Categories: Research Spotlight, Yeast and Human Disease

Smoothing Over an Extra Chromosome

August 22, 2013

Let’s say you had a rock you had to move that was way too heavy for you to lift. You could either start lifting weights until you could move it yourself or get someone to help you. Most of us would start texting our friends pretty quick.

Turns out our friend S. cerevisiae can be the same way. Many strains of this yeast can exist as either a fluffy colony or a smooth one. In a new study, Tan and coworkers show that some of these strains switch between the two by gaining or losing one of their chromosomes. They’d rather “get” an extra chromosome than try to gain a mutation that activates the necessary gene(s).

In this study, the authors found a strain where around one in a thousand yeast switched between fluffy and smooth colonies. As the smooth colonies grew, they developed “blebs” – little bumps on the smooth colonies.  Turns out these were yeast that switched back to the fluffy morphology.  The authors set out to explore why this strain switches at such a high rate and why it would want to. 

A first look showed that when this yeast strain went from fluffy to smooth, it gained an extra copy of chromosome XVI.  When the new smoother yeast lost this extra chromosome, it reverted back to its fluffy look.  A harder look showed that an extra chromosome XVI wasn’t the only way to a smoother yeast.  Occasionally the fluffy to smooth change could be caused by an extra copy of chromosome III, X, or XV, and an extra copy of V caused a slightly smoother colony.

These results suggest a couple of different ways that an extra chromosome might be leading to a smooth colony.  One is that just having extra DNA around causes the change.  The other is that a variety of genes can cause the change when present in higher than normal doses.  The researchers show pretty convincingly that the second reason is probably the right mechanism.

First off they show that not all extra chromosomes are created equal.  Some lead to a very sickly yeast while others have no effect on fluffiness.  Just having extra DNA around is probably not the culprit.

The authors next set out to figure out exactly what was going on with chromosome XVI.  Through a series of deletion studies, they found a single gene responsible for the fluffy to smooth shift – DIG1.  Overexpression of this single gene caused fluffy colonies to turn smooth.  Presumably there are other genes on some of the other chromosomes that serve a similar function.

They next set out to determine why yeast would ever want to do this.  Turns out that, as you might expect, each phenotype has an advantage in a different situation.  On a solid surface the fluffy strain did better, while the smooth one did better in liquid media.  

The “extra chromosome option” is actually a great way for a sedentary beast like yeast to quickly deal with a new situation.  Gaining an extra chromosome is much simpler than gaining a new mutation that up-regulates a gene under certain situations.

Figuring out this mechanism of fluffy to smooth transitions isn’t just fun biology either.  It may also point us in new directions for treatments for a variety of diseases, including drug-resistant cancers and microbial infections. 

In many cases, these cells become resistant because their chromosome number has changed from what is considered the norm.  If we could find a way to force cells to maintain the correct number of chromosomes, we might be able to make them more susceptible to drugs.  As usual, yeast studies are much more than fluff…they smooth the way to the future.

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

Categories: Research Spotlight

Tags: aneuploidy, Saccharomyces cerevisiae

Anchors Aweigh for Peroxisomes

August 15, 2013

Suppose you had a fleet of rowboats that you wanted to split up evenly between two shores.  You probably wouldn’t just set them free and hope they drifted to the right places. A better way might be to run some ropes from the distant shore and pull half the rowboats across.  That way you’d be sure to get the distribution of boats that you wanted.

Dividing cells face a similar problem with their organelles.  When cells divide, they need to make sure mother and daughter receive the right number of organelles.  Otherwise one or both could die!

This is obviously too important to just leave to chance, which is why cells have devised ways to precisely control how many organelles end up in mother and daughter.  But not every organelle is divided in the same way.  In our boat analogy, some are pulled over with ropes, others with chains, some with winches and so on. 

We don’t know a lot about how some organelles are distributed between mother and daughter cells.  For example, the details have not been worked out for peroxisomes, the organelles that contain enzymes for beta-oxidation of fatty acids. Until now, that is…

In a recent study in The EMBO Journal, Knoblach and colleagues looked in great detail at how yeast cells distribute their peroxisomes. During budding, some peroxisomes stay tied up in the mother cell while others are transported into the bud and re-tied there.  

The authors found that the structure that ties up peroxisomes is like a rope with hooks at both ends. One hook attaches to the peroxisome, while the other hook attaches to the cortical endoplasmic reticulum (ER) near the cell wall. Surprisingly, the same protein, Pex3p, acts as the hook at both ends of the rope, connecting it to both ER and peroxisomes.

The authors already knew that some peroxisomes stayed anchored around the edges of the mother cell while others were “mobile” and moved to the daughter when yeast cells divided. They also knew that the protein Inp1p was important for anchoring peroxisomes. In the inp1 null mutant all the peroxisomes are mobile and end up in the bud, while overexpressing Inp1p causes all the peroxisomes to be anchored in the mother cell and stay there.

Knoblach and colleagues suspected that Inp1p might act as the rope that tethers peroxisomes. To test this, they fused Inp1p to a protein that sits in the mitochondrial outer membrane, Tom70p. Now peroxisomes in this strain were attached to mitochondria! This established that Inp1p is the tether.

Another major molecular player in this process is Pex3p. The pex3 null mutant phenotype looks a lot like the inp1 mutant phenotype: the mother cell loses all its peroxisomes and they end up in the bud. Pex3p is an integral membrane protein that is channeled through the ER on its way to the peroxisomal membrane – so it can be present in both places. The authors found that both the N terminus and C terminus of Inp1p bind to Pex3p. All this suggested that together, Inp1p and Pex3p might form a structure that links peroxisomes to the ER.

They were able to show that Inp1p and Pex3p interact directly both at the peroxisome and at the ER using a neat trick called bimolecular fluorescence complementation. This simply means that if the two halves of green fluorescent protein (GFP) are brought close to each other, they can fluoresce like the intact protein.  The basic idea is that they fused the first half of GFP with Inp1p and the second half with Pex3p and looked for green spots to turn up in the right place of the cell.  Of course this is easier said than done!

To pull this off, the authors had to first make two haploid strains of opposite mating types. The first had a pex3 mutation that caused all Pex3p to be stuck in the ER, anchoring all its peroxisomes there. It also carried a version of INP1 that was fused to half of GFP.

The second strain had a different pex3 mutation that set all peroxisomes adrift, and this mutant pex3 gene was fused to the other half of GFP. This strain also had an additional marker that made peroxisomes glow red.

When the cytoplasms of these two strains had a chance to mix after mating, the zygote had red peroxisomes with glowing green spots, showing that Inp1p-half GFP from the first strain was interacting with Pex3p-half GFP from the second strain.  Because the Inp1p-half GFP of the first strain was bound to ER-localized Pex3p, and the Pex3p-half GFP of the second strain was localized to peroxisomes, this result showed that Inp1p connects peroxisomes with the ER.

The authors studied the kinetics of this process in a lot more detail and even tracked the wanderings of individual peroxisomes. The model that comes from all this work is that at start of budding, some peroxisomes are bound to Inp1p and others aren’t. Those that aren’t bound to Inp1p move to the bud, and the others stay anchored to the mother cell’s ER. Meanwhile, during budding Pex3p passes through the ER and re-emerges at the ER membrane at the bud cortex. It can then bind Inp1p, which in turn binds to the Pex3p on the surface of the migrating peroxisomes to dock them in the bud.

Not only is this cool just from a basic biology perspective, but it may also help us deal with some human peroxisome biogenesis disorders.  For example, Zellweger syndrome and infantile Refsum disease are associated with specific mutations in the human ortholog of PEX3. Once again, little S. cerevisiae is helping us navigate the inner workings of eukaryotic cells.  

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

Categories: Research Spotlight, Yeast and Human Disease

Tags: peroxisomes, Saccharomyces cerevisiae, yeast model for human disease

When One Spark Isn’t Enough

August 07, 2013

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

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

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

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

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

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

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

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

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

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

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

Categories: Research Spotlight

Tags: evolution, Saccharomyces cerevisiae

The Baby Bear of Proteins

July 31, 2013

In the story of Goldilocks and the Three Bears, Goldilocks always likes Baby Bear’s stuff best. Baby Bear has the most comfortable bed, the best porridge, and so on.

The reason Goldilocks likes Baby Bear’s things are that they are just right. They are neither too hard nor too soft, too hot nor too cold, too big nor too little.

Turns out that when it comes to certain proteins, the yeast S. cerevisiae is sort of like Goldilocks…it likes to have them at just the right levels. Too much or too little protein can throw things out of whack.

This idea is supported in a new study in GENETICS, where Sasanuma and coworkers find that a key helicase in yeast, Srs2p, needs to be present in just the right amounts for meiotic recombination to go off without a hitch. In particular, they show that this protein affects meiotic recombination by interfering with the assembly of filaments containing another protein, Rad51p.

Meiotic recombination starts off with Spo11p making a double stranded (DS) break in the DNA. This DS DNA is then trimmed back so that there is a 3′ overhang of single stranded DNA which is then coated with replication protein A (RPA), Rad51p, and Dmc1p. The coated single stranded DNA then invades a stretch of homologous DNA and recombination can begin. 

One of the first things Sasanuma and coworkers did was to show that toying with Srs2p levels has a negative effect on meiosis in general. Too little Srs2p brings spore viability down to 36.8% of wild type, and overexpressing it brings spore viability down to 22.4%.  Clearly Srs2p is a bit like Goldilocks…the amount has to be just right.

The authors next set out to determine how overexpressing Srs2p affects meiosis so profoundly. They showed that too much Srs2p delays the start of meiosis, causes chromosomes to end up in the wrong places, and stunts the repair of DS DNA breaks. Basically, extra Srs2p inhibits meiotic recombination.

They next looked at areas on the DNA where both Rad51p AND Dcm1p were bound, and found that too much Srs2p keeps Rad51p but not Dcm1p off the DNA. When either of these proteins binds to DNA, it forms foci that are visible as dots when the proteins are detected with fluorescent antibodies. While a wild type strain had roughly equal numbers of Dcm1p and Rad51p foci, there were four fold fewer Rad51p foci when Srs2p was overexpressed. Clearly Srs2p was keeping Rad51p-DNA complexes from forming. 

Srs2p can act as a translocase, and it can also bind Rad51p. Sasanuma and coworkers asked which of these functions is essential to its ability to disassemble Rad51p filaments on DNA. Using srs2 mutants that were blocked in just one of these functions, they showed that the translocase mutant was completely unable to remove Rad51p from DNA during meiosis. The Rad51p-binding mutant could still cause Rad51p to dissociate from chromosomes, although at a reduced rate compared to wild type. So the translocase activity is essential, while Rad51p binding is not.

Although it was known that in vitro Srs2p can cause Rad51p-DNA filaments to disassemble, this study is the first to establish that it actually happens in vivo during meiosis. The requirement for the translocase activity suggests that Srs2p may actually move along the filaments as it disassembles them. And this work also shows that just like Goldilocks with her bowl of porridge, the cell needs an amount of Srs2p that is not too big, not too little, but just right.

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

Categories: Research Spotlight

Tags: helicase, meiotic recombination, Saccharomyces cerevisiae

Size Isn’t All That Matters

July 23, 2013

Sometimes the pressures and stresses of everyday life can make some people go a little crazy…they lose a few of their marbles.  The same thing can happen to a cell too.  The only difference is that instead of losing their marbles, cells can lose their chromosomes!

There are all sorts of mechanisms in place to make sure that a cell has just the right number of chromosomes.  Still, sometimes all these systems fail and a chromosome is lost.  This can be catastrophic for a cell and, as an important part of cancer, catastrophic for the whole body too.

Given how important having the right number of chromosomes is, it is surprising how little we know about what makes a particular chromosome more likely to fly the coop.  Kumaran and coworkers set out to change this in their new study in PLOS ONE.

First off, they showed that some chromosomes in S. cerevisiae are indeed more likely to be lost than other ones and that there was a surprisingly wide range of stabilities.  For example, chromosomes XIII and XIV were thousands of times more stable than chromosome III.

One key factor in stability was chromosome size—the smaller the chromosome the more likely it was to be lost.  But chromosome III showed that size was not the whole story.  It was five times more likely to be lost than the smallest chromosome.

The authors next set out to determine what about chromosome III made it so flighty.  By creating a hybrid of chromosomes III and IX, they were able to show that there was no single site that made chromosome III so unstable.  They were also able to rule out the idea that HML, HMR, and the MAT locus made chromosome III more likely to be lost.

They next focused on the centromere because it is such an important player in chromosome segregation.  They created a series of plasmids using the centromeres from chromosomes III, IX, XII, XIV, and XV and found that the ones from chromosome III and chromosome XV were around 5-fold less stable than the other chromosomes. While they do not have a good explanation for why chromosome III and XV fared the same in their assay, the result did suggest that at least part of the instability of chromosome III could be explained by its centromere.

As a final experiment, they determined the frequency of chromosome loss in mad2 deletion mutants.  They did this because MAD2 is involved in the spindle checkpoint and so is a key mediator of chromosome stability.  They found that deleting this gene significantly increased the loss of other chromosomes, but chromosome III was 3-6 fold less affected by the loss of MAD2.  It was almost as if the centromere of chromosome III was already somewhat compromised for its interaction with the spindle.

The authors aren’t sure yet how chromosome III got to be so unstable. It could be that random mutations just made its centromere less effective. But another interesting possibility is that it might be under selective pressure. Carrying the mating type loci, chromosome III could be considered to be equivalent to a sex chromosome in larger eukaryotes, and we know that those chromosomes are under different evolutionary constraints from other chromosomes. Maybe S. cerevisiae just can’t take the pressure!

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

Categories: Research Spotlight

Tags: chromosome stability, Saccharomyces cerevisiae

Oxidation: Maybe Not SO Bad After All

July 11, 2013

You don’t have to be a scientist to get the message that oxidation is bad and antioxidants are good. Just go to the vitamin aisle of your local supermarket, or listen to the ads on late-night TV.  You’ll quickly find out that oxidation caused by free radicals is the reason for aging, and antioxidants are the fountain of youth.  Of course you shouldn’t believe everything you hear…

Things just aren’t that clear when you take a good hard look at aging. Yes, oxidation happens, but there actually isn’t solid experimental proof that it causes aging. In mice, this connection has not panned out at all: lowering the ability to sop up oxidants, by knocking out an antioxidant enzyme, does not shorten the mouse’s life.

In a recent eLife paper, joint first authors Brandes and Tienson and their coworkers used our favorite experimental subject, Saccharomyces cerevisiae, to see if oxidation is a cause or just a consequence of aging. They generated a ton of data about oxidation during aging and did not find any evidence for causation.  Instead they came to the surprising conclusion that the trigger for aging may actually be a sudden drop in the levels of the coenzyme NADPH.

The first step, published previously by this group, was to come up with a very sensitive assay for protein oxidation. The amino acid cysteine can act as a sensor for levels of oxidation, as its sulfur-containing thiol group can be oxidized and reduced. Their technique, known as OxICAT, detects the ratio of reduced to oxidized thiol groups on cysteine residues for individual proteins. They can do this for hundreds of proteins at the same time.

In the current study, they looked at the oxidation state of cysteine residues in about 300 different proteins and also measured the levels of several different metabolites related to the redox state of the cell. All of these data were collected over time in aging yeast cells, both under normal conditions and under conditions simulating caloric restriction or starvation.  These last conditions were included because a lower-calorie diet has been shown to slow down aging, in yeast as well as in animals.

Oxidation of proteins definitely did increase over time. But if oxidation were the cause of cell death, you would expect that it would increase steadily and at some maximum point, the cells would die. Surprisingly, that didn’t happen.

Instead, different groups of proteins were oxidized with different kinetics. The most sensitive proteins (about 10% of the set that they studied) were oxidized 48 hours before the cells started to lose viability. This set included some conserved proteins that are important in maintaining oxidation-reduction balance in the cell, such as the thioredoxin reductase Trr1p. 

But it wasn’t only those especially sensitive proteins that were oxidized. In a second wave of oxidation, almost all the remaining proteins (80%) were oxidized at 24 hours before death. And even with so many proteins oxidized the cells were still metabolically active, with ATP levels near normal. So massive oxidation did not equal instant death for these cells.

As predicted, a low-calorie diet slowed down the whole process. The pattern looked a lot like it did in cells on a normal diet, but there was more time between the waves of oxidation and before the end of viability.

The authors also looked at what happened to different metabolites during aging. One key metabolite is the coenzyme NADPH: it donates electrons to the thioredoxin system that helps balance oxidation and reduction. They found that even before any changes in oxidation are detectable, levels of NADPH decrease very suddenly. The authors speculate that this decrease starts the collapse in redox potential that ends in the death of the cell. The oxidation of protein thiols is an effect rather than a cause, and could actually be a way for the cell to sense its redox state and possibly regulate it. NADPH levels have been seen to decrease in aging rats as well, suggesting that this could be a universal part of the aging process.

The results of this study are too voluminous to describe fully here, but they raise a lot of intriguing questions. Some proteins never got oxidized – what protected them? Are NADPH levels really the trigger for aging, and if so, what causes the sudden decrease? Is oxidation of cysteines actually part of a sensory mechanism? And if that’s true, would preventing oxidation really be such a good thing? This may be another good reason to turn off late-night TV.

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

Categories: Research Spotlight

Tags: aging, oxidation, redox, Saccharomyces cerevisiae

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