New & Noteworthy

Slipping Through Haldane’s Sieve

May 22, 2014

Imagine you are panning for gold in a river and there are two kinds of nuggets.  One type is naked gold while the other is gold hidden inside of normal rock.  Pretty easy to figure out which nuggets you’ll gather first!

Now imagine instead that the process of panning is a rough one that knocks the shell off of the second type of nugget revealing the gold inside.  Now there won’t be any difference between the two.  You will be just as likely to keep both types of nuggets.

The same sort of situation applies to new beneficial mutations in a changing environment.  Back in 1927, J. B. S. Haldane predicted that the more dominant a mutation, the more likely it was to help a diploid beast adapt to a new environment.  The naked gold was more likely to be taken over the covered gold.

Gerstein and coworkers show in a new study that at least in the yeast Saccharomyces cerevisiae, Haldane’s sieve (as it is called) may not always apply.  The process of adapting to a new environment can strip away the dominant older allele, revealing the recessive one.  Loss of heterozygosity (LOH) uncovers the hidden gold of the recessive phenotype.

The authors had previously identified haploid mutants that were able to survive in the presence of the fungicide nystatin.  They mated these mutants to create either heterozygotes or homozygous recessive mutants and compared these to wild-type diploids growing either in the presence or absence of nystatin. 

Gerstein and coworkers found a wide range of effects of these mutations in the absence of nystatin.  Sometimes heterozygotes grew better than either homozygote, sometimes homozygous recessive strains did best, and sometimes wild type grew best.   Phenotypes were all over the map.

The story was very different in the presence of nystatin where only the homozygous recessives managed to grow.  This appears to contradict Haldane’s sieve.  Here there were no dominant mutations that allowed for survival.

Gerstein and coworkers found that some heterozygote replicates started to grow after a prolonged lag period.  A closer look at the heterozygotes that grew showed that they had lost the dominant allele so that they could now show the recessive phenotype and survive.  LOH had broken Haldane’s sieve. 

The authors found that the lower the nystatin levels, the more likely a population was to break through Haldane’s sieve.  They postulate that the populations survive longer at lower levels of nystatin, which increases the chances that a LOH will happen.  It is a race between survival and eliminating the dominant allele that keeps them from growing. 

The next step was to determine if LOH was common enough that populations with a small percentage of heterozygotes could survive.  They found that even in populations where only 2% were heterozygotes, around 5% of the 96 replicate populations managed to lose an allele and grow.  So even at low levels, a recessive mutation can give a population the advantage it needs to adapt and survive.

Combining the awesome power of yeast genetics with cheap sequencing is allowing scientists to test fundamental models of genetics that will unearth how populations adapt and survive in new environments.  We are finding those nuggets of scientific knowledge that have remained hidden.

Now of course, not every diploid is as numerous or as genetically flexible as yeast.  Cows, chickens, lizards, and people may all still be slaves to Haldane’s sieve.  We will need more studies to see if our recessive treasures can be uncovered in time to save us. 

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

Categories: Research Spotlight

Tags: antifungal resistance, evolution, Saccharomyces cerevisiae

Yeast: A One Man Band for Finding New Drug Leads

May 15, 2014

Imagine you are in a band and the only instruments you have are guitars.  Yes, you can play some beautiful music, but there will be a whole lot of music that your band won’t be able to play. 

In some ways, finding chemical leads to develop into drugs is similar to an all guitar band.  The compounds in available libraries all tend to have a lot in common.  They are like a vast array of subtly different guitars.

In a new study, Klein and coworkers use synthetic biology to have the yeast Saccharomyces cerevisiae make more varied libraries on its own.  As an added bonus, the authors also use the yeast to assay the new leads.  Not only have they expanded the range of instruments available to your band, but they’ve also made it so you can play all the instruments.  You are now a one man band!

The first step in all of this is to have an assay that can easily pick out the important leads.  Klein and coworkers use a galactose inducible Brome Mosaic Virus (BMV) system they had previously developed.

In this system, if one of the viral genes is on, then it produces a fusion protein that includes the Ura3 protein.  When the URA3 gene is expressed, yeast die in the presence of 5-fluoroorotic acid (5-FOA).  So any yeast that can make a compound that can inhibit viral expression will survive in 5-FOA.

The next step in creating these in vivo libraries was to randomly assemble various biochemical pathways into yeast artificial chromosomes (YACs) and to transform them into yeast.  These pathways were chosen because they have yielded important compounds before or because they come from medically important beasts.  This work was described in detail in a previous paper.

Specifically, Klein and coworkers randomly combined cDNA genes from eight biochemical pathways into YACs and transformed them into the BMV replication yeast strain.  They found 74 compounds that allowed the yeast to survive in the presence of 5-FOA.  Of these, 28 had activity in a secondary BMV assay. 

A close look at the 74 compounds showed that by and large, most had characteristics that put them in the right ballpark to be useful leads.  They had low molecular weight and the right hydrophobicity, and were chemically complex. In addition, many could easily be improved chemically (this last point is called optimizability).  Most importantly, they were pretty unique from a drug lead point of view. 

Over 75% of the compounds resembled nothing in known libraries.  And the compounds were not similar to one another.  Klein and coworkers had created a wide range of instruments other than guitars.

Of course, keeping a yeast strain alive is hardly reason to look for a new drug.  But that isn’t all these compounds can do.  At least some of these leads show excellent activity against two viruses related to BMV, Dengue and hepatitis C, and one looks particularly promising. 

With a random combination of genes from a variety of biochemical pathways, yeast has been coaxed into synthesizing chemical leads that can target two medically relevant viruses.  Scientists should be able to use a similar approach to tackle other diseases.  All they need is a yeast strain with the right assay.

Yeast can make our bread rise, get us drunk, and now maybe cure us of disease.  Is there anything yeast can’t do?  Well, they still can’t play a guitar. 

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

Categories: Research Spotlight, Yeast and Human Disease

Tags: drug discovery, Saccharomyces cerevisiae

The Curious Case of the Proteasome in the Nucleus

May 07, 2014

In the Sherlock Holmes mystery story “The Adventure of the Crooked Man,” a man and his wife are heard having an argument behind closed doors. There is a crash, and the doors are opened to reveal that the man is dead in a pool of blood and his wife has fainted. No one else is nearby, and it seems beyond any doubt that the wife has murdered her husband…of course, until Sherlock Holmes delves into the details and uncovers the real story.

Something similar is going on in the nucleus behind those nuclear pores. The proteasome is a huge molecular machine that recognizes and degrades ubiquitinated proteins. Scientists have seen key parts of the proteasome in the nucleus, and nuclear proteins are degraded by this complex.  Seems like an open and shut case that the proteasome degrades nuclear proteins in the nucleus.  But like our Sherlock Holmes story, things aren’t always as they appear. 

Chen and Madura applied some detective work to the details of our nuclear protein degradation mystery in a new study published in GENETICS and found that it probably doesn’t work this way at all.  Nuclear proteins need to be exported out of the nucleus to be degraded by the proteasome.  

The researchers first confirmed earlier work showing that the Sts1 protein is responsible for escorting proteasomes to the nucleus. In the temperature-sensitive sts1-2 mutant at the restrictive temperature of 37 degrees, two proteasome subunits from different subcomplexes of the proteasome (Rpn11p from the regulatory particle and Pup1p from the catalytic particle) didn’t make it into the nucleus.

They then looked at two nuclear proteins, Rad4p and Pol1p (also known as Cdc17p) that are substrates of proteasomal degradation. When the proteasome subunits Rpn11p and Pup1p didn’t make it into the nucleus because of the sts1-2 mutation, Rad4p and Pol1p were not degraded.

So the proteasome needs to get into the nucleus in order for nuclear proteins to get degraded. Sounds like the proteasome is guilty of degrading proteins in the nucleus. But like a good mystery novel, the story takes an interesting twist here.

Chen and Madura found that when they raised the temperature of the sts1-2 mutant cells, Rad4p and Pol1p were stabilized immediately. This didn’t really make sense though. Even if the temperature-sensitive mutation blocked import of proteasomes into the nucleus as soon as the temperature increased, the proteasomes already inside the nucleus should have been able to continue degrading their substrates.

Wondering whether the substrates might be exported from the nucleus to be degraded elsewhere, they tested what happened to Rad4p and Pol1p when nuclear export of proteins was blocked. Using a few different ways to prevent nuclear export (combinations of mutations, chemicals, and temperature), they showed that if Rad4p and Pol1p could not get out of the nucleus, they were not degraded by the proteasome.

So it’s clear that nuclear export is part of the degradation process for at least these two nuclear proteins. Chen and Madura also detected a general increase in multi-ubiquitinated proteins (tagged for proteasomal degradation) in the nucleus under conditions where export was blocked, suggesting that this mechanism may apply to other proteins as well. And it’s been shown in human cells that several specific proteins, including the tumor suppressor p53, need to get out of the nucleus to be degraded.

There are still a lot of details to be filled in about where degradation is really happening. An intriguing clue comes from the fact that Sts1p is distantly related to the Schizosaccharomyces pombe protein Cut8, which is a nuclear envelope protein that tethers the proteasome to the nuclear membrane. Might nuclear proteasomes work on the outside of the nuclear envelope?

More detective work is needed to answer this question. But it’s clearly a very important one. Nuclear export and protein degradation are highly conserved processes, and both are currently under study as potential targets of cancer treatment.

Let this be a warning to all of us not to take everything at face value.  Just because someone is holding the bloody knife, that doesn’t mean he is the murderer.  And just because subunits and subcomplexes of the proteasome machinery look to be in the nucleus, that doesn’t mean nuclear proteins are degraded there.  It is elementary, my dear Watson.  

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

Categories: Research Spotlight

Tags: nuclear export, proteasome, Saccharomyces cerevisiae

Modifications? Heterochromatin Don’t Need No Stinking Modifications (for Activation)

May 01, 2014

When a character is asked to show his badge in the movie The Treasure of the Sierra Madre, he famously says something along the lines of, “Badges? We don’t need no stinkin’ badges!*” If histones in yeast heterochromatin could talk they might say something similar, except instead of badges they’d bring up modifications. Maybe something along the lines of, “Modifications? We don’t need no stinkin’ modifications for activation!” At least, they’d say this if a new study by Zhang and coworkers holds up.

In this study, the authors show that two different genes in the yeast S. cerevisiae are activated in heterochromatin in the absence of any significant changes to the surrounding chromatin.  This result is surprising because most researchers think activation and changes in chromatin always go hand in hand.  Apparently, in at least some situations they do not. 

This isn’t to say that chromatin didn’t do anything here…it most certainly did.  It served as a general damper on transcription.  But in this study chromatin was by no means the major player; it had a relatively small influence on the levels of basal and activated gene expression.  The authors suggest that this may be true for other genes in the more transcriptionally active euchromatin as well.

In the first set of experiments, Zhang and coworkers used a model system where the heat inducible gene HSP82 is flanked by the HMRE silencer from the HMR mating type cassette.  These silencers cause a 30-fold reduction in transcription of this hsp82-2001 transgene.   

Using chromatin immunoprecipitation (ChIP) the authors show that their transgene is indeed embedded in heterochromatin.  They see a lot of Sir3p around the promoter, a high density of histones that lack any of the telltale modifications of euchromatin, and very little RNA polymerase II (Pol II) or the mRNA capping enzyme Cet1p around the promoter.  These are all hallmarks of heterochromatin in yeast.

Things change when the yeast is subjected to heat shock.  Consistent with the observed 200-fold increase in transcription, they suddenly see lots of Pol II and Cet1p around.  But there is not a big change in the number of histones around the gene nor in their modifications.

When HSP82 is in its normal place in the genome, its activation is accompanied by specific acetylation and methylation of H3 and H4 histones.   In heterochromatin, despite significant induction, there is none of this.  The histones remain looking the same whether there is significant transcription or not. 

One trivial explanation for this might be that the chromatin is unaffected because the levels of transcription are lower than normal.  In other words, the lower final activity in the induced state is affecting histone modification. 

Zhang and coworkers rule this out by using a TATA-less HSP82 gene in euchromatin and show that all the appropriate histone modifications still happen.  This is true even though the damaged gene has 5-fold less activity compared with their transgene.  The low level of transcription does not appear to explain activation in the absence of histone modification.

Of course another reason for this unexpected observation might be that this pretty artificial construct isn’t representative of natural genes.  This doesn’t change the fact that its transcription is activated in the absence of histone modification, but it does question its relevance in the real world.

To address this issue, the authors looked for an inducible gene in natural heterochromatin and with a little bit of detective work, found the subtelomeric YFR057W gene.  No one knows what this gene does, but a close look showed a possible Stb5p binding site in its promoter. 

When Stb5p heterodimerizes with Pdr1p, the resulting dimer activates genes involved in pleiotropic drug resistance.  Indeed the authors found that YFR057w was induced 150-fold with a small amount of cycloheximide.  And when they used ChIP to compare the induced and uninduced states, they again found almost no changes in the chromatin around this gene despite an increase in the amount of Pol II and Cet1p.

Taken together these results suggest that activation doesn’t always have to come with chromosomal changes.  Which, while a bit surprising today, wouldn’t have turned any researchers’ heads a few decades ago.

In the old days (1980’s and 1990’s), a lot of focus was on how transcriptional activators might affect the ability of Pol II to load onto the DNA and to pry it open and start transcribing.  A lot of this was based on prokaryotic work where there really isn’t very much in the way of chromatin and a lot of activation depends on improving the ability of the polymerase to transcribe.

These days when people think about turning up a gene, they think about changing nearby chromatin.  Various enzymes work to modify histones at specific places, which both loosens up the chromatin to allow access by Pol II and serves as a way for various coactivators to recognize the DNA. 

As usual, reality is probably a combination of the two.  Activators can activate transcription in lots of different ways, some of which probably include chromatin changes while in others chromatin changes are simply a consequence of activation.  Not all transcription activation needs stinkin’ histone modifications.  

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* This is actually a misquote that may have come from the Mel Brooks film Blazing Saddles.

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

Categories: Research Spotlight

Tags: heterochromatin, histone modification, Saccharomyces cerevisiae, transcription

Measure Twice, Cut Once

April 23, 2014

As any seasoned carpenter knows, if you are going to cut a piece of wood, you want to do it right the first time!  There is no second chance.

This means that good carpenters are very, very careful.  They use a clamp to hold the wood in place and measure where to cut not once but twice.  Now they have a good shot at getting a length of lumber they can use.

As shown in a new paper in Molecular Cell by Liang and coworkers, it turns out that our cells do something similar when cutting their RNA.  The yeast enzyme Rnt1p measures a piece of double stranded RNA twice to make sure it cuts in the right place.  And, like a second-rate carpenter, if it measures the RNA only once it often cuts the RNA in the wrong place.   

This is almost certainly not just a yeast thing.  Rnt1p is a member of the conserved RNAse III family, which is present in all domains of life except Archaea.

In higher organisms, RNAse III enzymes such as Dicer produce the small interfering RNAs (siRNA) and microRNAs (miRNA) that have important roles in gene regulation via RNA interference. S. cerevisiae doesn’t use RNA interference, but Rnt1p is still important for maturation of small nuclear RNAs, small nucleolar RNAs, and ribosomal RNA, and also for degradation of some specific mRNAs.

Most RNase III enzymes recognize the RNA they are to cut by certain secondary structures like loops.  Liang and coworkers used X-ray crystallography on Rnt1p in complex with an RNA substrate to learn how Rnt1p recognizes its substrate and “knows” where to cut it. The RNA had a double-stranded stem capped by a 4-nucleotide loop, a so-called tetraloop, that had a conserved G residue at the 2^nd position.

Rnt1p cleaves this RNA at a fixed distance from the tetraloop, and it cleaves the two strands unequally so that they have 2-nucleotide 3’ overhanging ends. The crystal structure showed that two of the five RNA-binding motifs (RBMs) in Rnt1p form a pocket that clamps down on the conserved G residue in the tetraloop. This clamp is fastened so tightly that the RNA structure is changed.  It is like the clamp distorting the carpenter’s piece of wood.

When Liang and coworkers deleted one of these two Rnt1p RBMs, or mutated the conserved G in the substrate, the substrate was no longer held or cleaved.  Clamping the RNA was critically important for the reaction. 

They also showed both by structural modeling and by mutational analysis that other parts of Rnt1p interact with the RNA stem structure. Clamping the RNA and interaction with the rest of the substrate puts the cleavage site at a fixed position relative to the Rnt1p active site.

This tight binding and measurement by protein-RNA interactions would seem to be good enough to ensure accurate cleavage. But it’s not the whole story.

Another domain of Rnt1p, the N-terminal domain (NTD), was known to contribute to substrate selection, but it was unclear exactly how it did this. Surprisingly, the crystal structure showed that it, too, contacts the tetraloop. When Liang and colleagues deleted the NTD, the RNA substrate was still cleaved but there was a mixture of products, cleaved at several different sites. So it too is needed for precise cleavage.

The overall conclusion is that two different domains contact the tetraloop, each acting like a ruler. The protein-protein and protein-RNA interactions stiffen each ruler such that the cleavage site is always precisely measured before cutting.  Just like our carpenter friend, to get the right cut, Rnt1p needs to measure twice before cutting. The same knowledge that is handed down through generations of carpenters is also deeply ingrained in our biochemistry! 

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

Categories: Research Spotlight

Tags: crystal structure, RNAse III, Saccharomyces cerevisiae

How Yeast Populations Make the Cut

April 15, 2014

Imagine that your dream is to be a professional basketball player.  Unfortunately for you, you are only five feet six inches tall and you can’t jump very high.  No matter how much you practice and work out, it is exceedingly unlikely you will be a starter for the Miami Heat.

Now imagine instead that you are six feet tall with a reasonable vertical jump.  Here, with enough effort you have a shot at beating out the guy with the genetic advantage of being six foot six inches high who doesn’t work as hard as you do.  Keep practicing and you might be passing the ball to LeBron James instead of him! 

In a new study in GENETICS, Frenkel and coworkers show that something similar can happen in yeast too.  If a population of yeast has some overwhelming advantage over a second population, the first will quickly outcompete the second every time.  But if the first population is just a bit better than the second, then the second can sometimes end up with a mutation that gives it an even better advantage than the first.  Now the first population is outcompeted and the second takes over.

Of course, when presented in a general way this is sort of obvious.  But Frenkel and coworkers set up their experiments in such a way that they got some hard numbers for just how much of an advantage one population needs to overcome to have a chance at winning.  If six feet is tall enough, what about five feet eleven inches?

The first step was to generate a number of mutants with different measured fitness advantages.  They selected mutant populations with advantages of 3, 4, 5, or 7%.  These populations were all tagged with a fluorescent marker.

They then seeded these mutants individually into 658 replicate reference populations that were tagged with a different fluorescent marker.  The mutants were seeded at a high enough level to prevent genetic drift from wiping them out.  The authors then followed each population for hundreds of generations by determining the levels of each population every 50 or so generations.

Their first finding was that mutants with a 7% advantage won out every time.  The reference population had no chance at getting a good enough mutation to beat it out.  No one is going to beat LeBron James out for his starting position with the Miami Heat.

Once the advantage was only 5%, around 16% of the time the second population won out.  As the advantage got smaller and smaller, the second population won out more and more often.   Even a genetically less gifted player has a shot at beating out the 12^th guy on the Heat’s roster!

These results can tell us quite a bit about the mutational landscape of haploid Saccharomyces cerevisiae.  For example, from these data Frenkel and coworkers figured out that only populations that get mutations that give at least a 2% advantage have a chance at outcompeting other populations.  By assuming a mutation rate of 4X10^-3, around 1 in 1000 mutations fit this bill, which might seem surprisingly high but is consistent with previous studies.  With a bit more hand waving, the authors hypothesize that disruption of something like 1 in 100 yeast genes is actually beneficial!

So yeast have a surprisingly level playing field.  Unless they are up against the equivalent of Kobe Bryant or Michael Jordan, they have a good shot at stumbling on a mutation that gives them an edge over their peers.    

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

Categories: Research Spotlight

Tags: evolution, mutation, population genetics, Saccharomyces cerevisiae

Redesigning Yeast One Chromosome at a Time

April 03, 2014

Everyone who reads our blog knows how awesome the yeast Saccharomyces cerevisiae is.  Without this little workhorse we would almost certainly not understand ourselves as well as we do now.  It is an indispensable tool in figuring out how eukaryotes work.

And of course yeast is much more than that.  It makes our bread fluffy and our drinks alcoholic.  It can be manipulated into making medicines like artemisinin, a powerful anti-malarial drug, or biofuels or whatever else we can think of.  It is the Swiss Army Knife of useful organisms.

Even with all of this fanfare, everyone knows yeast has its limitations.  It is a powerful tool but it could be improved.  For example, it would be nice if researchers could more easily manipulate its DNA to speed up the introduction of beneficial traits, add new biosynthetic pathways, or to do the kinds of experiments that will help one day cure cancer or Alzheimer’s disease.  This is where Sc2.0 comes in.

Sc2.0 is an idea that has been kicking around for the last decade or so.  First proposed by Ron Davis of Stanford University, the idea is to synthesize artificial yeast chromosomes to make yeast more useful.  Eventually the idea would be to recreate every yeast chromosome and intelligently redesign the genome for our own purposes. And maybe even to add new artificial chromosomes so we can easily add whatever genes we want.

In a new study out in Science, Annaluru and coworkers have taken a major step forward in the Sc2.0 project by replacing all 316,617 base pairs of yeast chromosome III with a 272,871 base pair synthetic version, synIII.   That leaves only 15 chromosomes and around 12.2 million base pairs before we have yeast with completely manmade DNA.

Annaluru and coworkers managed to do this with the help of a bunch of undergraduate students and yeast’s love of homologous recombination.  The first step was to have undergraduates synthesize around 30,000 base pairs each in the “Build a Genome” class at Johns Hopkins.  It took 49 students around 18 months to pull this off for synIII.

Basically they used 60-mer and 79-mer oligonucleotides to PCR up 750 base pair building blocks.  These pieces of DNA were designed so that they could be assembled into 2,000-4,000 base pair minichunks.  The final step was to transform yeast with an average of twelve of these minichunks and to let the yeast use homologous recombination to replace its native DNA sequence with the added DNA.  After 11 rounds of transformation, the yeast now had an artificial chromosome.

As you may have guessed, this chromosome is not exactly the same as the one it replaced.  To eventually free up a codon for repurposing later, all 43 of the TAG stop codons were converted to TAA.  When this is done with all of the chromosomes, researchers will now have a codon they can use to change this yeast’s fundamental genetic code.  This might allow for adding novel amino acids to proteins or even prevent viruses from infecting the new yeast.

Annaluru and coworkers also introduced 98 loxP sites which in the presence of estradiol will cause the yeast to undergo rapid DNA change.  The hope is that scientists will be able to harness SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution), as it has been named, to more quickly evolve useful traits in yeast for both study and biotechnological uses.

As a final step, the researchers cleaned up the chromosome by removing 21 retrotransposons and many introns and by moving 11 tRNA genes to a neochromosome.  They now had created a leaner, meaner chromosome III. 

The next obvious question was whether or not all of these changes affected the yeast.  Despite looking very carefully, Annaluru and coworkers could find little that was different between strains carrying natural and synthetic chromosomes.  They both grew similarly under 21 different conditions in terms of growth curves, colony size, and cell morphology, and had very similar transcription profiles.  But they weren’t identical.

For example, the strain with synIII grew slightly less well in the presence of high sorbitol, and showed differences in expression from wild type in 10 out 6,756 transcripts.  Of these ten, eight were intentionally altered in the creation of synIII and so were expected.  The two unexpected changes were a ~16-fold decrease in the expression of HSP30 on synIII and a ~16-fold increase in the expression of PCL1 on chromosome XIV.

Since all of these changes had such a small effect on the yeast, it is a green light for plowing ahead with creating yeast with completely manmade DNA.  Currently four other chromosomes, II, V, VI, and XII, are nearly done and the design work has been completed for chromosomes I, IV, VII, and XI (see an overview of the project).  It will only be a matter of time before we have a strain of yeast with completely synthetic DNA.  Scientists are making a powerful tool even better…who knows what this new strain will help us discover.

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

Categories: Research Spotlight

Tags: Saccharomyces cerevisiae, synthetic biology, teaching

Silly Sod’s Two Jobs

March 27, 2014

Most SGD users are probably too young to remember Saturday Night Live’s early years.  One very funny commercial parody involved Gilda Radner and Dan Aykroyd arguing over a product called Shimmer.  Gilda argues that it is a floor wax while Dan says it is a dessert topping.  In comes Chevy Chase to tell them that it is both.  Not quite as funny as Bassomatic, but still hilarious.

In a new study, Tsang and coworkers show something similar for the enzyme Sod1p.  Most people know Sod1p as an enzyme that protects the cell and its DNA by directly deactivating harmful reactive oxygen species (ROS) like superoxide.  Turns out that it may also be a transcription factor.

Now these two jobs aren’t quite as disconnected as a dessert topping and floor wax.  When Sod1p acts as a transcription factor, it is regulating genes that affect a cell’s response to ROS.  It is actually using its two functions to attack the same problem on multiple fronts.

Tsang and coworkers started out by looking at what happens to nuclear DNA under oxidative stress, using the Comet and TUNEL DNA damage assays. They found that endogenous and exogenous ROS caused DNA damage that was much worse in the sod1 null mutant – in other words, Sod1p protected the cells’ DNA. Using immunofluorescence, they also showed that Sod1p quickly went into the nucleus in the presence of ROS.  But if they restricted Sod1p to the cytoplasm by adding a nuclear export signal, the protein no longer protected the DNA.  In fact, it did no better than a strain deleted for SOD1.

In the course of these experiments one of the ways the researchers induced nuclear localization was with a burst of hydrogen peroxide.  But since hydrogen peroxide isn’t a substrate of the enzyme Sod1p, Tsang and coworkers next wanted to figure out how Sod1p got its signal to go nuclear.

Previous work had shown that SOD1 genetically interacted with MEC1, a yeast homolog of ATM kinases which sense oxidative stress.  They deleted MEC1 and found that Sod1p was trapped in the cytoplasm, unable to protect the cell’s DNA from damage.  This result was confirmed in human cells by showing that Sod1p only went nuclear if the cell made ATM kinase.

Tsang and coworkers suspected that this interaction might happen through a protein kinase called Dun1p, whose human homolog is a Mec effector. They confirmed a previous mass spectrometry result that showed Sod1p interacted physically with Dun1p.  And indeed, when DUN1 was deleted, Sod1p was again stranded in the cytoplasm.  Further work showed that Dun1p does its job by phosphorylating Sod1p on two serine residues, S60 and S99. When both these serines are mutated to alanine, preventing phosphorylation, less of the mutant Sod1p makes it into the nucleus. 

Using DNA microarrays, Tsang and coworkers next showed that SOD1 was required to activate 123 genes needed by the cell to respond to hydrogen peroxide.  These genes fell into five categories: oxidative stress, replication stress, DNA damage response, general stress response and Cu/Fe homeostasis.  The final experiment used chromosomal immunoprecipitation (ChIP) to show that in the presence of hydrogen peroxide more Sod1p was bound at the promoters of two of these genes, RNR3 and GRE2, but not the control gene ACT1. 

Of course, the authors have only looked at two of the 123 genes and an obvious next step is to figure out how many of the 123 have more Sod1p bound to their promoters in the presence of hydrogen peroxide.  Still, if these results can be confirmed and expanded they will suggest that Sod1p is able to combat oxidative damage in two completely different ways. 

In the first it uses its enzymatic activity to directly inactivate the ROS superoxide, while in the second it helps the cell respond to other ROS apparently by acting as a transcription factor.  While the jobs themselves are not as different as a floor wax and a dessert topping, how Sod1p goes about getting each job done is.  “Calm down you two, Sod1p is an enzyme AND a transcription factor.”

In addition to these two roles, we’ve written before about yet another regulatory role for Sod1p: it regulates glucose repression by binding to two kinases and stabilizing them. This is truly an overachiever of a protein!

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

Categories: Research Spotlight

Tags: oxidative stress, Saccharomyces cerevisiae, transcription

Guns vs. Butter in Cells

March 19, 2014

Life is a set of tradeoffs for people, countries, and even cells.  For example, governments need to decide how much money to dedicate to defense and how much to economic growth.  Too much on defense and your country fails, because defense spending sucks up so many resources that your country can no longer afford to pay for anything else.  And of course if you spend too little on defense, someone who spent a bit more can come and take you over.

No country lives in a vacuum though—how much to spend on defense and how much on growth depends on the country’s situation.  If you are the Ewoks living next to an Imperial shield generator, you’d better sacrifice some growth for defense.  But once the Death Star has blown up and the Empire is swept away, you probably focus more on growth (until a new Sith lord arrives). 

This guns vs. butter debate plays out at the cellular level too when it comes to protecting DNA from mutations.  If cells expend too much energy to protect their DNA they sacrifice growth, but if they spend too little, they develop too may harmful mutations to survive.  And just like with countries, how much protection a cell’s DNA needs depends on its environment.

If cells need to adapt quickly to a changing environment, a high rate of mutation is favored.  These cells are more likely to develop a mutation that gains them an advantage over their slower mutating brethren.

A new study by Herr and coworkers in the latest issue of GENETICS calculates the upper limit of the rate of mutation in a diploid yeast.  In other words, they figure out how little “spending” on defense these yeast can get away with and survive.

They find that diploid yeast can deal with a 10-fold higher rate of mutation as compared to haploid yeast.  This makes sense, since the extra gene copy afforded by being diploid can mask a recessive lethal mutation, but this study is the first to give this idea hard numbers.

The authors had previously generated a number of mutations in POL3, the yeast gene for DNA polymerase δ, that affect its ability to find and/or fix any mistakes made during DNA replication.  The study first focused on two mutations affecting accuracy, pol3-L612G and pol3-L612M, and one mutation affecting proofreading, pol3-01.  The accuracy mutations caused about a 10-fold increase in the mutation rate, while the proofreading mutation caused anywhere from a 20-100-fold increase.  Neither was enough to seriously affect a diploid’s growth.

The next step was to combine accuracy and proofreading mutations into the same gene to figure out if the combination resulted in a higher mutation rate.  The authors suspected that it did when they discovered that even though the heterozygotes were fine, their spores were inviable.  The POL3/pol3-01,L212M and POL3/pol3-01,L212G strains sporulated just fine, but none of the spores could germinate and grow. 

One way to explain this was that the double mutation increased the error rate to the point that it would kill off haploids but not diploids.  By looking at mutations in the hemizygous CAN1 gene they could see that the mutation rate in these diploids was indeed at around the haploid threshold. In terms of the CAN1 gene, this mutation rate was around 1X10-3 can1 mutations/cell division.

They next determined the mutation rate by sequencing the genomes of each mutant as well as the wild type.  They found a single T-G mutation in the wild type, 1535 point mutations in POL3/pol3-01,L212M and 1003 mutations in POL3/pol3-01,L212G.  From this they calculated a mutation rate of around 3-4X10-6/base pair/generation. 

Even though this level of mutation kills haploids but not diploids, this does not mean the diploids escaped unscathed.  When the heterozygous diploid colonies were subcloned the resulting colonies were variable in size, indicating that their higher mutation rate was catching up with them.  This high mutation rate was making them sick. 

Given this result, it wasn’t surprising that diploid homozygotes of each double mutant could not survive—the mutation rate was now too high.  The strains homozygous for pol3-01,L212M managed to get to around 1000 cells before petering out.  Strains homozygous for pol3-01,L212G did even worse—they only made it to around 10 cells.

In a final set of experiments Herr and coworkers used a variety of other mutations to tweak these mutation rates to find the threshold at which diploids fail to survive.  Some of these mutations were in POL3 while others were deletions of the MSH2 and/or DUN1 genes.  After testing many different combinations, they found that these yeast did pretty well up to around 1X10-3 can1 mutations/cell division (the haploid threshold rate).  Then, from 1X10-3 to 1X10-2 can1 mutations/cell division there began a rapid drop off with little to no growth at the end. 

So as might be expected, diploids can deal with a significantly higher mutation rate than can haploids.  But even though they can, wild type yeast in the lab still have a very low mutation rate.  It is like they are living near the Imperial city planet of Coruscant.  They are willing to expend the energy to keep their DNA protected.

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

Categories: Research Spotlight

Tags: DNA replication, mutation, Saccharomyces cerevisiae

A Heartfelt Need for Copper

March 06, 2014

Imagine the heater at your house is run by a homemade copper-zinc battery.  You are counting on a delivery of a copper solution that will keep the thing going.  Unfortunately it fails to come, which means the battery doesn’t work and you are left out in the cold. 

Turns out that something similar can happen in cells too.  The respiratory chain that makes most of our energy needs copper to work.  In a recent study, Ghosh and coworkers showed that if Coa6p doesn’t do its job delivering copper to the respiratory chain, the cell can’t make enough energy.

This isn’t just interesting biology.  In this same study, the researchers showed that mutations in the COA6 gene cause devastating disease in humans and zebrafish. And their discovery that added copper can cure the “disease” in yeast just might have therapeutic applications for humans.

The respiratory chain is a group of large enzyme complexes that sit in the mitochondrial inner membrane and pass electrons from one to another during cellular respiration. This process generates most of the energy that a cell needs.  Hundreds of genes, in both the nuclear and mitochondrial genomes, are involved in keeping this respiratory chain working.

Yeast has been the ideal experimental organism for studying these genes, because it can survive just fine without respiration. If it can’t respire for any reason, yeast simply switches over to fermentation, generating the alcohol and CO2 byproducts that we know and love.

Human cells aren’t as versatile though. Genes involved in respiration can cause mitochondrial respiratory chain disease (MRCD) when mutated. This is one of the most common kinds of genetic defect, with over 100 different genes known so far that can cause this phenotype.

Ghosh and colleagues wondered whether there were as-yet-unidentified human genes involved in maintaining the respiratory chain. They reasoned that any such genes would be highly conserved across species, because they are so important to life, and that the proteins they encoded would localize to mitochondria.

One of the candidates, C1orf31, caught their eye for a couple of reasons.  First, some variations in this gene had been found in the DNA of a MRCD patient.  And second, the yeast homolog, COA6, encoded a mitochondrial protein that had been implicated in assembly of one of the respiratory complexes, Complex IV or cytochrome c oxidase.

They first did some more detailed characterization of COA6 in yeast.  They were able to verify that the coa6 null mutant had reduced respiratory growth because it had lower levels of fully assembled Complex IV.

They also looked to see what happens in human cell culture.  When they knocked down expression of the human homolog, they also saw less assembly of Complex IV. This suggested that the function of this protein is conserved across species.

Next they turned to a sequencing study of an MRCD patient who had, sadly, died of a heart defect (hypertrophic cardiomyopathy) before reaching his first birthday. The sequence showed a mutation in a conserved cysteine-containing motif of COA6. To see whether this might be the cause of the defect, they created the analogous mutation in yeast COA6. The mutant protein was completely nonfunctional in yeast.

To nail down the physiological role of COA6 in a multicellular organism, they turned to zebrafish. The embryos of these fish are transparent, so it’s easy to follow organ development. Given the phenotype, the fact that they can live without a functional cardiovascular system for a few days after fertilization was important too.

When the researchers knocked down expression of COA6 in zebrafish, they found that the embryos’ hearts failed to develop normally and they eventually died. The abnormal development of the fish hearts paralleled that seen in the human MRCD patient carrying the C1orf31/COA6 mutation. And reduced levels of Complex IV were present in the fish embryos.

Going back to yeast for one more experiment, Ghosh and colleagues decided to see whether Coa6p might be involved in delivering copper to Complex IV. They knew that Complex IV uses copper ions as a cofactor, and furthermore Coa6p had similarities to several other yeast proteins that are known to be involved in the copper delivery.

They tested this by supplying the coa6 null mutant with large amounts of copper. Sure enough, its respiratory growth defect and Complex IV assembly problems were reversed.  The delivery of copper kept the energy flowing in these cells. And this result showed that Coa6p is involved in getting copper to Complex IV.

These experiments showcase the need for model organism research even in the face of ever more sophisticated techniques applied to human cells. The mutation in human C1orf31/COA6 was discovered in a next-generation sequencing study, but yeast genetics established the relationship between the mutation and its phenotype. The zebrafish system allowed the researchers to follow the effects of the mutation in an embryo from the earliest moments after fertilization. And the rescue of the yeast mutant by copper supplementation offers an intriguing therapeutic possibility for some types of MRCD. Just another testament to the awesome power of model organism research!

YeastMine now lets you explore human homologs and disease phenotypes.  Enter “COA6” into the template Yeast Gene -> OMIM Human Homolog(s) -> OMIM Disease Phenotype(s) to link to the Gene page for human COA6 (the connection between COA6 and disease is too new to be represented in OMIM).  To browse some diseases related to mitochondrial function, enter “mitochondrial” into the template OMIM Disease Phenotype(s) -> Human Gene(s) -> Yeast Homolog(s).

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

Categories: Research Spotlight, Yeast and Human Disease

Tags: respiration, Saccharomyces cerevisiae, yeast model for human disease, zebrafish

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