New & Noteworthy

When Heat is Not the Burning Issue

May 04, 2012

When is a temperature sensitive mutation not a temperature sensitive mutation? When the process being studied is affected by temperature too.

Mutations conferring temperature sensitivity – that is, a phenotype that only appears at higher- or lower-than-normal growth temperature due to the loss or alteration of function of a gene product at that temperature – have for decades been an invaluable tool in dissecting many biological processes in yeast and other model organisms. Now there is reason to question whether some temperature sensitive phenotypes might actually be the result of a more complex interaction between multiple genes.

This synthetic genetic interaction is easy to mistake for the effect of a single mutation. A scientist starts out with a mutation that weakens a gene in a certain process. Unfortunately for the scientist, the process being studied is itself weakened by higher temperatures. The effect of the higher temperature combines with the effect of the mutation to shut down the process. The mutation looks temperature sensitive even when it isn’t.

And this does not appear to be merely a theoretical concern. As Paschini and coworkers show in a new study out in GENETICS, something similar may have happened with key mutations used to study telomere function in yeast.

These researchers looked at several mutations but we’ll focus on the work they did with cdc13-1. A key experiment that had been previously done with this mutation dealt with the effects of the loss of cdc13 function in the absence of RAD9.

Basically, researchers had found that prolonged incubation of a cdc13-1/Δrad9 strain at 36° severely compromised viability. These results were used to infer CDC13 function based on its loss at 36°. However, Paschini and coworkers provide compelling data that cdc13-1 behaves equally poorly at 23° and 36°.

First off, they showed that prolonged incubation of wild type yeast at the temperatures used in these studies (36°) resulted in shorter telomeres. They found very little effect on telomere length at 32°.

Next they showed that biochemically, cdc13-1 didn’t behave like a temperature sensitive mutation. Strains with cdc13-1 produced around 4-fold less protein at both 23° and 36° and the protein that was made bound telomeres equally well at both temperatures.

They argue from these two pieces of data that the loss of viability comes from a combination of the compromised cdc13-1 mutation and the effects of higher temperature on telomere function. Something is going on in the rad9 experiment but it is not due to an increased loss of CDC13 function at higher temperatures. There is some other factor involved that is being inhibited.

Of course it could be that cdc13-1 still confers temperature sensitivity, but that they didn’t have the right biochemical assay to see it. To address this issue, they generated five new mutations in cdc13 that behaved more like traditional temperature sensitive mutations.

They focused on one, cdc13-S611L, that was compromised for protein production at temperatures of 32° and above. They then repeated the rad9 double mutant experiment at 32° and 36°. They found that viability was compromised only at 36° even though Cdc13p was equally absent at both 32° and 36°. These results suggest that the loss of viability at 36° is not only the result of cdc13-1.

If this and other results hold up, scientists will need to rethink what previous experiments meant and they may need to modify their models. This should also get other researchers thinking about their temperature sensitive mutations.  It is important to confirm biochemically that a mutation indeed makes a specific gene product temperature sensitive. Because sometimes even if it quacks, it isn’t a duck…

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

Categories: Research Spotlight

Tags: cdc13, synthetic lethal, telomere, temperature sensitive, yeast

SGD Spring 2012 Newsletter

April 30, 2012

SGD sends out its quarterly newsletter to colleagues designated as contacts in SGD. This Spring 2012 newsletter is also available online. If you would like to receive this letter in the future please use the Colleague Submission/Update form to let us know.

Categories: Newsletter

Idling Transcription Factors

April 24, 2012

A new study by Lickwar and coworkers suggests that many transcription factors fidget on and off the DNA, waiting for some signal to get to work.  Once they get that signal, they clamp down and start affecting the activity of nearby genes.

If true this would help explain some perplexing results researchers have been getting with chromosomal immunoprecipitation (ChIP) assays.  Transcription factors appear to be bound at many places where they are not affecting any nearby genes.  Now we might have an idea why.

These researchers came up with this model through the use of an elegant, in vivo competition study.  What they did was to set up a yeast strain that contained two different versions of the transcription factor Rap1p.  One version was tagged with a FLAG epitope and was under the control of RAP1’s endogenous, constitutive promoter.   The other version was tagged with a Myc epitope and was under the control of an inducible promoter.

They started out seeing where Rap1p was bound in the absence of the inducer by using an antibody against FLAG.  This is the equivalent of a typical ChIP experiment.  They found Rap1p was bound in many places throughout the genome including sites where it did not appear to affect any nearby genes.

Then they added the inducer galactose and at various time points repeated the ChIP experiment with antibodies against either FLAG or Myc.  They were basically looking for how quickly the Myc-tagged Rap1p replaced the FLAG-tagged Rap1p with the idea that less stably bound transcription factors would be replaced more quickly.

They indeed found that some sites were better able to withstand the onslaught of Myc-tagged Rap1p.  And more importantly, that these sites were near genes most influenced by Rap1p.  In other words it appears that the more stably bound the Rap1p, the bigger the effect it has on nearby genes.

They then went on to show that more stable binding correlated with lower nucleosome occupancy and stronger in vitro binding.  From this data they propose a model where the level of the effect on transcription is the result of a competition between nucleosome and transcription factor binding.  Stronger transcription factor binding keeps nucleosomes away so transcription can proceed.

They took the model one step further and proposed that transcription factors are idling on the DNA, waiting for a signal to bind more tightly and influence the activity of nearby genes.  In other words, transcription factors are ready to have an effect at a moment’s notice.

This part of the model still has to be proven though.  All that has been shown so far is that a slow off rate is required for effective transcription activation by Rap1p.  What we don’t know is whether this translates to other transcription factors or if idling Rap1p is ever more stably bound.

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

Categories: Research Spotlight

Tags: ChIP, chromosomal immunoprecipitation, competition, off rate, stable binding, transcription factor, yeast

New data tracks added to GBrowse

April 23, 2012

SGD has added a new mix of data tracks to our GBrowse genome viewer from seven publications covering transcriptome exploration via tiling microarrays (David et al. 2006), genomic occupancy of RNA polymerase II and III and associated factors (Kim et al. 2010; Ghavi-Helm 2008), 3′ end processing (Johnson et al. 2011), histone H2BK123 monoubiquitination (Schulze et al. 2011) and high-resolution ChIP by a novel method called ChIP-exo (Rhee et al. 2011; Rhee et al. 2012). Download data tracks, metadata and supplementary data by clicking on the ‘?’ icon on each data track within GBrowse or directly from the SGD downloads page. We welcome new data submissions pre- or post-publication and invite authors to work with us to integrate their data into our GBrowse and PBrowse viewers. Please contact us if you are interested in participating or have questions and comments. Happy browsing!

Categories: New Data

Tags: ChIP-exo, histone modifications, RNA polymerase II, RNA polymerase III, transcriptome

Yeast to the Rescue (of Personalized Medicine)

April 09, 2012

Genomic scientists are quickly being overwhelmed by all of the data they are generating.  As trillions of A’s, T’s, C’s and G’s come pouring out of sequencers all over the world, how is anyone going to make sense of it all?

One idea is to use yeast to quickly figure out what effect certain differences have on a gene’s function.  Now this won’t be that useful for differences outside of genes or in genes that aren’t shared by yeast and humans.  But that still leaves an awful lot of SNPs that we might be able to better understand using the awesome power of yeast genetics.

In the most recent issue of GENETICS, Mayfield and coworkers use yeast to study a large number of variants in the human cystathione-beta synthase (CBS) gene.  They chose this gene because it is involved in the metabolic disease homocystinuria, different variants respond to treatment in unpredictable ways, and it can substitute for the yeast homolog, CYS4.

The hope was that they would be able to group CBS variants based on their phenotype in yeast and that this would let them predict which treatments would work for novel variants.  They were definitely able to group variants based on phenotype.  Time will only tell whether they can use this to better treat patients who come into the clinic with novel variants of the gene.

They looked at 84 known alleles of CBS that affected an amino acid with a single base pair change (81 were from homocystinuria patients).  They grouped these alleles based on growth phenotypes in yeast under varying conditions.  For example, they determined how well each grew in the absence of glutathione.  Only those alleles that were still functional would support growth.  They also varied the amount of glutathione, looked at the effect of heme and vitamin B6, studied metabolite profiles with mass spectroscopy and so on. 

From this they were able to group many of the alleles in clinically meaningful ways.  This means that when a novel allele comes up in a patient, they can screen it in this yeast assay to see if it falls within a known group.  At least 38 never before seen missense mutations have been found in the CBS gene since 2010 and undoubtedly new ones will keep appearing as more DNA is sequenced.

The study also revealed alleles that were more difficult to interpret in this assay.  For example, some alleles known to cause disease did not affect yeast growth.  This might mean that their particular mutation needs something human and/or patient specific to manifest itself or that the enzyme function is fine but something else is wrong. 

This study provided a powerful proof of principle.  The next step will be to see how well it works in practice and if any patients can benefit.

Benjamin deals with his homocystinuria

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

Categories: Research Spotlight, Yeast and Human Disease

Tags: CBS gene, CYS4, high throughput screen, homocystinuria, personalized medicine, yeast

Studying Aging in Yeast Just Got Easier

April 02, 2012

Watching a yeast cell age can be a real pain.  In budding yeast like Saccharomyces cerevisiae, the buds quickly outnumber the mom.  Which means scientists need to remove the buds as they appear.

Up until now, scientists have had to use a 50-year-old method that involves removing the buds by hand.  Not only is this labor intensive, but the field is held back by the inability to use high resolution microscopy to investigate the aging process. 

These technical limitations may soon be swept aside with a new microfluidic dissection technique described by Lee and coworkers in a recent study out in PNAS. These researchers were able to monitor 50 aging yeast at once with a variety of microscopic techniques without having to remove the buds by hand.  And unlike the older technique, they were able to keep a constant environment for the yeast cells (i.e. no decrease in nutrients and/or build up in wastes).

Basically Lee and coworkers tucked the yeast mother cells under a micropad which they then washed with a constant flow of nutrients.  Because the daughter cells are smaller than the mother, they are washed away as they emerge.  So no manual bud removal is required.

Sounds convenient but the researchers needed to show that this new technique gave similar results as compared to the old one.  And they did.

They showed that mutant strains behaved similarly with both techniques.  So a SIR2 deletion mutant still had a shorter lifespan and a FOB1 deletion mutant still lived longer with microfluidic dissection.  Not only that, but the number of divisions in an average yeast’s lifetime was comparable with both techniques.  At first blush the techniques do seem comparable.

Now they were ready to take their new technique out for a spin to see what it could do.  First they were able to show heterogeneity in how yeast cells age.  Some cells died as spheres around their 12th division while others died as ellipsoids after their 25th division.  The shape of the yeast later in life correlated with how long that yeast lived.

The researchers were also able to use GFP to explore the vacuoles of aging yeast.  They found three classes of vacuoles: tubular, fused, and fragmented.  The tubular vacuoles were only found in the longer-lived ellipsoid yeast.

Researchers could not have discovered these properties of aging yeast without the new microfluidic dissection technique.  And these findings are really just the tip of the iceberg of what can now be learned about aging by studying yeast.  It will be exciting to see what else scientists will be able to learn about the twilight of a yeast cell’s life.

Life and Death of a Single Yeast

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

Categories: Research Spotlight

Tags: aging, microfluidic, vacuole, yeast

Sopping up Uranium with Yeast

March 23, 2012

Yeast may be good for more than making bread and beer or understanding how eukaryotes like humans work.  They may also be useful for cleaning up high volume, low concentration waste uranium (think uranium waste water).

The idea would be to add yeast to the contaminated area, have the yeast take the uranium up, put the yeast into radioactive waste and repeat with new yeast.  This would be a relatively cheap, simple way to detoxify this form of radioactive waste.

An obvious way to improve on this idea is to identify yeast strains that can accumulate more uranium than the wild type strain.  In a new study out in Geomicrobiology Journal, Sakamoto and coworkers have started down this path by identifying genes that allow yeast to grow in the presence of uranium and those involved in uranium accumulation.

They did this with two different screens using a set of 4,098 non-essential gene deletion strains.  In the first they identified 13 strains that grew more poorly than wild type at 0.5 mM uranium.  And in the second, they identified 17 strains that accumulated less uranium than wild type.

There was very little overlap between the two sets of strains suggesting different pathways (or sets of pathways) may be involved in accumulation and growth.  However, there were two deletion strains that showed up in both screens.  Both of the identified genes, PHO86 and PHO2, are involved in phosphate metabolism.

These genes definitely make sense.  A number of previous studies had hinted strongly that uranium accumulates on the surface of yeast in the form of insoluble uranium-phosphate complexes. 

The idea behind the importance of these genes is that yeast deals with higher uranium levels by scavenging more phosphate.  When genes involved in this process are knocked out, the yeast can’t get the extra phosphate it needs to form the insoluble uranium phosphate complexes.  Now it grows poorly and has less uranium on its surface. 

It will be interesting to see how the other genes are involved in uranium survival or accumulation.  Perhaps one day researchers will be able to turn yeast into a grade A uranium sponge.  Here’s hoping they can!

For those really interested, here is a list of the genes identified in each screen:

Uranium sensitive: PHO2, PHO84, PHO86, PHO87, VPS74, ENT5, CPR1, GLO2, OPI1, ATG15, PTC6, SLC1, and uncharacterized ORF, YPR116W.

Uranium accumulation: OPI1, PHO86, APL4, PEX10, VPS74, PHO2, SPT20, GAL11, SWP82, IVY1, FLO1, DIT2, RPL2A, and uncharacterized ORFs, YGL214W, YJR098C, YNL035C, and YPR116W.       

A nice lecture on bioremediation (using biology to clean up toxic waste)

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

Categories: Research Spotlight

Tags: bioremediation, nuclear waste, Saccharomyces cerevisiae, uranium, yeast

Not any Cyclin Will Do

March 16, 2012

Getting through a cell cycle is a complicated process. All sorts of proteins need to work and stop working at the right times in the right places to get the DNA copied, get the cells growing larger, have the cells divide and so on.

Key regulators in this process are the cyclins and their dependent kinases. Different cyclins are expressed at different points in the cell cycle, at which time they direct their cyclin-dependent kinase (CDK) to the appropriate subset of proteins to be phosphorylated. A big part of cell cycle regulation, then, comes from when a cyclin is expressed. But this is not the whole story.

In a study out in this month’s issue of GENETICS, DeCesare and Stuart showed that at least for the B-type cyclin Clb5 in the yeast S. cerevisiae, timing isn’t everything. And even more unexpectedly, they found that a key part of this cyclin’s specificity comes from its N terminus.

In yeast, Clb5 is involved in premeiotic DNA synthesis. Many researchers had previously argued that any B-type cyclin expressed at the right time would be sufficient to promote this function. DeCesare and Stuart were able to show that this was not the case by putting two different cyclins, Clb1 and Clb3, under the control of the CLB5 promoter. These cyclins were now expressed at the right time but neither could substitute for Clb5.

The authors next set out to discover what part of Clb5 conferred this specificity by creating chimeric versions of Clb3 and Clb5. They identified two regions in Clb5 important for premeiotic DNA synthesis — a hydrophobic patch and the N terminus.

The hydrophobic patch was expected; this region is highly conserved in all cyclins and has previously been shown in to be involved in interacting with protein substrates. But the N terminus was a surprise. It was thought to be involved primarily in cyclin stability and/or subcellular localization and not protein-protein interactions.

The authors were not able to identify which specific part of the N terminus of Clb5 was involved in conferring specificity. In their experiments, there was a gradual decline in the ability of the Clb3-Clb5 chimera to promote premeiotic DNA synthesis as more and more of Clb5 was replaced with Clb3. It is as if the whole region is involved in determining specificity.

And the decreasing ability of the Clb3-Clb5 chimera to induce premeiotic DNA synthesis was not due to the loss of kinase activity. When paired with Cdc28 (also known as Cdk1), all of the chimeras in the experiment were equal or even more active than the wild type Clb5/Cdc28 pair.

What it looks like is happening is that Clb5 uses both its hydrophobic patch and its N terminus to bring appropriate proteins to Cdk1 for phosphorylation. Different parts of each region are used to interact with different subsets of proteins involved in premeiotic DNA synthesis. At least for Clb5 and premeiotic DNA synthesis, it looks like not any cyclin will do.

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

Categories: Research Spotlight

Stellar Strain Yields Increased SAM Production

March 10, 2012

Yeast geneticists often go the extra mile to get their mutant. But Huang and coworkers went hundreds of extra miles to get theirs. Hundreds of miles straight up, that is.

In a recent study published online in the Journal of Applied Microbiology, Huang and coworkers identified and improved upon a strain of Saccharmoyces cerevisiae that makes increased amounts of S-adenosyl-L-methionine or SAM. This is an important chemical in many pharmacological and medical uses and is primarily made via microbiological synthesis. Increased production would be an obvious boon to researchers and the pharmaceutical industry.

What makes this study interesting is that the researchers obtained their initial strain from outer space. They shot cultures up into space on a satellite where the poor yeast had to endure the harsh environment there for 18 days. Researchers then collected the samples when the satellite returned to Earth.

Out of six hundred random clones from the flight, researchers found 43 that made at least 10% more SAM than their wild type counterpart. A second round of selection yielded strain H5M147 which made 84% more SAM than the wild type strain.

Unfortunately the researchers were not able to (or did not report in this study) why the strain made extra SAM. They used a technique called AFLP that allowed them to see that there were differences in the new and host strain’s genome, but it did not allow them to pinpoint what those differences were nor which ones were significant. That will have to wait for a future study.

They did manage to ramp up SAM production even further in this strain though. First they added an extra copy of a key player in SAM production, the MAT2 gene. Researchers have tried to coax other strains of yeast to make more SAM by adding MAT2 but to no avail. This space strain apparently has genetic mutations that allow extra MAT2 to increase SAM production. The new strain with the integrated version of MAT2 was called H5MR38.

Finally the researchers tinkered with culture conditions to optimize SAM production in H5MR38 further. They found that using sucrose as the carbon source and adding peptone to yeast extract and urea yielded the most SAM.

In the end, Huang and coworkers managed to get 7.76 grams of SAM per liter of culture after 84 hours. This compares to the previous high in Sacchromyces cerevisiae of 5.7 gram per liter after 120 hours and 3.6 grams per liter from a strain of Pichia pastoris in 100 hours. Clearly their space-derived yeast strain is an improvement over anything else identified so far.

Huang and coworkers aren’t the only ones putting yeast into space

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

Categories: Research Spotlight

Engineering Magnetic Yeast

March 02, 2012

With a bit of genetic manipulation and a hearty diet of iron, Nishida and Silver report in the latest issue of PLOS Biology that they have caused yeast cells to become magnetic. And this isn’t just a parlor trick. Their research could one day help other scientists create new therapies for the sick and new applications for research and industry.

The first step in the process was to load the yeast up with magnetic iron. The authors took a couple of different approaches.

The simplest was to grow the yeast in lots of iron. Surprisingly, without any other manipulation, this was enough to make the yeast a bit magnetic. But the authors wanted more magnetism.

To accomplish this goal, they needed to keep yeast from transporting excess iron to their vacuole where it is nonmagnetic. They did this by knocking out the gene encoding the vacuolar iron transporter, CCC1.

When grown in lots of ferric citrate, the ccc1Δ strain was about 1.8 times more magnetic than wild type. Nice, but to get even more magnetic yeast, Nishida and Silver added back the three human genes necessary to reconstitute human ferritin. This new strain was now about 2.8 times more magnetic than wild type.

None of this was really earth-shattering yet. Scientists knew that iron was needed to make a cell magnetic and that ferritin-iron complexes were a bit magnetic. What made these initial studies important was that they gave Nishida and Silver the tools to study the underlying mechanisms of magnetism.

The authors took a directed approach to study this problem and knocked out genes known to be involved in iron homeostasis or oxidative stress. Of the 60 knockout strains tested, tco89Δ was the only one to consistently be less magnetic than the wild type strain. On average it was about two fold less magnetic.

Tco89p is a nonessential part of TORC1, a complex involved in the regulation of cell growth in response to nutrients, stress, and redox states. As might be predicted from TORC1 function, the authors determined that nutrients and the redox state of the medium affected the yeast’s magnetism. They then expanded their screen to look for genes involved in carbon metabolism and mitochondrial redox that might affect magnetism and discovered several (POS5, YFH1, SNF1, and ZWF1).

The current model is that the redox state within the cell and in particular, within the mitochondria, impacts the amount of iron precipitation and hence magnetism in yeast. This is consistent with the iron deposits the authors saw in electron micrographs of the mitochondrial membrane of the magnetic yeast.

These findings should help point researchers in productive directions for engineering magnetic cells in other systems but it is only a first step. Science has a long way to go before therapies based on cell magnetism are helping patients.

More details on these magnetic yeast

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

Categories: Research Spotlight

Tags: ferritin, magnetic, MRI, yeast

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