New & Noteworthy

Lessons from Yeast: Poisoning Cancer

April 06, 2016

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In the book Dune, the mentat Thufir Hawat is captured by the evil Harkonnens and given a residual poison. He can only stay alive by getting a constant dose of the antidote. Once it is withdrawn, he will die.

A new study in the journal GENETICS by Dodgson and coworkers shows that the same sort of thing can happen to yeast that carry an extra chromosome. In this case, certain genes on the extra chromosome turn out to be like the residual poison. And a second gene turns out to be the antidote.

Once that second gene is deleted, the yeast cell dies. It has been deprived of its antidote.

This synthetic lethal phenotype isn’t just a cool finding in yeast either. Cancer cells invariably have extra and missing chromosomes. If scientists could find similar “antidote genes” in specific types of cancers and target them, then the cancer cell would die. And this would happen without damaging the other cells of the body that have a typical number of chromosomes.

The first thing these researchers did was to make separate yeast strains each with an extra chromosome I, V, VIII, IX, XI, XII, or XVI. The next step was to see what happens when every gene was deleted individually, one at a time, from each strain.

As expected, these yeast did pretty well when a gene on the extra chromosome was deleted. So, for example, a strain with an extra chromosome I tolerated a gene deleted from chromosome I. This makes sense as this just brings that gene back to its normal copy number.

But this was not the case with chromosomes VIII and XI. Here deleting genes on the extra chromosome often had a negative effect. This suggested that the screen probably had a high number of false positives and these researchers later confirmed this.

Likely reasons for the high number of false positives include the strain with the extra chromosome being W303 and the deletion strain being S288C, errors in the deletion collection itself, and what they refer to as neighboring gene effects. Basically this last one is the effect that deleting a gene has on nearby genes.

Once Dodgson and coworkers corrected for these problems, they found two broad sets of phenotypes – general and chromosome specific.

The general ones were the ones shared by most or all of the strains. These were deletions that affected the yeast no matter which chromosome they had an extra copy of.

For the most part, these genes were enriched for the Gene Ontology (GO) term vesicle-mediated transport, indicating that they have something to do with the transportation of substances in membrane-bounded vesicles. For example, deletion of MNN10, HOC1, and MNN11, genes all involved in protein transport and membrane-related processes, had a negative effect on many of the yeast strains with an extra chromosome. Consistent with this, brefeldin A, a drug that targets protein trafficking, negatively affected most of the strains.

Another gene that affected many of these strains when deleted was TPS1. This gene encodes a subunit of trehalose-6-phosphate synthase, a key enzyme for making trehalose, a molecule that helps yeast deal with stress. Perhaps not surprisingly, having an extra chromosome is stressful!

In addition to the genes that affect many strains with an extra chromosome, there were also genes that were chromosome specific. The best characterized of these was the EDE1 gene in the strain with an extra chromosome IX. Deleting EDE1 in this strain increased its doubling time by more than 80 minutes while only causing an increase of 5 minutes in the doubling time of wild type yeast. This was a severe phenotype in their assay.

They next tried to find which gene on chromosome IX might be responsible for the severe effect of deleting EDE1. Since EDE1 is known to be involved in endocytosis, they looked for genes involved in the same process. And they found one – PRK1.

The strain with a deleted EDE1 gene and an extra chromosome IX was rescued by deleting one copy of the PRK1 gene. The extra PRK1 gene was the poison and the EDE1 gene was the antidote.

If a similar pair could be found in cancers that often have the same set of extra chromosomes, then perhaps scientists could develop drugs that target an antidote gene. Now the cancer cells would die and the “normal” cells would be fine. Thanks again, yeast, for pointing us toward new ways to treat human disease.

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

Categories: Research Spotlight, Yeast and Human Disease

Tags: aneuploidy, cancer, synthetic lethal

Apply Now for the 2016 Yeast Genetics & Genomics Course

March 30, 2016

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For almost 50 years, the legendary Yeast Genetics & Genomics course has been taught each summer at Cold Spring Harbor Laboratory. (OK, the name didn’t include “Genomics” in the beginning…). The list of people who have taken the course reads like a Who’s Who of yeast research, including Nobel laureates and many of today’s leading scientists.

The application deadline is April 15th, so don’t miss your chance! Find all the details and application form here.

This year’s instructors – Grant Brown, Maitreya Dunham, and Marc Gartenberg – have designed a course (July 26 – August 15) that provides a comprehensive education in all things yeast, from classical genetics through up-to-the-minute genomics. Students will perform and interpret experiments, learning about things like:

• How to Find and Analyze Yeast Information Using SGD
• Isolation and Characterization of Mutants
• Transformation of Plasmids & Integrating DNAs
• Meiosis & Tetrad Dissection as well as mitotic recombination
• Synthetic Genetic Array Analysis
• Next-Gen. whole-genome and multiplexed DNA barcode sequencing
• Genome-based methods of analysis
• Visualization of yeast using light and fluorescence microscopy
• Exploring synthetic biology with CRISPR/CAS9-directed engineering of biosynthetic pathways

Techniques have been summarized in a completely updated course manual, which was recently published by CSHL Press.

Scientists who aren’t part of large, well-known yeast labs are especially encouraged to apply – for example, professors and instructors who want to incorporate yeast into their undergraduate genetics classrooms; scientists who want to transition from mathematical, computational, or engineering disciplines into bench science; and researchers from small labs or institutions where it would otherwise be difficult to learn the fundamentals of yeast genetics and genomics. Significant stipends (in the 30-50% range of total fees) are available to individuals expressing a need for financial support and who are selected into the course.

Besides its scientific content, the fun and camaraderie at the course is also legendary. In between all the hard work there are late-night chats at the bar and swimming at the beach. There’s a fierce competition between students at the various CSHL courses in the Plate Race, which is a relay in which teams have to carry stacks of 40 Petri dishes (used, of course). There’s also a sailboat trip, a microscopy contest, and a mysterious “Dr. Evil” lab!

Last year’s Yeast Genetics & Genomics Course was loads of fun – don’t miss out!

Categories: Announcements

Updated Genome Browser

March 27, 2016

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In an effort to provide a comprehensive view of sequence-based functional elements in Saccharomyces cerevisiae, we have upgraded our genome browser, and added new data tracks, to allow users to quickly and easily browse the information-rich yeast genome. We invite authors to work with us to integrate published data into our new JBrowse genome viewer pre- and/or post-publication. Please contact us if you are interested in participating or have questions and comments. Watch for the regular addition of new tracks to SGD’s JBrowse in the future!

Take a look at our newest video tutorial to get acquainted with JBrowse, and let us know if you have any questions or suggestions.

For more SGD Help Videos, visit our YouTube channel, and be sure to subscribe so you don’t miss anything!

Categories: Data updates, Website changes

Sometimes Simple is Better

March 23, 2016

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Diagnosing why something has gone wrong in a complicated system can be difficult. There are so many bells and whistles that you can easily get lost.

That’s why it can sometimes help to turn to a simple system and then apply what you have learned to the more complicated one. This will, of course, sound familiar to any scientists studying that marvel of a model organism, Saccharomyces cerevisiae.

For example, it is amazing what you can glean from this yeast about human brain and blood diseases. Even though, of course, baker’s yeast has neither blood nor a brain!

This becomes very clear in a study out in PLOS Genetics by Fernandez-Murray and coworkers. In this study they use yeast to help figure out why mutations in the SLC25A38 gene in people leads to something called congenital sideroblastic anemia. And even better, their work hints at a possible treatment.

People with sideroblastic anemia make too little hemoglobin in their red blood cells and have too much iron in the mitochondria close to the nucleus (perinuclear mitochondria). The current treatment for this condition is not ideal, involving lots of transfusions and iron chelation.

Sometimes people are born with this anemia and sometimes people get it later in life. One subset of the inherited version happens because a gene with an unknown function, SLC25A38, isn’t working correctly. This group of patients is the focus of this study.

Fernandez-Murray and coworkers started out by using yeast to figure out what the yeast homolog, HEM25, does in a yeast cell. When the gene was deleted, the cells made about 50% less heme than wild type yeast and adding back the human gene, SLC25A38, to this deletion strain restored heme levels. Looks like they had made a yeast model of this inherited anemia.

Previous work had suggested that SLC25A38 might be a glycine or serine transporter and the next set of experiments confirmed it as a glycine transporter in a couple of ways. In both, they took advantage of cases in which yeast can use glycine as their sole nitrogen source if the glycine can make it into the mitochondria.

In the first case, they showed that yeast cells deleted for HEM25 grew poorly on plates where glycine was available as the only nitrogen source. In the second case, they showed that cells deleted for both SER1 and HEM25 grew poorly on plates where again glycine was the only nitrogen source. This last result confirms HEM25 as a glycine transporter since yeast deleted for the SER1 phosphoserine aminotransferase can only grow in the absence of serine if they can get glycine into their mitochondria. (There isn’t space to go into it here, but they also showed that HEM25 was not a serine transporter.)

OK so now they had created a yeast cell that mimicked the effect of sideroblastic anemia and figured out why people with a mutated SLC25A38 gene had the condition. Now it was time to find a treatment.

The researchers came up with three possibilities. The first treatment was just to give the yeast extra glycine, the second was to drive glycine synthesis within the cell by adding a lot of serine, and the third was to add a downstream precursor of heme synthesis, 5-aminolevulinic acid (5-Ala).

They tested each scenario on yeast cells deleted for HEM25 and found that both glycine and 5-Ala worked to restore heme synthesis, but that added serine had no effect. Both glycine and 5-Ala returned heme levels to that seen in wild type.

Of course we aren’t yeast, so they next tested their treatment on something a bit more complicated — zebrafish. By using morpholino technology to knock down both copies of the zebrafish SLC25A38 homolog, SLC25A38a and SLC25A38b, the researchers managed to lower a zebrafish’s heme levels to about 50% of normal.

When they gave these zebrafish extra glycine or 5-Ala, their heme levels did not improve. They were still anemic!

After a bit of thought, the researchers realized that folate might be what the zebrafish were missing. In work that we didn’t have time to go over before, the researchers had shown that a folate dependent pathway was critical for getting heme levels up to normal.

Yeast could get away without added folate in these experiments because they make their own. However, zebrafish, like people, do not.

So the final step was to try to add both glycine and folate to these fish. Now the zebrafish’s heme levels returned to about 80% of normal.

These results suggest a better treatment for some people with sideroblastic anemia — added folate and glycine. And it all came from studying the problem in the simpler, bloodless S. cerevisiae. Nice work again yeast.

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

Categories: Research Spotlight

Tags: anemia, human disease, model organism, zebrafish

Join SGD at The Allied Genetics Conference

March 22, 2016

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SGD will be attending The Allied Genetics Conference (TAGC) in Orlando, Florida, July 13–17, 2016! For the first time ever, the meetings of the yeast, C. elegans, ciliate, Drosophila, mouse, and zebrafish model organism communities will be united under one roof, along with a new meeting on population, evolutionary, and quantitative genetics.

Submit your abstracts now! Abstract submission closes March 23, 2016, but advance registration is available until June 29. If you want GREAT science and access to the leaders of the field, then TAGC is the place for you. SGD will be there, will you?

Categories: Conferences

New SGD Help Video: YeastMine Scenario

March 17, 2016

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If you’re not already using YeastMine to answer all your questions about the Saccharomyces cerevisiae genome and the gene products it encodes…you should be! YeastMine enables slicing and dicing of data from SGD in any way you choose. Ask questions like “Which genes can mutate to confer oxidative stress resistance, and what biological processes are they involved in?” or “Are there any undiscovered subunits of the mitochondrial ribosome?”

Take a look at our newest video tutorial to dig into YeastMine, and let us know if you have any questions or suggestions.

For more SGD Help Videos, be sure to visit our YouTube channel!

Categories: Tutorial

SGD March 2016 Newsletter

March 15, 2016

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SGD periodically sends out its newsletter to colleagues designated as contacts in SGD. This March 2016 newsletter is also available on the community wiki. If you would like to receive the SGD newsletter in the future please use the Colleague Submission/Update form to let us know.

Categories: Newsletter

Budding Yeast Diversifies its Phosphatase Portfolio

March 10, 2016

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You’ve probably heard the old saying, “Don’t put all your eggs in one basket.” The idea of course is that the wise thing to do is to spread out your possessions so when something happens to one set, you still have the rest. (See what Homer and Marge Simpson think of this saying.)

If it really is wise to follow this saying, then according to the results of a new study just published in GENETICS by Kennedy and coworkers, the budding yeast S. cerevisiae is wiser than the fission yeast S. pombe. Well, at least as far as for one part of entry into mitosis.

To enter mitosis, every eukaryote tested so far needs to increase the activity of cyclin dependent kinase 1 (Cdk1). Dephosphorylation of a key tyrosine residue in Cdk1 is an important part of this increased activity.

One of the big players in this dephosphorylation is the phosphatase Cdc25 in S. pombe or Mih1p in S. cerevisiae. In fact, it is so important in S. pombe, that deleting it is lethal. These poor cells arrest in G2 and eventually die.

The same is not true for S. cerevisiae. Deleting MIH1 has only mild effects—a slight delay in entering mitosis and starting anaphase. The phosphorylation on the key tyrosine on Cdk1p, Y19, remains for a longer period of time in this strain, but does eventually clear, explaining the delayed mitotic entry.

One interpretation of this result is that S. cerevisiae has spread its Cdk1 phosphatase activity over multiple proteins. Knocking out MIH1 still leaves enough Cdk1 activity to allow the cell to enter mitosis, albeit more slowly.

One likely suspect in S. cerevisiae is Ptp1p. Previous work had shown that in S. pombe, Pyp3, the homologue of Ptp1p, can also dephosphorylate Cdk1-Y19.

Kennedy and coworkers found that deleting both MIH1 and PTP1 in S. cerevisiae had a more severe effect on mitotic entry and exit from anaphase compared to deleting only MIH1. In addition, the level of Y19 phosphorylation on Cdk1p remained for an even longer period in the mih1 ptp1 deletion strain. But it was still not lethal and the cells did eventually manage to pass through mitosis.

These results suggest there is still another player involved. The next suspect these researchers focused on was protein phosphatase 2A (PP2A). Previous work had shown that mutation of the B-regulatory subunits of PP2A, Cdc55p and Rts1p, both affect Cdk1p phosphorylation.

Because of the multiple routes by which PP2A can affect entry into mitosis, the authors designed an in vivo phosphatase assay to accurately measure the level of phosphorylation of Y19 of Cdk1p. The results of this assay suggested that PP2A^Rts1 and not PP2A^Cdc55 affected the phosphorylation state of Y19.

Kennedy and coworkers finally managed to kill off their yeast by deleting MIH1, PTP1, and PP2A^Rts1! They had finally found enough of this yeast’s phosphatase activity to mimic the effects of just Cdc25 in the fission yeast S. pombe.

Using immunopurified protein complexes, Kennedy and coworkers were able to show that both Mih1p and Ptp1p could dephosphorylate Y19 of Cdk1. They could not, however, see dephosphorylation by PP2A^Rts1. It could be that their in vitro assay did not detect it for this protein or that PP2A^Rts1 works on a different phosphatase that affects Cdk1.

Bottom line is that the budding yeast has evolved such that the phosphatase activity needed to enter mitosis is spread out over multiple proteins. The fission yeast evolved in a way that kept all of its phosphatase eggs in the same basket, Cdc25. We’ll let you decide which yeast you think is the wiser.

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

Categories: Research Spotlight

Tags: Cdk1, mitosis, phosphatase, PP2A, redundant regulation

The Benefits of Sex

March 03, 2016

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While you doggedly swipe right and left or wait night after night at that club, you may be wondering whether it is all worth it. Biologists have been wondering something similar.

Now they haven’t been wondering about the value of sex…since everything from amoebas to zebras has sex, it must be pretty important. No, the hard part has been figuring out why it is so beneficial.

On balance it can seem that the minuses of disease risk and passing on only half of your DNA outweighs the benefits of the combining two individual sets of DNA for some brand new combination. A new study by McDonald and coworkers in Nature using our old friend S. cerevisiae provides compelling evidence for a couple of ways that sex is good for a species.

First it is a way of combining individual beneficial mutations into a single individual. Now rather than having a couple of well adapted individuals battling for supremacy, the mutations can merge into one super beast that can outcompete everyone else.

This benefit, recombination speeds adaptation by eliminating competition among beneficial mutations, had been predicted and goes by the name of the Fisher-Muller effect. But this is the first time scientists have actually seen it playing out at the DNA level.

The second big benefit of sex is freeing good mutations from a bad genetic background. Now the beneficial mutation is not weighed down by other negative mutations. It’s like finally getting rid of that concrete block tied around your ankle.

Yeast is an ideal system for studying the benefits of sex because it can happily exist as a sexual or asexual creature. This means that researchers can directly compare the two in the same experiment. Which is just what McDonald and coworkers did.

They followed 6 sexual and 12 asexual populations through about 1000 generations of adaptation. The only difference between the asexual and sexual populations was, as you might have guessed, sex.

The sexual populations included 11 bouts of sex. In other words, every 90 generations or so, an ‘alpha’ cell would swipe left and find an ‘a’ cell to hook up with.

As expected and has been seen before, the sexual populations were much better adapted to their environment than were the asexual populations. Sex is clearly a good thing! The next step was to tally up the mutations in each population to try to figure out why.

What McDonald and coworkers found was that there wasn’t a lot of difference in the mutations that crop up in each. Over time, both groups had about the same number and ratio of intergenic, synonymous, and nonsynonymous mutations.

The big difference between the asexual and the sexual populations was in the mutations that became fixed. In the sexual group, most mutations were weeded out over time. In their experiment, 78% of mutations became fixed in the asexual population while only 16% hung around in the sexual population.

Sheer numbers wasn’t the only difference between the two either. The kinds of mutations that became fixed differed significantly in both as well.

In the asexual population, each of the three kinds of mutations fixed at around the same rate. Around 75-80% of intergenic, synonymous and nonsynonymous mutations became established in this population.

It was a different story in the sexual population. Here, 22% of the nonsynonymous, 11% of the intergenic and none of the synonymous mutations became fixed. It seems like only mutations that make a difference end up getting selected for.

Further analysis revealed two big reasons why the two populations differed. First, good mutations ended up getting stuck with other bad mutations in the asexual population. This blunted the positive effects of the beneficial mutation.

And second, the various good mutations tended to be spread out among different groups in the asexual population. The end result was that instead of working together, these groups battled each other for supremacy resulting in some beneficial mutations being lost.

So no need to wonder anymore about the benefits of sex to a species. It is a strong purifier, weeding out unimportant or damaging mutations and a powerful aggregator, squirrelling all the good ones into one group. No wonder most every beast does it!

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

Categories: Research Spotlight

Tags: Fisher-Muller effect, mutation, nonsynonymous, selection, synonymous

Not Quite The Same

February 24, 2016

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Imagine a world where you either make your own bread from scratch or have it delivered to your doorstep. Not much of a difference, right? Either way you’re eating bread.

Except of course that the two are pretty different. Having your bread delivered frees up time to do other things.

It turns out that something similar may be going on in our old friend, Saccharomyces cerevisiae. According to a new study in Nature Microbiology by Alam and coworkers, a yeast able to make its own amino acids or nucleobases works very differently than one that can’t but is supplied all the nutrients it can use.

This is important for yeast studies because these sorts of auxotrophic markers are used all the time. It means researchers need to be very careful about comparing a wild type yeast strain with a yeast strain deleted for, say, URA3, but grown in the presence of plenty of uracil. The two are not equivalent.

And the study may even have implications for other folks as well. For example, cancer cells have many mutations, some of which can be in metabolic genes. These mutations may affect how these cancers respond to drug treatment.

This all might not matter much if the effects were small. But they weren’t in this study. The changes were profound.

Alam and coworkers compared 16 different strains that were identical except that four different metabolic genes were deleted in various combinations. These genes included HIS3, LEU2, URA3 and MET15 (also known as MET17).

Using mRNA sequencing, they found that 5,011 out of 5,923 transcripts were affected in one strain or the other. This is 85% of the coding genome of yeast!

While not all of these changes were huge, 573 of them differed by 2-fold or more. In other words, around 10% of the genome is significantly affected when a yeast cell is provided a nutrient instead of having to make it itself. Not surprisingly, the affected genes were enriched for those involved in metabolic activity and enzymatic function.

The authors next looked at some publically available gene expression experiments that used auxotrophs in the same BY4741 background. These studies primarily looked at the how the knocking out of a specific gene affected global gene expression. The vast majority of deleted genes were not metabolic.

Alam and coworkers found that a sizeable minority of changes overlapped with the ones they saw with deleting HIS3, LEU2, URA3 or MET15. In other words, on average, at least 18% of the changes in the genes identified in these studies were not due to the gene deletion they were studying. They were instead due to the deletion of a “housekeeping” metabolic gene.

This all might be less of a big deal if the affected genes were always the same. Then you could just be on the lookout for these genes when using a specific auxotroph.

Unfortunately, it isn’t so easy. Different combinations of deleted metabolic genes yield different changes in gene expression patterns with very little overlap.

So, for example, when the authors compared a his3 deletion strain to one deleted for both HIS3 and URA3, a very different set of genes was affected. And these were different from a strain deleted for HIS3 and MET15, and so on. Looking at all the possible combinations confirmed that universal gene targets were rare.

The bottom line from these experiments is that researchers need to be very careful about the strains they compare because they may not be as equivalent as they think. Just because the older Star Trek films and the newer ones have a Spock, that doesn’t mean the half Vulcan is the same in both. Just ask Uhura.

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

Categories: Research Spotlight

Tags: auxotrophic markers, auxotrophy, gene expression, metabolic background, supplementation

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