New & Noteworthy

Yeast Genetics Meeting Abstract Deadline Extended

April 25, 2014

The deadline for abstract submission for the Yeast Genetics Meeting has been extended. Abstracts will now be accepted until 4 PM EDT, Monday April 28th. Don’t miss this chance to showcase your work for the yeast research community!

Categories: Conferences

Tags: Yeast Genetics Meeting

Yeast Genetics Meeting Abstracts Due Tomorrow

April 23, 2014

The Yeast Genetics Meeting abstract submission deadline is tomorrow: Thursday, April 24.  Be sure to get your abstract in by the deadline so that it will be programmed and included in the online abstract search. IMPORTANT NOTE: NEW THIS YEAR: The full text of all abstracts submitted by the deadline date will ONLY be available online and will not be printed in the program book. The program book will still contain the full schedule information, including platform and poster session date, time, title, authors, gene index and the listing of exhibitors. Late abstracts will only be accepted if space permits and will not be included in the online search.

This meeting is an exceptional opportunity for you to present your work to an international audience. There is no better way to advance your career than meeting people in the field. Financial Aid is available and presenting authors are given first priority.

Plan now to attend YGM to hear the latest research that is happening around the world, re-connect with fellow yeast researchers, network for a job, meet future collaborators, see the latest equipment available from the exhibits, and enjoy Seattle in July.  Last but not least, you’ll have the opportunity to talk with SGD staff about what’s new at SGD, get individual instruction on tools and resources, and contribute your thoughts about SGD’s future development! A preliminary schedule for the YGM is now available. 

SGD staff join with the YGM organizers in looking forward to seeing you in July.

Categories: Conferences

Tags: Yeast Genetics Meeting

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

SGD Spring 2014 Newsletter

April 17, 2014

SGD periodically sends out its newsletter to colleagues designated as contacts in SGD. This Spring 2014 newsletter is also available on SGD’s 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

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

Apply Now for the CSHL Yeast Genetics & Genomics Course

March 31, 2014

For more than 40 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 many of today’s leading scientists and two Nobel laureates (Randy Schekman in 1975 and Jim Rothman in 1985, who both won the 2013 Nobel Prize). 

Now it’s your chance to attend this summer course (July 22 – August 11) and get a comprehensive education in all things yeast, from classical genetics through up-to-the-minute genomics and the latest cytological techniques. 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.

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).

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

Categories: Conferences

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

Explore a Large New Chemogenomics Dataset Via SGD

March 26, 2014

What happens when you cross two comprehensive deletion mutant collections with a library of more than 1800 structurally diverse chemicals? HIP HOP happens. Not the music, but a whole lot of very informative phenotype data – over 40 million data points!

The response of S. cerevisiae mutant strains to a chemical can tell us a lot about which pathways or processes the chemical affects. This is not only interesting for yeast biologists, but also has important implications for human molecular biology and disease research. So a group at The Novartis Institutes of Biomedical Research decided to test the sensitivity of nearly 6,000 mutant yeast strains to a panel of about 1,800 compounds. 

Hoepfner and colleagues have published these results and have also generously offered them to SGD.  They used the HIP and HOP methods (HIP, HaploInsufficiency Profiling, using diploid heterozygous deletion mutant strains; HOP, HOmozygous deletion Profiling, using diploid homozygous deletion mutant strains) that have proven very useful in yeast since the creation of the systematic deletion mutant collections.

To do this mammoth series of experiments they obviously needed to set up an automated pipeline. These sorts of experiments have been done before, but in this study Hoepfner et al. improved on existing procedures in many ways: the physical techniques, the controls and replicates included, and the methods for data analysis.

Phenotype annotations in SGD. We’ve incorporated a subset of these results into SGD as mutant phenotype annotations. Why a subset? Some of the chemicals that were used in these experiments are un-named proprietary compounds, so the individual phenotypes would not be very informative in the context of SGD. We’ve added the phenotypes that involve named chemicals to SGD – more than 5,500 annotations. These may be viewed on Phenotype Details pages for individual genes (see example), retrieved as a set using Yeastmine, or downloaded along with all SGD mutant phenotype annotations in our phenotype data download file.

Easy access to the full dataset and analyses. We’ve also added a new set of links to SGD that take you directly from your favorite gene to the authors’ website, which provides full access to all of the data and interesting ways to look at it (see below). When you click on a “HIP HOP Profile” link from the Locus Summary page or the Phenotype Details page of a gene in SGD, the landing page at the authors’ website allows you to explore data for mutants in that gene or for chemicals affecting that mutant strain. You can see which chemicals had the greatest effects, which other mutant strains have a similar range of phenotypes, and much more. And if a chemical that has interesting effects is proprietary, don’t worry; Hoepfner and colleagues have stated that they “encourage future academic collaborations around individual compounds used in this study.”

Information about mutant strains. In the course of this study, the authors also generated some very useful data about particular mutant strains in the deletion collection. Some of them were hypersensitive to more than 100 different chemicals. Others turned out to be carrying additional background mutations that could affect the phenotypes of the mutant strain. We are planning to display this kind of information (from this and other studies) directly on SGD Phenotype Details pages in the future.

We thank Dominic Hoepfner and colleagues for sharing these data with SGD and for helping us to incorporate the data.  And we encourage you to explore this new resource and contact us with any questions or suggestions.

Categories: New Data

Yeast Genetics Meeting Abstract and Registration Sites Now Open

March 24, 2014

The 2014 Yeast Genetics Meeting will be held July 29 – August 3 at the University of Washington, Seattle, Washington. Abstract submission and registration sites are now open.  The abstract submission deadline is April 24.   Be sure to get your abstract in by the deadline so that it will be programmed and included in the Program Book.*

The Genetics Society of America is pleased to announce the following awards and lectures will be presented:

• George Church, Harvard University – Lee Hartwell Lecture
• Olga Troyanskya, Princeton University – Ira Herskowitz Award
• Jeremy Thorner, University of California, Berkeley – Lifetime Achievement Award
• Anita Hopper, Ohio State University – Winge-Lindegren Address

In addition, there will be two special presentations: Jon Lorsch, Director of the National Institute of General Medical Sciences, NIH will discuss the future plans of the Institute and Gerry Fink, MIT, will give a retrospective look at Fred Sherman’s life and his impact on the field of yeast genetics research.

As usual, SGD staff will be at the meeting and we look forward to meeting and talking with yeast researchers. Don’t miss this premier conference of the yeast community!

*IMPORTANT NOTE: NEW THIS YEAR: The full text of all abstracts submitted by the deadline date will ONLY be available online, as a pdf and in the abstract search program,  and will not be printed in the program book. The program book will still contain the full schedule information including platform and poster sessions date, time, title, authors, gene index and a listing of exhibits. Late abstracts will only be accepted if space permits and will not be included in the online search.

Questions? Contact Anne Marie Mahoney: mahoney@genetics-gsa.org

Categories: Conferences

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