New & Noteworthy

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

Unlocking Chromatin

February 10, 2016

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In Die Hard, Hans Gruber and associates need to break through seven locks in the right order on a safe to get to bearer bonds worth 640 million dollars. Of course the hero John McClane foils the plot and beats the villains.

Nothing so exciting in yeast, but some genes are nearly as hard to turn on as that safe was to open. One of the most stubborn is the HO gene. It requires three locks or gates be opened in the right order to start making the HO endonuclease.

A new study in GENETICS by Yarrington and coworkers shows that the second lock for HO is a set of nucleosomes that blocks the binding of the transcription activator SBF. When they rejiggered this promoter so that these nucleosomes were removed, the HO gene needed fewer steps to get activated.

It is as if Hans Gruber and his gang only had five or six locks to get through to open their safe. And the 7th, hardest one was removed.

The HO gene is usually turned on in three sequential steps. First the Swi5p activator binds to a region called URS1, which recruits coactivators that then remodel the chromatin at the left half of URS2 (URS2-L). This allows SBF to bind its previously hidden binding sites which then remodels the chromatin again. Now a second set of SBF sites is revealed in the right half of URS2 (URS2-R).

These authors set out to provide direct proof that nucleosome positioning over URS2-L is the key to the second lock. They did this by making a set of chimeric promoters between HO and CLN2.

Both of these promoters are activated by SBF. A key difference between the two is that the CLN2 promoter, like 95% of yeast promoters, is in a nucleosome depleted region (NDR).

The idea then is to make an HO promoter in which the usual URS2-L is replaced with the NDR region of CLN2. If the nucleosomes matter over URS2-L, then this construct should be activated in two instead of three steps.

Or, to put it another way, Swi5p binding to URS1, the first lock, will no longer be needed to open the second lock. HO activation will now be Swi5p independent. This is what the authors found.

When they looked at their chimeric protein that lacked nucleosomes over URS2-L, they found that using a strain deleted for SWI5 had very little effect on activity. There was only around a 2-fold difference in activity with this construct in the wild type and SWI5-deleted strains. This is very different than the wild type HO promoter where there was around a 15-fold difference between the two strains.

The authors then did an additional experiment where they took their chimeric reporter and mutated the nucleosome depleted region such that nucleosomes could bind there. This construct was now more Swi5p-dependent: there was around a 5-fold difference in activity between the wild type and SWI5 deletion strains. They had at least partially rebuilt that second lock.

Yarrington and coworkers continued with ChIP experiments to confirm that their chimeric construct was indeed depleted for nucleosomes, as well as other experiments to tease out more subtle details about the regulation.

Given that it switches a yeast cell’s mating type, it isn’t surprising that the HO gene is under such lock and key. The yeast cell wants to make sure it only turns on when needed. Just as the Nakatomi Corporation wanted to make sure only the right people could get to that fortune in bearer bonds.

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

Categories: Research Spotlight

Tags: chromatin remodeling, HO endonuclease, nucleosomes, transcription regulation

Of Medieval Market Townes and Wasp Guts

February 03, 2016

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Back before trains, planes and automobiles, people didn’t get around as much. And for the people of medieval Europe, this could be a real problem genetically.

At this time there were a lot of small, isolated villages scattered across Europe. If people in these villages stayed put, inbreeding might have gotten as bad as the poor Spanish Hapsburgs. Their last king, Charles II, was infertile, riddled with genetic diseases and his royal line died out with him.

One reason (among many) that this didn’t happen to people all over Europe was market towns. These were centrally located places where villagers came to sell goods. And where they also found partners to bring home to freshen up the gene pool.

Turns out that out in the wild, our friend yeast is in an even worse predicament than medieval Europeans. Because they are all clones of each other, they exist in isolated colonies with almost no genetic diversity.

Yeast are also way less mobile than people. They do have spores but these don’t tend to travel very far without help.

And yet, looking at yeast DNA shows that yeast definitely get around. There are all sorts of signs of various DNA mixing over time. So where are all these yeast hooking up?

A new study by Stefanini and coworkers in PNAS suggests that yeasts’ market towns are in the guts of wasps. It is there that various yeasts can meet and mate before heading back to their “villages.”

This makes sense in a lot of ways. First off, as we described in an earlier blog, there is good evidence that yeast winter in wasp guts.

So there are definitely a variety of yeast hanging around for months, waiting for warmer weather. The gut is also the kind of harsh place where spore dissolution, the first step in yeast mating, can happen.

When the authors looked at the yeast isolates from a wasp’s gut they saw a lot more outbreeding compared to other sources. This suggests that a lot of mating is indeed going on there.

The next step was to directly test how much mating can actually happen in a wasp gut. Stefanini and coworkers tested this by having the wasps eat five different yeast strains and then analyzing the isolates genetically over time. They compared the results from this experiment to the amount of mating that happens in wine must and under ideal lab conditions.

What they found was a whole lot of mating going on.

After two months, around 1/3 of the yeast in the wasp’s gut were outcrossed. This is OK but pretty comparable to what is found in wine must.

It was a different story after four months. Now 90% of the yeast were outcrossed. This is an even better result than scientists typically get in the lab. Clearly the wasp gut is a great place for a yeast to find a partner.

The authors also found that the S. paradoxus strain had to mate to survive in the gut. The only time they found this strain in yeast isolates was in hybrids with S. cerevisiae.

The next steps will be to see if this kind of mating actually has a big effect on yeast diversity in the wild. And of course what, if anything, the wasp gets out of hosting these cavorting yeast.

A market town was great for both the town and the visitors. People met up, sold goods, found partners and the towns prospered from all of this traffic. I can’t wait to find out if the wasp/yeast situation is so mutually beneficial as well.

Jerry Lee Lewis has a whole lot of shakin’ going on, just like a wasp’s gut has a whole lot of matin’ going on.

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

Categories: Research Spotlight

Tags: inbreeding, mating, outcrossing, Saccharomyces cerevisiae, Saccharomyces paradoxus

Unfrying An Egg

January 20, 2016

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Eggs start out as slimy and awful, but can end up warm, firm and wonderful. All it takes is some heat to denature the egg proteins and voilà, a tasty breakfast.

Not that anyone would want to do it, but of course it is impossible to do the reverse. You can’t take a fried egg and turn it back into a raw one. The denaturation is pretty much permanent.

When a cell is hit with high temperatures, its proteins start to denature as well. And scientists thought that most of the denaturation of many of these proteins was as irreversible as the eggs. The thought was that many or most of these denatured proteins were “eaten” through proteolytic degradation. Although cellular chaperones are capable of disaggregating and refolding some heat-denatured proteins, it wasn’t known which aggregated proteins met which fate in a living cell.

A new study out in Cell by Wallace and colleagues shows that at least in yeast, most eggs get unfried. After a heat shock, aggregated proteins in the cell return to their unaggregated form and get back to work.

Now those earlier scientists weren’t crazy or anything. The proteins they looked at did indeed clump up and get broken down by the cell after a heat shock. But these were proteins introduced to the cell.

In the current study, Wallace and colleagues looked at normal yeast proteins being made at their normal levels. And now what happens after a brief heat shock is an entirely different story.

The first experiment they did looked at which endogenous yeast proteins aggregated after they were shifted from their normal 30 to 46 degrees Celsius for 2, 4, or 8 minutes. The researchers detected aggregation using ultracentrifugation—those proteins that shifted from the supernatant to the pellet after a spin in the centrifuge were said to have aggregated.

Using stable isotope labeling and liquid chromatography coupled to tandem mass spectroscopy (LC-MS/MS), they were able to detect 982 yeast proteins easily. Of these, 177 went from the supernatant to the pellet after the temperature shift. (And 4 did the reverse and went from the pellet to the supernatant!)

After doing some important work investigating these aggregated proteins, the researchers next set out to see what happened to them when the cells are returned to 30 degrees Celsius. Are they chewed up and recycled, or nursed back to health and returned to the wild?

To figure this out they did an experiment where proteins are labeled at two different times using two different labels. The researchers first grew the yeast cells at 30 degrees Celsius in the presence of arginine and lysine with a “light” label. This labels all of the proteins in the cell that have an arginine and/or lysine.

Then the cells are washed and a new media is added that contains “heavy” labeled arginine and lysine. The cells are shifted to 42 degrees Celsius for 10 minutes and then allowed to recover for 0, 20, or 60 minutes.

After 60 minutes of recovery, the ratio of light to heavy aggregated proteins looked the same as proteins that hadn’t aggregated. In other words, aggregation did not cause proteins to turn over more quickly.

It looks as if aggregated proteins are untangled and allowed to go about their business. So after a heat shock the cell doesn’t throw its hands in the air and simply start things over.

Other experiments done by Wallace and coworkers in this study, that we do not have the space to tackle here, suggest that the cell has an orderly process for dealing with heat stress. After a heat shock, certain proteins aggregate with chaperones in specific areas of the cell. Once the temperature returns to normal, these stress granules disassemble and the aggregated proteins are released intact.

None of this will help us unfry an egg — a denatured egg protein is obviously significantly different than an aggregated protein protected by chaperones in a stress granule. But this study does help us better understand how our cells work. And that’s a good thing.

Unlike Mr. Bill’s dog, most aggregated yeast proteins can return from a heat shock.

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

Categories: Research Spotlight

Tags: chaperones, heat shock, protein aggregates, protein aggregation

Join SGD at The Allied Genetics Conference

January 12, 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 will be open until March 23, 2016. 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: Announcements, Conferences

Clearing Customs in the Nucleus

January 06, 2016

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Going through customs at the airport is a necessary evil. Once off the plane, you need to stand in line, scan for an open station, have various forms looked over and possibly stamped before you can pass through the airport doors and get into a new country.

And of course if there is anything wrong, you can be sent back to get your papers in order. A pain but it does help protect people.

Things work pretty similarly in the nucleus. The mRNA disembarks off the DNA, gathers up a set of proteins, and heads for the nuclear pore. There its proteins are checked and if everything is in order, it is allowed to proceed to the cytoplasm. And if there are problems, it is denied entry.

A couple of new studies out in the Journal of Cell Biology use imaging microscopy to give us a close up view of the bustling airport that is the nucleus of a yeast cell. It is utterly fascinating.

Both studies showed that mRNAs often hang out at the nuclear envelope, pausing at a nuclear pore and then sometimes moving to a new one. And that factors both in the nuclear pore and bound to the mRNA affect this scanning of the nuclear envelope.

The basic strategy with both studies is to fluorescently label specific mRNAs in a live yeast cell and follow its journey from the nucleus to the cytoplasm. To do this, they also needed to fluorescently label the nuclear pores, the custom stations in the nuclear envelope.

They labeled the mRNA using the bacteriophage PP7 RNA-labeling system. Basically, they load up the untranslated region (UTR) of a specific gene with sequences that form specific loops. Once transcribed, these loops are then bound by fluorescently labeled PP7 coat protein. Now they can track this labeled mRNA.

To more easily track mRNAs, they chose low expressing genes. That way they could follow a single mRNA more easily. They also needed to get rid of the yeast cell wall so they could see inside the cell better.

Overall they found that at least in yeast, the mRNA takes around 200 milliseconds to get exported to the cytoplasm. Very little of this time is spent in the nucleoplasm; the mRNA very quickly makes its way to a nuclear pore.

Once there things slow down. The mRNA stays at a nuclear pore or slides along the nuclear envelope to a different pore in a process the authors call scanning. Eventually the lucky successfully make it through the pore to the cytoplasm where they can seek out a ribosome for translation. Around 90% of the mRNAs they studied made it through.

They had a couple of different ideas about why the mRNA hangs around the nuclear envelope for so long. One is that the extended stay at the pore is to make sure everything is in order with the mRNA. It can’t pass through customs unless all of the right forms have been filled out properly.

Another possibility is that by scanning it is looking for a nuclear pore that is competent for exporting. It has to search for an available customs agent.

Now that the authors had established a system to look at mRNA export, they next set out to see which factors play important roles. As you might guess, mucking with parts of the nuclear pores or the proteins that bind the mRNA can throw a monkey wrench into the process.

In the first study, Smith and coworkers looked at what happens to the process when one of the key mRNA binding proteins, Mex67p is mutated. This protein is known to interact with the nuclear pore.

It has also been proposed that Mex67p is important in making sure the trip through the pore is one way. Once the mRNA goes through, it releases Mex67p which makes the mRNA let go of the cytoplasmic side of the nuclear pore. The imaging studies here confirmed that Mex67p is indeed important for mRNA directionality.

Using a temperature sensitive mutant of Mex67p the researchers found that the mRNA they tracked stayed at the nuclear envelope about three times longer than in a wild type strain. The process was also much less efficient with only 32% making it to the cytoplasm instead of the 90% seen in the wild type strain. And of the 14 mRNAs which failed to make it through the pore, 7 headed back through the pore to the nucleus.

In the second study, Saroufim and coworkers concentrated on a part of the nuclear pore called the nuclear basket. This is the first part of the nuclear pore that the mRNP, the mRNA plus its proteins, encounters.

They found that deleting or mutating two key parts of the nuclear basket, MLP1 and MLP2, made the mRNA linger for a shorter time at the pore. The mRNA no longer scans the nuclear envelope.

But that didn’t mean the mRNA passed through to the cytoplasm more quickly. No, it just tended to fall back into the nucleoplasm and then have to reattach more often.

It is as if you had to deal with a customs agent who keeps sending you back into the airport. Or agents who keep putting up the “Out to Lunch” sign as soon as you get to the head of the line.

These two studies give researchers a way to study mRNA export in live cells in real time. As we piece together which proteins play what role, we will get a better handle on this important part of gene expression.

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

Categories: Research Spotlight

Tags: mRNA export, nuclear pore, Saccharomyces cerevisiae

SGD December 2015 Newsletter

December 18, 2015

SGD periodically sends out a newsletter to colleagues designated as contacts in SGD. This December 2015 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

Happy Holidays from SGD

December 17, 2015

We want to take this opportunity to wish you and your family, friends and lab mates the best during the upcoming holidays. Stanford University will be closed for two weeks starting at 5:00 p.m. PST on December 18th, reopening on January 4th, 2016. Although SGD staff members will be taking time off, please rest assured that the website will remain up and running throughout the winter break, and we will attempt to keep connected via email should you have any questions.

Categories: Announcements

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