New & Noteworthy

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.

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.

Yeast on Their Best Behavior

December 10, 2015

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As the holidays approach, many of us are getting ready to crowd around the table for a big family dinner. Some of us may behave differently around family than we might with friends or coworkers.

For example, with your relatives, you might bite your tongue if your political views vary greatly from theirs. Where we are and with whom we interact can sometimes affect what we do.

Turns out that yeast growing in a colony can be the same way. Though of course they aren’t keeping their opinions to themselves. (Well, we don’t think they are…)

A yeast cell can end up acting differently depending on where it is in a colony. For example, only a narrow band of cells gets to sporulate while all the others are left to plod through mitosis.

A new study out in GENETICS by Piccirillo and coworkers shows that these cells sporulate because nearby cells “encourage” them to. They are being influenced to sporulate because of the cells around them. Just like your relatives might influence you to change your behavior at the dinner table.

The first step in showing that one set of cells signals a second set to sporulate was to find the genes involved in setting up this pattern. Since the authors were looking at Saccharomyces cerevisiae, it was pretty easy to get mutants to study. They just had to open their freezer and pull out their yeast homozygous diploid deletion library.

Initially, they looked for strains where the usual pattern of sporulating cells was disrupted. They then took these candidates and looked for those that could still sporulate normally in suspension. They wanted mutants that could sporulate but couldn’t do it in the right place.

They found seven strains that fit the bill. Three of the deleted genes, MPK1/SLT2, BCK1, and SMI1, were in the cell-wall integrity pathway (CWI). They also showed that mutation of three other genes in the pathway, SLG1/WSC1,TUS1 and RLM1, all impacted colony sporulation as well.

Further work showed that the transcription factor RLM1 was induced 1-2 days before the master regulator IME1 was turned on. IME1 is a key player in getting meiosis started so that yeast cells can sporulate.

So the story seemed to be that RLM1 is turned up which then turns on IME1, which kick starts meiosis. Makes sense except it is unlikely that Rlm1p is directly activating IME1. There is no obvious Rlm1p site in the IME1 promoter.

A close look at the colonies showed that RLM1 is upregulated in a layer of cells just under the ones where IME1 is upregulated. Deletions in the CWI pathway seemed to have disrupted a group of “feeder” cells whose job it is to get nearby cells to sporulate.

To show this, the authors used a chimeric colony assay that consisted of two strains. The first strain, which had functional Rlm1p, had a reporter, either RFP or lacZ, under the control of the IME1 promoter. The second strain was either wild type or deleted for the transcription factor RLM1.

They created colonies with equal amounts of each strain and looked at IME activation. The idea is that if RLM1 is important in the cells that sporulate, then the second strain shouldn’t matter. You should get the same number of cells in which the IME1 promoter is activated whether or not adjacent cells express RLM1.

But if it is important for RLM1 to be expressed in nearby cells, then there should be a falloff in activation if adjacent cells are deleted for RLM1. This is just what the authors found.

And it wasn’t just the artificial reporter system that was affected either. There was also a drop off in the number of cells that sporulated in the case where some of the cells lacked RLM1.

In a further set of experiments, Piccirillo and coworkers showed that these feeder cells became more osmosensitive compared to the ones that go on to sporulate. While they did not find the signal that prompted the meiosis of nearby cells, this change in osmosensitivity is consistent with the cells preparing to release something into the environment.

So it looks like activating the CWI pathway in one set of cells causes a second set to start down the road of sporulation. And if the CWI pathway is disabled in these cells, then the second set of cells no longer changes their behavior and begin to go through meiosis.

This all seems weird at first until you realize that the cells in a colony usually all share the same DNA. What is good for one set of cells is good for the survival of the DNA even if it is at the expense of other cells in the colony.

Yeast cells tend to sporulate when food grows scarce. But sporulating takes a lot of energy. Colonies may get around this paradox by having some of the cells in the colony give up nutrients or energy to a few cells that go on to sporulate. The feeder cells deprive themselves so that other cells have a better shot at survival.

Now the DNA, shared by all the cells, can live on for the next round of holiday dinners….

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

Keeping Gen(i)e Drives in Their Lamps

December 2, 2015

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Everyone knows about genies. They have almost infinite power, can grant you three wishes, and are kept under control by the owner of their lamp.

And as we saw in Disney’s Aladdin, it is a good thing that the lamp is around! When the evil sorcerer Jafar was given the powers of a genie, he began to take over the world. Until, that is, Aladdin forced him back into his lamp where he could be kept under control.

In the last few years, scientists have come up with their own genies. While not as powerful as the “real” ones, these gene drives can still pack quite a punch. And maybe even grant us a few wishes.

Gene drives can force genes to spread quickly through a population whether those genes are good for a species or not. This means we might be able, for example, to force a “bad” gene to spread through the mosquitoes that transmit malaria. By causing the mosquito population to crash, our wish to save hundreds of thousands of lives each year would be granted!

But just like a genie, we need to keep gene drives under control. We do not want something that overrides natural selection to escape and wreak havoc with ecosystems.

Which is where, as usual, our friend yeast can help! In a new study out in Nature Biotechnology, DiCarlo and colleagues use yeast to test two different strategies to make gene drives safe enough to use. And, they argue, safe enough to research.

Gene drives are based on the idea of homing endonucleases. Basically, if a gene associated with a gene drive is on just one of the two chromosomes in a pair, the gene drive will copy and insert the gene into the other chromosome through a precise DNA cut.

Now both chromosomes end up with a copy of the gene. Which of course means all of the offspring will get the altered gene too. This copying will happen generation after generation until the new gene has swept through the population.

The idea for gene drives has been around since 2003 but really only became practical with the discovery of the CRISPR/Cas9 system. This genome editing tool, which is ludicrously simple to program to target most any DNA sequence, allows scientists to create most any gene drive they want.

The CRISPR/Cas9 system has two parts. One part is the guide RNA which leads the second part, the endonuclease Cas9, to the right spot in the genome to cut. What makes the system so powerful is that you just need to make a different guide RNA to target different sequences in the genome.

One easy way to help control a gene drive is to keep these two parts separate. Do not have the guide RNA and the Cas9 on the same piece of DNA. Then, if one part were to escape, it couldn’t do anything on its own.

This is of course easy to do in yeast. Just integrate one part into a chromosome and keep the second part on a plasmid.

This is just what DiCarlo and coworkers did. And they showed that this separation can be very effective.

They integrated a guide RNA into the ADE2 gene of a haploid yeast to create a gene drive designed to disrupt ADE2. As expected, this strain produced red colonies on adenine limiting media.

They next mated this strain to a wild type haploid. All of the resulting diploids were cream colored. This is what would be expected as both copies of ADE2 need to be disrupted to see red colonies in a diploid.

When these diploids were sporulated, the researchers got the expected 2:2 ratio of red to cream colored haploids. This all changed when they introduced a Cas9 containing plasmid into the experiment.

In the presence of Cas9, more than 99% of the resulting diploids were red. And when sporulated, these diploids produced all red haploid colonies.

The two parts of CRISPR/Cas9 together drove the disrupted ADE2 through the population. But importantly, just having the guide RNA integrated into ADE2 had no effect on how the two alleles were passed down. Once one part is removed, the gene drive stalls out.

The same system also worked when the ADE2 gene drive included the URA3 gene so that URA3 spread through the population as well. It also worked when the essential gene ABD1 was targeted.

And genetic background did not significantly affect how well this ADE2 gene drive worked. When they mated their haploid to six different strains of yeast they saw no loss in efficiency.

So separating the two parts of the gene drive is a pretty good failsafe. But of course nothing is perfect.

Ideally we need some way to shut the system down if all of our safety features fail. We want to be able to get rid of Jafar and the lamp entirely if possible.

DiCarlo and coworkers showed that they could create a gene drive that could overwrite and correct the ADE2 they had disrupted with the guide RNA. This new gene drive targeted a synthetic sequence in the original gene which means that it would only affect altered yeast. So even if things go awry, we may be able to erase the changes we made.

These two strategies should help keep gene drives in check both in the wild and the lab. But of course, again, it is important to keep in mind that nothing is foolproof.

At the end of Aladdin, they buried Jafar and his lamp deep in the desert to keep him from causing any more trouble. But his lamp was found and Jafar reemerged to wreak havoc in the second Aladdin movie, reminding us that we must be very careful when unleashing powerful forces.

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