New & Noteworthy

Cheerio Containment

September 11, 2017


In the store the other day I saw a kid in a stroller covered in Cheerios, an opened, near empty Ziploc-type plastic bag next to him. It looked like one of those cheap, knock off brands that are much harder to close properly.

p_101384655

Just like a poorly closed zipper means more spilled Cheerios, a looser H1 clamp means more transcripts. Image from http://www.parents.com.

Other than keeping the Cheerios out of reach, his parents had two options here. The first was to use two bags, putting the cheaper re-sealable bag into another one. The odds are much better that at least one of them would be closed properly.

The second option was to get a re-sealable bag with a better zipper, like an actual Ziploc. With a better baggie, the parents had a much better chance of making sure that the bag was sealed.

In a new study out in GENETICS, Lawrence and coworkers show that yeast cells go for the second approach when it comes to the histone H1 and gene expression regulation. Rather than regulating gene expression by controlling the number of molecules of H1 in a cell, the yeast instead regulates transcription by controlling how well H1 can bind DNA. And it looks like the strength of binding is influenced by the acetylation of H1’s more famous histone cousins—the histones found in the nucleosome.

Almost everyone has heard of the histones that make up the nucleosome, that structure of DNA wrapped around a core of eight histone proteins. H1 is a bit less famous. It binds the linker DNA, or the DNA between some of the nucleosomes.

When bound, H1 tends to repress nearby genes. It acts like a clamp that keeps the nucleosome in place.

This gene repression is probably why our friend Saccharomyces cerevisiae has only one H1 per 4-37 nucleosomes as compared to the one H1 per nucleosome in vertebrates. After all, S. cerevisiae’s genome has a much higher gene density. If it had one H1 for every nucleosome it might not have any genes turned on!

You may have noticed that scientists don’t have a good handle on an exact number of H1 molecules in a yeast cell. One of the first things these authors did was a careful study to try to nail down just what that number is. They came up with 18.9 +/- 1 nucleosomes/H1.

The next step was to figure out how a yeast cell controls H1 to regulate gene expression. There are two general ideas about how a cell might go about this.

In the first, the cell controls the amount of H1 it makes. The more H1, the more genes get repressed.

In the second, H1 is controlled by the acetylation of the core histone proteins. The acetylation loosens the core histone’s grip on the DNA making it harder for H1 to bind.

It would be very hard to do experiments in most beasts to try to tell which model is correct. For example, mammals have 11 genes for H1 proteins. This makes changing the overall amount of H1 nontrivial to say the least.

This is where the ultimate model organism, S. cerevisiae, can come in and save the day yet again (#APOYG). Since this yeast has only one H1 gene, HHO1, it is much simpler to tweak the amount of H1 protein in a cell.

Lawrence and coworkers put HHO1 under the control of the GAL promoter and found that in the presence of galactose, the extra H1 had a severe effect on growth. These yeast were unhappy with around three times as much H1 protein, even though there was not much of an effect on chromosomal structure as measured by micrococcal nuclease assays.

cheerios_in_ziploc

Low acetylation on this Ziploc. Image from General Mills.

The researchers next looked for genes which when deleted, rescued the growth defect of yeast making too much H1. They did this by transforming their GAL-HHO1 plasmid into the collection of ~4700 nonessential gene knockout strains.

Histone deacetylases (HDACs) were the most interesting class of genes they found that could rescue the growth defect. By deleting an HDAC gene, histone acetylation increases (because the deacetylation decreases), implying that H1 can be regulated by the acetylation status of histones. The loss of the HDAC increased the amount of acetylation, which made it harder for H1 to bind, and so repress transcription.

They confirmed this finding in a couple of ways with the most interesting one using a couple of different mutants of histone H3. One mutant had its acetylation sites in the H3 tail changed to glutamines, which mimics a histone in a perpetual state of acetylation. This mimic of an acetylated H3 tail partially rescued the growth defect of too much H1.

So it looks like the effect of H1 can be regulated by the acetylation status of its more famous histone kin. More acetylation results in a looser grip, which makes for more transcription. Like a cheap plastic baggie with a zipper with a looser grip making Cheerios more likely to spill, H1’s looser grip due to more acetylation results in more transcription and more transcripts being let loose into the cell.

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

Categories: Research Spotlight

Tags: HHO1 , nucleosome , H1 , histones , gene regulation

That Sweet Spot of Mistranslation

August 03, 2017


Anyone at a party knows that a little alcohol can make you charming, but a lot can doom any relationship from blossoming (just listen to Drunk Uncle!). In fact, too much can destroy a party! You need to have enough alcohol to curb social inhibitions, but not so much you overwhelm them.

Drunk man

Just as drinking too much alcohol can be toxic to people, too much mistranslation can be toxic to a cell. from pbs.twimg

According to a new study out in GENETICS by Berg and coworkers, something similar sometimes seems to be true when the genetic code evolves. A variety of beasts, including the yeast Candida albicans, have slightly different genetic codes—one or a few codons code for a different amino acid than usual. This is often the result of a mutated tRNA, the molecule that carries the right amino acid to the right codon.

These authors found that mutating only the anticodon, the part of the tRNA that recognizes the codon, is not a great way to head down the path to a new genetic code. That mutated tRNA leads to too many of one amino acid being replaced with another. Like the boorish drunk who kills the party because he has had too much to drink, the cell is overwhelmed with too many of the wrong amino acids scattered across its proteins and dies.

What Berg and coworkers found was that having fewer of these tRNAs with a mutated anticodon allowed for a tolerable level of amino acid substitutions. This means that this tRNA can hang around until it is helpful, like when it can suppress a new mutation of a key amino acid in a key protein. 

The authors dubbed these low level mutated tRNAs as “phenotypically ambivalent intermediate tRNAs”—tRNAs that are in the process of changing the genetic code for at least one codon. It may be that the variants of the genetic code found in nature arose this way.

They started out with a strain of Saccharomyces cerevisiae with a mutation that inserted a proline in the wrong place of the TTI2 protein.  This strain does extremely poorly in the presence of 5% ethanol.

The authors then tried to create and/or isolate suppressor mutations in a serine tRNA that could allow the strain to grow in the presence of 5% alcohol. The idea is that this tRNA would now carry a serine to that troublesome proline codon.

They started off by changing the anticodon of a serine tRNA to UGG. Now, the cell would put a serine in at CCA proline codons.

When they transformed a plasmid carrying this tRNA into their yeast strain, they got very few colonies. This obnoxious tRNA overwhelmed the cell by changing too many prolines to serines. It ruined the party!

 They next set out to find a way to bring this bad boy under control. They mutated the serine tRNA with the proline anticodon using UV mutagenesis and found four mutants that allowed this yeast strain to grow in 5% ethanol.

Each mutant tRNA had a single mutation: G9A, A20bG, C40T, and G26A. Berg and coworkers set out to figure out why the cells now tolerated the mistranslation they couldn’t handle before.

What they found was that at least for two of them, G9A and G26A, the cells dealt better with the mutated serine tRNA because there was less of them around. The toxic drunk had become the tipsy charmer!

I can teach you lots of things about biology but you’ll need to learn about drinking alcohol on your own. Beer mug from Max Pixel (http://maxpixel.freegreatpicture.com/).

Well, maybe not quite charming, but at least something that could be dealt with. Both mutated tRNAs affected cell growth in the absence of ethanol, with the G26A version having the more severe effect—a reduction in growth by 70%.

Most likely there was less of the G26A variant because it was a victim of the rapid tRNA decay (RTD) pathway. The G26A variant affected the growth rate much less in the absence of alcohol in a strain deleted for MET22, a key gene in the RTD pathway.

By looking at the crystal structure of a serine tRNA in complex with its aminoacyl tRNA synthetase from Thermus thermophilus, Berg and coworkers predicted that the G9A mutation should result in a poorly folded tRNA. They found this was indeed the case when they compared the melting curves of the G9A mutant and the tRNA lacking the G9A mutation.

So what we have are some tolerable, but by no means benign mutations. For example, the G26A is quickly selected against in the absence of ethanol.

This makes it hard to imagine how these sorts of mutations might arise and one day permanently alter a genetic code. The key to understanding how this might happen is a set of experiments Berg and coworkers did that showed that both G26A and G9A have little or no effect on cell growth in the absence of a mutated anticodon. In other words, tRNAs can exist in a poised state, ready to easily adapt with a single change to the anticodon if need be.

And it turns out that poised tRNAs may exist in the real world. For example, human tRNAs have a lot of variation. Perhaps these are around to one day save a cell with a mutation that would normally be deadly.

As this work (and real life) shows, too much of a good thing can be bad. This is true of alcohol (remember high school or college?) and true of some mutant tRNAs. Yeast can teach us about the tRNAs, the rest we need to learn on our own. #APOYG

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

Categories: Research Spotlight

Tags: mistranslation , tRNA , MET22 , TTI2

Now Yeast Even Finds Fungal Pathogens!

July 27, 2017


Some products have lots and lots of uses. Sometimes they are designed that way like the hypothetical Shimmer, a floor wax and dessert topping from Saturday Night Live. And sometimes they can be used in more situations than they were originally designed for like Windex in the movie My Big Fat Greek Wedding, or the most versatile thing out there in real life, duct tape. (Click here and here for some fun duct tape uses).

Positive Pregnancy test results

Yeast might one day be used to identify fungal pathogens with an assay as easy to use as a pregnancy test. from Wikimedia Commons

Wait, did I say duct tape was the most versatile thing out there? Maybe I spoke too soon.

Our friend Saccharomyces cerevisiae is in contention with duct tape for that title. Of course it is used to make bread, beer and wine, but it has lots of other uses too. Just a very few include using it to: make antimalarial drugs, understand how our cells and a surprising variety of human diseases work, understand how evolution can happen, understand how cancer forms and progresses, and make ambergris (whale puke). In the near future, it may even help deal with climate change by making biofuels out of agricultural waste, and pain management by making opioids.

And now a new study by Ostrov and coworkers adds another use—the simple and inexpensive detection of fungal pathogens. This is a big deal because if it works the way the authors think it will, this new use could one day save the lives of a significant fraction of the two million or so people who die from fungal pathogens each year.

In a nut shell, these authors have engineered S. cerevisiae to be attracted to different kinds of fungus by monkeying with its pheromone detection system.

S. cerevisiae comes in two mating types, a and α. The a-type cells make the secreted mating pheromone “a-factor” and STE2, a receptor that responds to “α-factor.” And α-type cells make the secreted mating pheromone “α-factor” and a receptor, STE3, that responds to the “a-factor.” So, thea-type cell makes something that makes it attractive to α-type cells and vice versa.

The end result of the factor/receptor interaction is that a number of genes are regulated so that the two haploid cells, a-type and α-type, can merge to become an a/α diploid. Fertilization yeast-style.

What these authors have done is to switch out the STE2 receptor from S. cerevisiae with the receptors from other fungal species in a-type cells so that the newly engineered cell responds to the presence of the new fungal species’ pheromone. For example, they replaced S. cerevisiae STE2 with the STE2 from Candida albicans so that the newly engineered cell now responds to pheromones from C. albicans.

Now of course that doesn’t make much of a sensor! No, they also need a readout to know that the engineered yeast has detected the presence of C. albicans.

They started out using a fluorescent reporter to see whether their engineered system responded to C. albicans mating pheromone. The cells fluoresced when the C. albicans mating pheromone was around at low levels but not with any of nine other fungal mating pheromones. So the system is definitely working—it can specifically identify C. albicans.

But to make it more useful in the developing countries where fungal pathogens are the biggest problem, they wanted to create a system with a more visible readout. For this they turned a bright red pigment called lycopene.    

They needed to add three genes from Erwinia herbicola to get yeast to make lycopene: crtE, crtB, and crtI. They placed the first two under constitutive promoters (pADH1 for crtE and pTEF1 for crtB) and the third gene, crtI, under the control of the pheromone-inducible promoter from FUS1. So, lycopene is not made unless the appropriate pheromone is around to turn on the crtI gene.

This new system worked as well as the fluorescent one.

For the next step, they replaced the S. cerevisiae STE2 with STE2 genes from nine other fungal species involved in human disease and/or food or agricultural spoilage and showed that their system worked for all of them. These new pathogens included: Candida glabrata, Histoplasma capsulatum, Lodderomyces elongisporus, Botrytis cinerea, Fusarium graminearum, Magnaporthe oryzae, Zygosaccharomyces bailii, and Zygosaccharomyces rouxii.

And they didn’t stick to just the well-characterized fungal species either. They were also able to create a system that worked for Paracoccidioides brasiliensis, a less-well studied fungus that causes paracoccidioidomycosis (PCM), a disease endemic to Latin America.

It may be that just understanding a fungal species’ mating system is enough to create this sort of biosensor. More species will have to be tested to see just how universal the simple swapping out of a single gene will be.

hammock made of duct tape

Yeast is giving duct tape a run for its money in terms of versatility (although it can’t yet be used to make a hammock like this). From Ally’s Place (http://moonshadowangel.blogspot.com/2007/01/duct-tape-ho.html).

In their final step, Ostrov and coworkers tested whether they could create a simple, inexpensive dipstick test by spotting their engineered strains onto filter paper. For the initial test, they focused on two of their engineered strains: the one that detects the presence of C. albicans and the one that detects the presence of P. brasiliensis. They found that the system worked by simply putting the yeast-spotted filter paper into soil, urine, serum, and blood that had been supplemented with synthetic pheromone.

The yeast with C. albicans STE2 turned red with C. albicans pheromone, but not with P. brasiliensis pheromone and vice versa. And to increase its utility further, the authors found that the filter paper stayed functional even after being stored for 38 weeks at room temperature.

The impact of fungal pathogens on human health is understudied and underappreciated despite the toll they take on people’s lives, especially in the developing world. For example, as one recent review points out, “…at least as many, if not more, people die from the top 10 invasive fungal diseases than from tuberculosis or malaria.”

The basic research done on S. cerevisiae has allowed these scientists to devise an ingenious way to identify fungal pathogens. That magical combination of basic research and the most versatile eukaryote out there, S. cerevisiae, is poised to help humanity yet again.  #APOYG

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

Categories: Research Spotlight

Tags: crtI , MATALPHA , crtB , fungal pathogens , crtE , STE2 , STE3 , MATA , mating

Make Some More Room for Lamarck

July 17, 2017


For most people, a move to Tibet or other high altitude places is a real struggle. They suffer the many nasty symptoms of high altitude sickness while they are there.

Some people though, like natives of Tibet or of the Andes, have adapted to the extreme altitudes through natural selection and do just fine. How they adapted is a typical Darwinian story.

Princess Bride, mostly dead

Lamarck’s theories are only mostly dead which means they are slightly alive. from the Brick In the Sky blog (https://thebrickinthesky.wordpress.com/).

Those who happened to have the right set of DNA did better than those who didn’t and so had more kids. Over time, the beneficial DNA became more common until the population was able to tolerate the low oxygen of the higher altitudes. (In Tibet, they may have acquired this helpful DNA by having kids with our close relatives the Denisovans.)

Imagine instead that the story went a bit differently. In a Lamarckian twist, let’s say that people who live in low oxygen were more likely to gain the traits needed to do well in this environment, strictly as a result of there not being enough oxygen around. In other words, the environment would make it more likely for the beneficial changes to happen.

Turns out this might have been the way the story went if people were more like yeast. And if dealing with low oxygen environments relied on a gene near a place in the DNA where replication forks often stalled.  And if that the gene was upregulated in low oxygen. 

It is under these conditions that Hull and coworkers found that yeast could preferentially develop the right mutations in an environment-dependent way. Instead of low oxygen though, these authors studied the yeast growing in high levels of copper.

One way that yeast deal with toxic levels of copper is to turn up the CUP1 gene. Or more precisely, turn up their tandem arrays of multiple copies of CUP1.

This last point is important because it hints at how yeast can increase the likelihood of beneficial mutations at CUP1 in the presence of copper. The increased transcription of the CUP1 genes increases the likelihood of a change in the copy number of these genes. Those yeast with increased copy number thrive in their new copper-tainted environment.

Now of course not every gene ends up with an increase in beneficial mutations just because it is induced. No, the gene also seems to have to be near where a DNA replication fork is more likely to stall. It is this combination of high rates of transcription and stalled DNA replication that can lead to changes in gene copy number.

The first thing the authors did was to map out where replication forks tend to stall in the yeast genome. These sites are marked by the presence of S139-phosphorylated histone H2A (γH2a).

Using chromatin immunoprecipitation sequencing (ChIP-seq) for γH2a they showed that likely stalling spots were within 1,000 base pairs of around 7% of the genes of Saccharomyces cerevisiae. These genes tend to be expressed at low levels under optimal conditions and higher levels under less ideal growth conditions. One of these genes is CUP1.

It is well known that when yeast are put into a high copper environment, the end result is yeast with more copies of the CUP1 gene. But this could simply represent the few cells that happened to have extra copies that then outgrow their compatriots with fewer copies. Ordinary old Darwinian selection.

What these authors wanted to show is that it is increased transcription that leads to increased copy number and not the selective pressure. They get at this a couple of different ways.

In the first approach, they introduce multiple copies of a Gal-inducible reporter at the CUP1 locus. In this system there is increased transcription in the presence of galactose, but no selection.

They found multiple colonies with changes in the copy number of the reporter gene with galactose and no differences in copy number with glucose. So, transcription matters.

Lamarck

Lamarck was only mostly wrong when it comes to evolution. Occasionally beasts can pass on traits they acquired over their lifetime. From Wikimedia Commons.

The second way they attacked this problem was by killing off any daughter cells to get rid of the problem of selection. In this strategy copy number mutants do not get a chance to outgrow their lower copy number sisters. Only the original mother cells remain.

Any increase in copy number would not be due to run of the mill Darwinian selection. Instead, they would be due to the presence of the factor in the environment the cells need to adapt to. And this is just what these authors saw happen.

They eliminated daughter cells using a mother-enrichment system in which daughter cells are killed in the presence of beta-estradiol. They treated a population of cells with beta-estradiol and then put half in normal media and half in media with 1 mM copper sulfate.

They found about a 9-fold increase in the number of copy number variants (CNV) in the presence of copper (27% CNV events compared to 3%). They did follow up experiments to show that copper was not acting as a mutagen—the copper had to induce transcription to cause the copy number variation. And judging by the bud scars, it looks like the cells divided more in the absence of copper, meaning this 9-fold increase is an underestimate.

So growing in the presence of copper increased mutations in the CUP1 gene that allowed the yeast to grow better in copper. Calling Doctor Lamarck!

We don’t have time to go into the rest of the experiments they did to flesh out their findings. For example, they show that this copy number variation can still happen even when they use tandem arrays that are much shorter than the usual 13 CUP1 copies. And that deletion of the H3K56 acetyltransferase RTT109 completely eliminates the effect of copper on the expansion of CUP1. And so much more! Anyone interested should definitely read the article.

These findings show us another example of Lamarckian selection. The environment itself causes the adaptations needed to prosper and these adaptations can be passed on. Not quite the ancestors of giraffes passing on their stretched necks to the next generation but still pretty cool.

The awesome power of yeast genetics again shows us something new about how life adapts and evolves. #APOYG

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

Categories: Research Spotlight

Tags: copper , replication fork , copy number variants , histone , Lamarck , RTT109 , CUP1-1 , CUP1-2 , Darwin , ChIP-seq

How Histones Use FACT(s) to Find Their Way

July 05, 2017


Looking at map

The map is like FACT helping histones get to the right place. from publicdomainpictures.net.

Some people (like me) have no sense of direction. Send me to the store and who knows where I’ll end up!

Tools like maps, a GPS system, and my iPhone all help to make sure I get to where I need to be. And seat belts, airbags and working brakes keep me safe while I am getting there.

Histones are similar. These proteins, which help to organize and run our DNA, can get lost without a variety of helpers to show them the way. They also need to be kept safe as they travel.

Instead of an iPhone, histones get to where they need to go with the help of histone chaperones like Nap1p and the FACT complex. A new study by Hodges and coworkers in GENETICS helps to figure out which parts of histones interact with the FACT complex and, to a lesser extent, Nap1p.

Turns out that a few residues in an acidic patch on H2A/H2B dimers are critical for interacting with FACT. This makes sense given that previous work had shown that this acidic patch interacts with other proteins (although no one had shown it interacts with the FACT complex or Nap1p). Hodges and coworkers also identified other residues outside of the acidic patch that were important for FACT complex binding.

The first step was to analyze residues in this patch known to be lethal when mutated to alanine—H2A: Y58, E62, D91, and H2B: L109. The authors used co-immunoprecipitation (co-IP) assays against either Nap1p or Spt16p (a subunit of the FACT complex) to identify which, if any of these essential residues, was important for interacting with these histone chaperones.

As expected, wild type Nap1p and Spt16p interacted with both H2A and H2B in their assay. Of all of the essential residues of the acidic patch, only L109 on H2B significantly affected H2B’s ability to interact with the FACT complex. There was about a 4-fold decrease in the amount of Spt16p brought down with H2B: L109A compared to wild-type H2B in these experiments.

The authors decided to broaden their search for residues important for interactions by looking at those in the acidic patch that were not lethal when mutated to alanine. Recent work suggested two other residues, H2A: E57 and H2A: E93, might be important for getting the FACT complex to actively transcribed genes in yeast. Hodges and coworkers were able to confirm the importance of H2A: E57 in their co-IP experiments.

They now expanded to other residues on H2A and H2B that have been shown or hypothesized to be important for binding to the FACT complex—H2A: R78A, and H2B: Y45A, M62E. These authors found that only H2B: M62E significantly impacted binding to the FACT complex (H2B: Y45A had a small effect). They also found that H2A: R78A affected binding to Nap1p, but not the FACT complex.

shepherding

Histones need to be shepherded to the genome by histone chaperones. by Mclaire MClaire, Wikimedia Commons

OK, so there is good co-IP data that H2A: E57, H2B: L109A, and H2B: M62E each affect binding of the FACT complex to the H2A/H2B dimer. These authors also provide good evidence that these mutants affect nucleosome occupancy and have nucleosome-based effects on transcription as well.

They used chromosomal immunoprecipitation linked to qPCR (ChIP-qPCR) against H2A and H2B to show decreased occupancy of H2A and H2B with the H2B: L109A mutant at four different promoters. Occupancy was around 3-4 fold lower than with wild type H2B.

They were also able to show that some of their H2A and H2B mutations mimicked the effects of partial loss of function mutations in the Spt16p part of the FACT complex. For example, just like mutations in SPT16 make a cell more sensitive to hydroxyurea, so too do H2B: Y45A, H2B: M62E, and H2A: E57A. These three mutants also induce cryptic transcription from the FLO8 gene like an Spt16 mutant.

Key residues in the acidic patch of H2A/H2B are critical for making sure histones get to the right place in the genome. Mutating them is similar to me not hearing the direction I need to go from my iPhone. Just like a histone not able to hang onto its chaperone, I will end up at the wrong place and not able to do what I needed to do.

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

Categories: Research Spotlight

Tags: map , NAP1 , histone , FACT complex , H2A , H2B

Yeast’s Skynet Against Salt

June 27, 2017


Skynet

Like Skynet, PI3,5P2 signaling is a rapid defense response. from Wikipedia.

 In the Terminator franchise, the U.S. creates an artificial intelligence (AI)-based defense system called Skynet to, among other things, react more quickly to threats than any general or politician could. What starts out as an interesting idea almost dooms mankind to extinction once Skynet becomes conscious and decides to eliminate its greatest threat—humans. 

Our friend Saccharomyces cerevisiae has its own version of Skynet for when it is “attacked” by too many salt ions. No, the system isn’t conscious and it does not threaten this yeast’s very existence but like Skynet, it is designed to react more quickly than more conventional systems based on gene regulation. It basically buys yeast enough time to allow the cells to more stably adapt to their new high salt environment. 

Within 1 -5 minutes of being plunked down into high salt, yeast activates Hog1p, a key MAP kinase. The activated Hog1p heads into the nucleus and within 30-60 minutes, it tweaks the expression of a bunch of genes so the yeast can now better deal with its new environment.

This is a lot of time to be languishing in high salt. Luckily, yeast’s “whole hog” approach to high salt is not limited to just Hog1p. According to a recent study by Jin and coworkers in the Journal of Cell Biology, there is another, faster reaction to the high salt. And at least in these experiments, it is critical for yeast’s survival when it is assaulted by too much salt.  

This rapid response involves a signaling lipid found in the vacuole called phosphatidylinositol 3,5-bisphosphate, or PI3,5P2. The amount of this lipid goes way up within just five minutes of the high salt shock.

Sarah Connor

One way to give Sarah Connor an easier life might have been to make Skynet’s control of defense transient. from cdn.movieweb.com.

PI3,5P2 is synthesized in yeast by a single enzyme, Fab1p. It stands to reason that if PI3,5P2 is critical to yeast survival in high salt, then deleting FAB1 should affect yeast’s ability to deal with all of those extra ions in its environment. This is just what Jin and coworkers found.

They compared the viability of wild type, hog1Δ, and fab1Δ strains under normal conditions and after a four hour exposure to 0.9M NaCl (high salt). Under low salt conditions, the fab1Δ strain was less viable than the other two. Around 30% of the fab1Δ yeast were dead.

At high salt, less than 10% of the wild type yeast and around 30% of the hog1Δ yeast were dead after four hours. This compares to the greater than 80% dead fab1Δ yeast.

The next steps in the study were to identify how the high salt increases the amount PI3,5P2. They reasoned that they needed something fast and that kinases just might fit the bill, so they started looking for strains that dealt poorly with high salt in the “…knockout haploid yeast mutant collection of 103 nonessential protein kinases.” They found a likely candidate in Pho85p and further work showed that its partner cyclin Pho80p was also involved.

Both the pho85Δ and pho80Δ strains had enlarged vacuoles (a common phenotype in yeast that cannot make PI3,5P2). More importantly, both strains could not make PI3,5P2 either under normal or high salt conditions and were also less viable than wild type under high salt conditions.

Additional experiments provided strong evidence that Pho85p phosphorylated Fab1p and that this phosphorylated Fab1p was important for synthesizing PI3,5P2 under high salt conditions. The final experiments confirmed that something similar happens in mammalian cells.

Jin and coworkers showed that the Pho80p-Pho85p equivalent in mammalian cells, CDK5-p35, phosphorylates the Fab1p equivalent, PIKfyve, in vitro. They also showed that CDK5-p35 is important for mouse fibroblasts to make more PI3,5P2 when exposed to high salt.

These studies suggest that yeast and probably mammals have at least two systems for dealing with high salt. The first is a rapid increase in PI3,5P2 that protects the cells from the environmental insult which gives the cells time to set up the second system—a longer term, more stable adaptation.

If only the folks in the Terminator world were as smart as yeast and had made Skynet a transient system set up to protect the U.S. while humans had time to respond in a more stable way. Think how much easier Sarah Connor’s life would have been! 

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

Categories: Research Spotlight

Tags: FAB1 , PIKfyve , 5-bisphosphate , high salt , phosphatidylinositol 3 , PHO80 , HOG1 , Terminator , PHO85 , CDK-P35

Making the Best of a Sticky Situation

June 05, 2017


Lemonade stand

Like turning lemons into lemonade, Hope and coworkers turned gummy yeast into a useful strain. from Pixabay.

 Back in 1915, writer Elbert Hubbard coined the phrase, “When life gives you lemons make lemonade.” (His actual quote was “He picked up the lemons that Fate had sent him and started a lemonade-stand.”)

The idea of course is to take something bad and make it into something good. Like, if your research gives you terribly weak glue, invent Post-It notes.

Or as Hope and coworkers show in a new study in GENETICS, when your yeast experiment gets gummed up because the yeast evolves a sticky trait, do additional experiments to learn about the evolution of that complex trait. And “invent” a way to make the yeast less likely to flocculate, or stick together.

This new “invention” will be very useful for anyone trying to evolve yeast to create new products or to study how evolution works. It also showed that for this trait, under these conditions with this strain, there was one major way to get to flocculence—up-regulating the FLO1 gene. This meant they could greatly reduce the risk of this trait popping up by simply deleting FLO1.

Studying evolution in yeast often involves using a chemostat, an automated way to keep the yeast growing through repeated dilutions with fresh media. Scientists use this method to study how yeast evolves under varying conditions over hundreds of generations.

An unfortunate side effect of this method is that it also tends to select for yeast that stick together. These yeast are diluted away less, often meaning they become more common over the generations.

In this study, Hope and coworkers ran 96 chemostats under three different conditions for 300 generations and found that in 34.7% of the cultures, the yeast ended up aggregated. This was even though they used a strain of S288c in which the FLO8 gene was mutated. This strain flocculates less often than wild type!

These authors picked the 23 most aggregated cultures to study in more detail. They found that 2 out of 23 strains aggregated because of a mother/daughter separation defect. The rest were more run-of-the-mill flocculent strains. 

They next used whole genome sequencing to try to identify which genes when mutated caused flocculence in the strains. They saw no FLO8 revertants.

The two strains with a mother/daughter separation defect both had mutations in the ACE2 gene, which encodes an important transcription factor for septation as well as other processes.

Flos

By CBS Television (eBay item photo front photo back) [Public domain], via Wikimedia Commons. Progressive insurance facebook flo advertising failsBy woodleywonderworks, via Flickr
No, these Flos don’t cause flocculence, FLO1 does.

FLO1-related mutations dominated the other 21. They found a couple of different Ty insertions in the promoter region of FLO1 in 12 of the strains and mutations in TUP1 in 5 more. Tup1p is a general repressor known to repress FLO1, so it looks like up-regulating FLO1 leads to flocculent cultures. Other candidate mutations were found in FLO9 and ROX3.

They wanted to try to identify the responsible gene(s) in strains where there was no obvious candidate gene and also to confirm that the genes they identified really caused the trait, so they next did backcrosses between each mutant strain and a wild type strain.

The backcrosses identified two other ways to get flocculence— by mutating either CSE2 or MIT1. The researchers also confirmed that the mutations they found, including these two new ones, were probably the main cause of the flocculence in each individual strain.  This trait co-segregated with the appropriate mutation in a 2:2 pattern for 20/21 strains as is predicted for a single causal mutation.

The results are even more FLO1-heavy than they appear. Further studies showed that ROX3, CSE2, and MIT1 all require a functional FLO1 to see their effects.

So FLO1 appears to be the main route to flocculence in this strain. And the next set of experiments confirmed this.

Hope and coworkers ran 32 wild type and 32 FLO1 knockout strains in separate chemostats for 250 generations and found that 8 wild-type strains flocculated while only 1 FLO1 knockout strain did. Knocking out FLO1 seems to make for a more well-behaved yeast (at least in terms of evolving a flocculent trait in a chemostat).

And the strain can probably be improved upon even more. For example, researchers may want to limit Ty mobility as this was a major way that FLO1 was up-regulated, increase the copy number of key repressors or link those repressors to essential genes. Another possibility is to also mutate FLO9 as its up-regulation was the cause of that one aggregated strain in the FLO1 knockout experiment.

Researchers no longer need to “settle” (subtle, huh?) for big parts of their experiments being hampered by gummy yeast. Hope and coworkers have created a strain that is less likely to flocculate by simply knocking out FLO1. Not as ubiquitously useful as a Post-It note but a potential Godsend for scientists using yeast to understand evolution.

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

Categories: Research Spotlight

Tags: FLO9 , FLO8 , flocculation , FLO1 , evolution , ROX3 , MIT1 , TUP1 , CSE2 , ace2

Creating an Ethanol-Making ‘Super Bowl’ Championship Team

May 24, 2017


New England v. Miami NFL

Tom Brady makes a team better by making the players around him play better. The same thing can be said for a mutant RPB7 gene that makes other genes work together better as an ethanol-making team. By Paul Keleher [CC BY 2.0 (http://creativecommons.org/licenses/by/2.0)%5D, via Wikimedia Commons.

There are a few ways to turn a failing sports team around. One is to tailor individual training to make each player better. Now, the team is better overall because of the changes each player makes.

Another way to improve a team is to change a player in a key position who makes everyone better. A classic example of this is the American football team, the New England Patriots.  

On September 23, 2001, Drew Bledsoe, then the starting star quarterback of the New England Patriots, took a savage hit from New York Jets linebacker Mo Lewis. The Patriots replaced Bledsoe with his backup, Tom Brady, and some might argue, the team (whom Brady led to their first Super Bowl win that year) and the NFL, has not been the same since.

Quarterback Tom Brady, along with head coach Bill Belichick, makes whomever the New England Patriots bring in better. Wide receivers, tight ends, and running backs can be replaced in the lineup without the team missing a beat. He just makes the players around him better than they might be on another team.

In a new study, Qiu and Jiang take a “Patriots” approach to ethanol production in the yeast Saccharomyces cerevisiae. Rather than improving individual genes on their own, these authors instead decided to “bring in” a new version of RPB7, a gene that encodes a key subunit of RNA polymerase II, the molecular machine responsible for making messenger RNA (mRNA).

They hoped that changing this pivotal transcriptional player would cause lots of other genes to do “better” so that “team” yeast would make a lot more ethanol.  Their hopes were realized in their Tom Brady equivalent—a mutant they called M1. Yeast bearing this mutant RPB7 gene became the Super Bowl champs of ethanol production.

One of the keys to increasing ethanol production in yeast is to find strains that are more tolerant of high levels of ethanol. The more ethanol they can withstand, the more they can make.

These authors used error prone PCR mutagenesis of the RPB7 gene to find their game-changing mutant. They then took their library of ~108 clones and cultured them in increasing amounts of ethanol, selecting for more ethanol-resistant strains.

After 3-5 rounds of subculture, they plated the cells onto media containing ethanol. Around 30 colonies were picked and sequenced with the best mutant being the one with two mutations—Y25N and A76T. They named this mutant M1.

This mutant grew a bit better than the parental strain background, S288C, in the absence of ethanol, but where M1 really shined was when ethanol was around. It grew around twice as fast in 8% ethanol and could grow at 10%, a concentration that completely inhibited the parental strain from growing.

Being able to withstand high levels of ethanol is important, but it isn’t all that yeast have to deal with. There are multiple other stressors around when you are swimming in 20 proof media.

For example, yeast can suffer from high levels of reactive oxygen species (ROS). M1 not only tolerated 3.5 mM hydrogen peroxide, a proxy for ROS, better than the parental strain, but it also had around 37% of ROS levels inside cells than that of the parental strain. M1 can deal with high levels of ethanol and ROS.

The authors then tested how this mutant dealt with other potential fermentation problems. For example, acetate, a fermentation byproduct, and high levels of NaCl both inhibit yeast growth. M1 tolerated 80 mM acetic acid and 1.5 M NaCl better than the parental strain did.

drunk Gingy

A couple of mutations in the RPB7 gene makes yeast able to tolerate alcohol way better than this guy. By jerome Chua [CC BY 2.0 (http://creativecommons.org/licenses/by/2.0)%5D, via flickr.

M1 appeared to be a champion mutant for making ethanol, and the fermentation studies bore this out.

Under a wide variety of conditions, M1 outperformed the parental strain in terms of growth rate, cell mass, and amount of ethanol made. For example, after 54 hours, yeast containing the M1 mutation of RPB7 managed to make 122.85 g/L of ethanol, 96.58% of the theoretical yield. This is a 40% increase over the control strain. Quite the ethanol producer!

Finally, Qiu and Jiang used microarray analysis of the parental and M1 strains at high levels of ethanol to discover the genes that M1 affected. They found 369 out of a total of 6256 genes behaved differently between the two strains. Of the 369, 144 were up-regulated and 225 were down-regulated.  

I don’t have time to go over all the genes they found but a great many of them make sense. As the authors write, “…a significant set of genes are associated with energy metabolism, including glycolysis, alcoholic fermentation, hexose transport, and NAD+ synthesis.”  M1 seems fine-tuned for making ethanol.

A mutant subunit in RNA polymerase II has made yeast better at making high levels of ethanol, most likely by affecting many key genes at once. It is a fascinating way to quickly affect a whole suite of genes involved in a process. In the ethanol-making Super Bowl, we have a new champion yeast strain, M1.

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

Categories: Research Spotlight

Tags: RPB7 , Super Bowl , NFL , RNA polymerase II , Patriots , Tom Brady , ethanol , fermentation

Mass Production in Yeast

May 11, 2017


Workers on first moving Ford assembly line

This approach changed everything when it came to manufacturing in factories. Perhaps the ideas in this new study will change things for manufacturing in cells.Image from Wikimedia Commons

After Henry Ford invented the moving assembly line, manufacturing was never the same. With it, his workers were able to push out a car every 2 ½ hours instead of the 12 it used to take. (Another website said it was reduced to 90 min!)  The technology quickly spread to every factory.

Now of course, an assembly line is only as fast as its slowest worker. If someone is taking extra time to bolt down that part, then everyone downstream will have to go slower too, resulting in fewer cars being made.

But, you also can’t go too fast. If you do, someone can get injured, shutting down the whole line. (Or the worker has to eat all the candy to keep up, like Lucy.)

And you want to make sure things happen in the right part of the factory. You don’t want the paint sprayer out in the open, poisoning factory workers. So, that needs to happen in a special room.

This also applies to cell processes where something complicated is built, step by enzymatic step. All the enzymes need to be at the right levels and in the right place to maximize the productivity of the whole process.

This all becomes very obvious when you try to move an enzymatic process from one beast to another. What worked perfectly before, now barely works at all.

One way to fix this is through trial and error, trying to optimize one part of the process at a time. This is incredibly time consuming!

In a new study out in Nature Communications, Awan and coworkers show one way to tweak all of the enzymatic steps involved in making penicillin at the same time in the yeast Saccharomyces cerevisiae. While this isn’t that useful for making this antibiotic (there are better ways available right now), it does show how researchers can apply the same techniques to perhaps identify and produce new antibiotics. And, it can also be applied to other unrelated enzymatic processes.

Penicillin is made in a five-step process in filamentous fungi. In the first part of the process, two enzymes create a tripeptide precursor using alpha-aminoadipic acid, cysteine, and valine, called ACV. This part of the process had been previously recapitulated in yeast, so Awan and coworkers used this as a starting point for their penicillin producing strain.

The next part of the process uses the last three enzymes and takes place in peroxisomes in filamentous fungi. These authors found that they only got penicillin when these enzymes were tagged to be sent to the peroxisome in yeast. Like a special room for spray painting cars, these enzymes need to be in the right place to make penicillin.

But this was by no stretch of the imagination an efficient penicillin-making machine. The thing managed only 90 pg/ml in the media. As Ursula from Little Mermaid might say, “Pathetic.”

Ursula from The Little Mermaid

From Tumblr

Still, it is a starting point. The next step is to get the yeast to crank out more penicillin. To do this, they used a combinatorial approach to optimize the process all at once. Well, not really all at once.

First, they set out to optimize how much of the precursor ACV the yeast made. Then, they optimized how much ACV was converted to penicillin.

Awan and coworkers created a library of low copy plasmids that had the genes for the first two enzymes, pcbAB and npgA, under the control of different pairs of promoters. One plasmid, with the pTDH3 promoter driving pcbAB expression, and the pPGK1 promoter driving npgA expression, outperformed all of the others. As measured by Liquid Chromatography-Mass Spectrometry (LCMS), the yield of ACV increased from 20 to ~280 ng/ml.

Next, the authors used this new strain as a starting point for optimizing the activity of the final three enzymes using a similar approach. They used a “…one-pot combinatorial DNA assembly using Golden Gate cloning…” to make a library of around 1000 high copy plasmids where each gene was under the control of one of ten different promoters of varying strength. Using LCMS they found strains that could make 3 ng/ml of penicillin, a significant improvement over the original 90 pg/ml.  

The 3 ng/ml of penicillin in the media should be high enough concentration to inhibit the growth of bacteria like Streptococcus pyogenes. So, they confirmed that their penicillin was active using growth inhibition assays.

After sequencing the plasmids, the authors saw that the best strains tended to have strong constitutive promoters driving one of the genes, pclA, and medium strength promoters driving another one of the genes, pcbC. They used a minION DNA sequencer to confirm that this was not the result of a biased library.

As a final step, they set out to optimize penicillin production and to increase the throughput of their assay. They created another library that swapped six different promoters that varied in strength from medium to high for each of the last three genes in the pathway, pclA, pcbC and penDE. Instead of using LCMS to screen for penicillin production, they used a 96 well plate-based assay that looked for inhibition of Streptococcus pyogenes growth for their 120 new strains.

They selected 12 of the highest performing strains and confirmed by LCMS that they made lots of penicillin. Five of the strains made more than 5 ng/ml, a more than 50-fold increase over their original strain.

As this concentration is still three orders of magnitude below what other organisms can currently do, this new yeast strain will not go into penicillin production any time soon. But this study gives us a way to quickly optimize antibiotic production using growth inhibition assays instead of the more cumbersome LCMS.

And it isn’t restricted to just antibiotic production. Similar combinatorial approaches can be used for almost any stepwise enzymatic process. Researchers can create libraries of plasmids where levels of enzyme vary and use the long reads of minION DNA sequencing technology to confirm that their results are not skewed by a biased library.

As usual, this is only possible as a simple, easy procedure because of the awesome power of yeast genetics (#APOYG). Researchers have the tools to use yeast to find new antibiotics and to manufacture them at a high rate, like inventing the car and the assembly line at the same time.

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

Categories: Research Spotlight

Tags: penDE , minION , penicillin synthesis , liquid chromatography-mass spectrometry , ACV , pcbC , pcbAB , pclA , npgA

Meiotic Fail Safes

May 04, 2017


minuteman in silo

Launching into meiosis too soon is as dangerous (for a cell) as launching a nuclear missile. Luckily both have protocols to make sure each can only happen in the right circumstances. (Hopefully never for the nuclear missile.) Image from Wikimedia Commons.

If the movie WarGames is anything to go on, the US government does not make it easy to launch a nuclear missile. Two soldiers have to do many things simultaneously and in the right order before that missile can take flight.

This makes perfect sense as you do not want to launch a nuclear attack unless you absolutely have to. The continued existence of the human race depends on these fail safes being in place and working. The same goes for a cell that is heading into meiosis.

Meiotic fail safes are in place to ensure the survival of a cell during the dangerous, early part of meiosis, when there are lots of double-strand breaks in the DNA. These all need to be resolved before a cell is allowed to continue through meiosis to create gametes. If the cell moves on while the breaks are still there, gamete production will fail and the cells will die.

While the exact sequence of events needed to launch World War III is known (at least by a few people), the exact details of getting a cell safely through meiosis are a bit murkier. With the help of good old Saccharomyces cerevisiae, we have the broad outlines, but are still investigating the finer points.

A new study by Prugar and coworkers in GENETICS has helped clear up a bit of the murk in yeast. They have uncovered a connection between the meiosis-specific kinase Mek1p and the transcription factor Ndt80p that may explain how a cell “knows” when it is safe enough to emerge from prophase and keep progressing through Meiosis I.

Mek1p is known to be active when there are lots of these double-strand breaks around and to lose activity as these breaks are resolved. Ndt80p, on the other hand, is inactive when there are lots of these breaks and active when they are resolved. So it makes sense that their activities might be related to each other.

In this study, the authors show that once Mek1p activity falls below a certain level, it can no longer keep tamping down Ndt80p activity. Once unleashed, Ndt80p can go on to activate many genes, including the polo-like kinase CDC5 and the cyclin CLB1. This round of gene activation allows the cell to progress through meiosis.

The key to teasing this out was a set of experiments where Prugar and coworkers were able to control the activities of Mek1p and Ndt80p independent of the cell’s DNA state. It is like circumventing the set of protocols to get those missiles launched.

To independently control Ndt80p activity, they used a form of the protein that requires estradiol to be active. And they controlled the activity of Mek1p by using a mutant, mek1-as, that is sensitive to the purine analogue 1-NA-PP1. In the presence of this inhibitor, Mek1p stops working.

They looked at the targets of these two proteins to infer activity. For example, they determined if Ndt80p was active by looking for the presence of CDC5. And to see if Mek1p was active, they looked for phosphorylated Hed1p.

In the first experiment, they showed that in the absence of both estradiol and 1-NA-PP1, Hed1p stayed phosphorylated. Mek1p was constitutively active in the absence of Ndt80p even as double-strand breaks were resolved.  (They used phosphorylated Hop1p as an indirect measure of double-strand breaks.)

War Games (1983)

In the end, as yeast relies on MEK1 to prevent a meiotic disaster, humans, not computers, kept the world safe in the movie WarGames. Image from flickr

When Ndt80p was activated through the addition of estradiol, CDC5 was turned on and Hed1p lost its phosphorylation. This loss of Mek1p activity did not happen as quickly as with 1-NA-PP1.

These results suggest a negative interaction between Mek1p and Ndt80p. When Ndt80p is active, Mek1p is not and when Mek1p is active, Ndt80p is not. The resolution of the DNA breaks as indicated by the loss of phosphorylated Hop1p was not sufficient to shut off Mek1p activity. It took the activation of Ndt80p for this to happen.

Well, Ndt80p did not directly cause Mek1p’s inhibition. A second set of experiments suggested that a target of Ndt80p, CDC5, was responsible.

For this they made Cdc5p activity independent of Ndt80p induction by making it dependent on estradiol, similar to what they did with Ndt80p. Using a strain deleted for NDT80, they found that inducing Cdc5p activity was enough to eliminate Mek1p activity.

I don’t have the space to go into the rest of the experiments in this study, but I urge you to read it if you want to learn about more of the details of the cell’s protocol for know when it is OK to progress through meiosis.

With the help of the awesome power of yeast genetics (#APOYG), Prugar and coworkers have added to our knowledge about the safeguards that are in place to keep a cell from launching into meiosis too soon. Turns out they are even more complicated than the ones that prevent accidental thermonuclear war.

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

Categories: Research Spotlight

Tags: double-strand breaks , MEK1 , NDT80 , meiosis , DSB , CDC5

Knock out YME1, Luke

April 20, 2017


Death Star Explosion & Millenium Falcon

Instead of an exhaust port, one of a mitochondrion’s fatal flaws may be the YME1 gene. Image from Manoel Lemos, flickr.

In the original Star Wars, Luke destroys the Death Star with a precise strike of proton torpedoes down a small thermal exhaust port. For him it was as easy as bullseyeing “womp rats in my T-16 back home.”

Luke and the rest of the Rebel Alliance learned of this engineered fatal flaw from Jyn and her friends in the prequel Rogue One. With this information the Rebel Alliance was able to keep the rebellion alive long enough to finally bring down the Empire by the end of Return of the Jedi.

It turns out that our friend Saccharomyces cerevisiae has taught us about a fatal flaw in mitochondria. Like proton torpedoes in an exhaust port, when the gene YME1 is inactivated, mitochondria become unstable. But instead of bits of Death Star raining down on nearby planets, mitochondrial DNA (mtDNA) is released into the cytoplasm.

Sometimes this mtDNA can end up in the nucleus and find its way into nuclear DNA. And if the conclusions of a new study in Genome Medicine by Srinivasainagendra and coworkers turns out to be right, this numtogenesis (as the authors call this process) can have profound consequences when it happens in people. Their data suggests that it might lead to cancer or possibly cause cancers to spread.

These researchers searched through whole genomes of colon adenocarcinoma patients and found that these cancer cells had 4.2-fold more mtDNA insertions compared to noncancerous cells from the same patient. They also found that patients with more of these insertions tended to do worse (although the sample sizes were too small to say this definitively).

Why is this happening in the cancer cells? What has caused the mitochondria to give up their DNA?

Srinivasainagendra and coworkers turned to previous work that had been done on the YME1 gene in the yeast S. cerevisiae to find one possible reason. YME1 had been shown to be an important suppressor mtDNA migration to the nucleus. Perhaps this was true in mammalian cells as well.

A search through the genomes of cancers suggested that this seemed to be the case. Around 16% of the colorectal tumors they looked at had a mutated YME1L1 gene, the human homologue of YME1. And mutated YME1L1 genes were found in other tumors as well.

If only destroying gene function was as fun.

They used CRISPR/Cas9 to directly test the effects of knocking out YME1L1 in the breast cancer cell line MCF-7. The knock out cells had a 4-fold increase in the amount mtDNA in the nuclear fraction compared to cells that still had working YME1L1.

As a final experiment, they used a yeast strain, yme1-1, in which YME1 function was inactivated, to show that the human homologue, YME1L1, could suppress the migration of mtDNA to the nucleus.

This yme1-1 strain has a TRP1 gene encoded in the mtDNA instead of the nucleus. Since the gene cannot be read by the mitochondrial transcription machinery, the only way this yeast strain can survive in the absence of tryptophan is if the TRP1 gene moves from the mitochondrion to the nucleus. 

In their experiment, with vector alone, they got around 1000 TRP+ colonies with yme1-1. When they added back yeast YME1, this number dropped to less than 50 compared to the 100 or so they got when they added the human homologue, YME1L1. So YME1L1 can suppress mtDNA migration to the nucleus.  

Given that YME1L1 was mutated in just a subset of the cancers, it is unlikely that it is the only player in the mtDNA these authors found in the nuclei of cancer cells. But it does look like it is one way this can happen.

And it would have been very hard to fish out the human gene without the critical work that had been done in yeast previously. Yeast shows us the way again. #APOYG

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

Categories: Research Spotlight

Tags: nuMt , nuMtogenesis , cancer , YME1L1 , MCF-7 , mtDNA , YME1 , colon cancer , CRISPR/Cas9

The Dark Yeast Rises

April 04, 2017


Sometimes in life you need to take risks to survive and prosper. Maybe you need to take a leap of faith like Bruce Wayne does to escape that pit in The Dark Knight Rises. Or you need to try a risky business strategy to put your company out in front.

While these are critical things to do at the time, chronic risk-taking is not usually a good idea. Even Bruce Wayne retires to Florence at the end of The Dark Knight Rises, his risky life choices done now that he has saved his beloved Gotham City. He can now settle down with Selina Kyle (Catwoman) and live happily (and safely) ever after.

Something similar can happen in the budding yeast, Saccharomyces cerevisiae. In the right environment, certain yeasts can build up mutations like mad, hoping to hit on one that lets them make it out of that pit.

But then, over time, yeasts with the successful mutation lose that frenetic mutation rate—they take fewer risks with their DNA. One way they can do this is by ending up with a mutation that suppresses the high mutation rate. This is like Bruce Wayne retiring to a nice villa on the Arno.

Another way they can reestablish their old mutation rate is to mate with a nonmutator, a yeast strain that does not risk its DNA with a high mutation rate. Now, the next generations have the beneficial mutation and a lowered mutation rate as well. It is as if Bruce Wayne settled down with an accountant instead of Catwoman and had risk-averse children to carry on the Wayne name.

Bui and coworkers investigate this phenomenon in yeast in a new study out in GENETICS. They show that natural isolates exist that can sporulate into one of these mutator strains. And that at least one strain predicted to have a high mutation rate has a number of suppressor mutations that tamp down that higher rate.

Previous work had shown that a high mutation rate can happen with mutations in two genes in the mismatch repair (MMR) pathway, MLH1 and PMS1. This was discovered when researchers saw that some of the haploid strains from a mating of two laboratory strains, S288C and SK1, showed this mutator phenotype. A closer look revealed that S288C has a mutation in MLH1, and SK1 has a mutation in PMS1.

Bui and coworkers show that these mutator haploid strains outcompete other strains in high salt media. The strain hits upon mutations that allowed for better survival in high salt faster than its slower mutating brethren. But this advantage does not last.

After 120 generations these mutator strains start to lag behind strains with more typical mutation rates. Their DNA becomes as beat up as Bruce Wayne’s body by the third movie.

The researchers next looked at the MLH1 and PMS1 genes of 1,010 natural isolates of S. cerevisiae to see if there were any that might be able to sporulate into a mutator strain. Or that were already mutator strains themselves.

Since the mutations that lead to the mutator phenotype are recessive, strains that are heterozygotes for mutations in both genes should be able to form haploid spores with the higher mutation rate. They found 18 that fit the bill.

They also found one strain, YJM523, that was homozygous for both mutations and so would be predicted to have a high mutation rate. They further investigated this strain.

broken

The first thing they did was to test the YJM523 mutant genes in an S288C background. They found that these two mutant genes did indeed give S288C a mutator phenotype. In fact, it had a higher mutation rate than the original genes from S288C and SK1 did, suggesting there were other mutations in one or both YJM523 genes that further increased the mutation rate.

These researchers next wanted to determine if YJM523 had a higher mutation rate or not, but needed to first develop a new assay that could be used in natural isolates. They came up with a reporter system that is not dependent on the typical markers used in yeast experiments, but instead relies on two antibiotic markers.

The reporter uses the NatMX antibiotic resistance marker for selection and the KanMX marker to identify revertant mutants. By scoring the number of colonies resistant to both antibiotics, they could determine the mutation rate.

When the researchers did this experiment, they found that YJM523 did not have a particularly high mutation rate. Sporulation experiments showed that this was most likely due to multiple suppressor mutations.

So out in the wild, the potential for mutator strains exists. And a close look at the genome of YJM523 showed that if it did have a mutator phenotype, it was for a short time. This yeast at least quickly lost its high mutation rate (assuming it ever had one).

This study helps to explain how yeast can obtain that mutator phenotype that researchers have seen before. And how yeast can escape that mutation pit to get back to the safety of a reasonable DNA repair pathway. To quote the prisoners in The Dark Knight, “Deshi Basara”, or ‘rise up’ yeast!

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

Categories: Research Spotlight

Tags: experimental evolution , genetic incompatibility , DNA mismatch repair , mutator phenotype , adaptation , natural yeast isolates

Too Big for the Oven

March 23, 2017


I love lucy, Pioneer Women episode picture

Just like baking bread, “oven” size matters when trying to increase yields of fatty acids in yeast.

In a classic skit from I Love Lucy, Lucy bakes a loaf of bread that is so big it ends up coming out of the oven, pushing her against the far wall. Definitely one of the funniest moments from early TV.

In a new study in Nature Communications, Gajewski and coworkers show that the yeast enzyme fatty acid synthase (FAS) complex, is a bit less comical when it comes to the fatty acid chains it makes. When these authors engineer the enzyme complex to “shrink” the oven, the “bread” it makes becomes smaller. A more constrained active site that holds onto its growing fatty acids less tightly makes shorter-chained fatty acids.

This is an important finding because these shorter-chained fatty acids are so useful as precursors for products like biofuels. Engineered yeast like these may, among other things, one day help us get to a carbon neutral future.

Gajewski and coworkers were able to pull this off because the 3-D structure of this enzyme complex is so well understood. They engineered changes that would be predicted to restrict the growth of the fatty acid chain.

For example, a key player in the elongation step happens in the condensation domain (KD) of the complex. They focused initially on the amino acid methionine at position 1251 (M1251).

This amino acid sticks out into the active site and needs to rotate out of the way when the fatty acid chain elongates. The authors reasoned that if they made it bigger and harder to rotate, the enzyme complex  wouldn’t be as good at elongating the fatty acid chains it makes. The loaf would stop growing when it ran out of room.

They were right. When they mutated a nearby glycine to serine (G1250S), the yeast now made 15.3 mg/liter of fatty acids with 6 carbons (C6 fatty acids), a 9-fold increase over wild type. FAS usually makes C12-C18 fatty acids.

two chemostats with yeast cultures

Tomorrow’s oil wells? By (Image: Maitreya Dunham) [CC BY 2.5], via Wikimedia Commons

They got even better results when they bulked up position 1251 by changing the methionine to a tryptophan (M1251W). Now the yeast made 19.1 mg/liter of C6 fatty acids, a 12-fold increase, and 32.7 mg/liter of C8 fatty acids, a 56-fold increase. They made a third mutation, F1279Y, that ended up affecting growth adversely when combined with G1250S.

This was good, but not good enough. To be commercially viable, they needed higher yields. They next wanted to make mutations that would cause the enzyme complex to hold onto the fatty acids less tightly, like having the baker remove the bread from the oven before it got too big.

For this, they focused on the malonyl/palmitoyl transferase (MPT) domain. They reasoned that by making the complex less able to bind malonyl, it would also bind the growing fatty acid chain less well too. Again, they were right.

When they replaced an arginine with a lysine at position 1834 (R1834K), the yeast made 100 mg/liter of mostly C8 fatty acids, a 23-fold increase. They made an additional mutation, I306A, that had little effect on its own.

Things got really interesting when they looked at different combinations of these mutations. When they mixed and matched them, they pushed yields up even more. And different combinations made different proportions of various sized fatty acids. Scientists can pick the mutant that gives them their desired product.

The best yield was with yeast carrying the triple mutant, I306A-R1834K-G1250S. These yeast managed to make 118 mg/liter of shorter-chained fatty acids.

Gajewski and coworkers boosted this mutant’s yield even more by changing out the promoter. Doing so resulted in the yeast making 464.4 mg/liter of the enzyme.

We now have a strain of yeast that can make a lot of the precursors needed for making biofuels and other chemicals. And scientists can pick the mutant that gives them more of their desired precursor. If they want more C6, they should use one mutant and if they want more C8 they should use a different one.  

#APOYG is helping to create designer molecules that could, among other things, help steer us toward a more carbon neutral future.  

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

Categories: Research Spotlight

Tags: precursor , biofuels , fatty acid synthesis

In Yeast, Being Old Can Be a Good Thing

March 13, 2017


Like this marathon runner, some older yeast are able to win out over their younger counterparts. In the right environment, that is! Image from Wikimedia Commons.

A square slab can make an excellent door stop. Over time, though, the corners can get chipped, making the slab a bit rounded. This new bit of rock makes a less useful doorstop, but a much better wheel! The chipping and aging of the original stone has made it worse in some situations, but better in others.

A new study by Frenk and coworkers in Aging Cell shows that something similar can happen in yeast. Young yeast are much better at utilizing glucose, but older yeast have them beat with galactose (as well as with raffinose and acetate).

One way to think about this is that age turns yeast from a glucose specialist into a sugar generalist. Aging chips away at a yeast cell’s ability to use glucose, but this loss results in a gain in its ability to use galactose.

So at least in the right environment (i.e., when there’s lots of galactose around), with our old friend Saccharomyces cerevisiae, there can be advantages to getting older.

What makes this particularly fascinating is that at least in yeast, this suggests that there may be a positive selection for aging because of the advantage it can give in certain environments. Those yeast who are ageless would compete less well compared to their aging counterparts when their glucose was taken away. The aging process wins out over immortality!

Frenk and coworkers used a relatively simple experimental set up. Take young cells and old cells, mix them together, and see which outcompetes the other using various sugars.

They used yeast that had been aged for 6, 24, and 48 hours in glucose. This is a nice range as 6-hour “old” yeast are fully viable, 24-hour “old” yeast are starting to suffer a bit in the reproductive viability department, and the 48-hour “old” yeast have passed the median lifetime of a yeast cell. Young adult, middle aged, and elderly yeast.

In the first experiment, they compared these yeast to log-phase yeast which the authors refer to as young (vs. the other three which are referred to as aged).

While the 6 hour yeast could hold its own against the young yeast in glucose, the 24-hour and 48-hour yeast grew much more slowly. This is what you would expect, the younger yeast growing faster than the older yeast. The young guns outdoing the older generations.

The situation was different in galactose. Here, the elderly, 48-hour yeast, ran circles around the young yeast. They blew them out of the water.

And it appears to be an age thing. When they compared 6- and 48-hour yeast that were aged in galactose instead of glucose, the more aged yeast still won. So, it wasn’t the shift in environment that caused the difference, it was in the older yeast cells all along.

The change is also not permanent. The offspring of the older yeast weren’t any better at growing in galactose than the younger yeast were. Only the cells that had lived a longer life could use galactose so well.

flintstonemobile

The cylindrical stone may not be as good a doorstop, but it makes a much better wheel! Image from flickr.

A concern here is that yeast as old as 48 hours are pretty rare in the wild. But when they changed assays and looked at colony size as opposed to competition, they saw that even 18-hour yeast had an advantage over the young whippersnappers.

This was such a surprising result that they also looked at cell cycle times of individual aged cells and their daughters. The older mother cells cycled faster in galactose than their daughters. And the opposite was true in glucose.

So it really looks like there are advantages to growing older. Things break down a bit, but that breakdown uncovers new talents that had previously lain dormant.

If you’re a yeast, growing old is not a one-way decline into dotage. You gain new abilities that, under the right conditions, let you outcompete your children! The older cells are selected for in the right environments. #APOYG shows us something good about growing older.

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

Categories: Research Spotlight

Tags: evolution of aging , aging , generalist , specialist

Feed a Cold, Starve a Cancer?

March 02, 2017


Unlike a fever, starving some cancers is actually a good treatment. And our friend yeast may be able to help. Image from http://maxpixel.freegreatpicture.com.

You may have heard the old wives’ tale of feed a cold, starve a fever. Turns out that this isn’t particularly good advice (although some studies do suggest that with a fever, you shouldn’t force-feed yourself). It also turns out to have probably originated in the 19th century and not from Chaucer in the 14th as many websites claim.

But while starving a fever is probably never a good idea, starving a cancer can be. Not by following the medical myth that since cancers use a lot of sugar, you can starve them by cutting down on sugar in your diet. Instead you can starve some cancers by denying them the amino acid asparagine (Asn).

On their way to becoming cancerous, acute lymphoblastic leukemia (ALL) cells lose their ability to make Asn. This means that unlike the cells around it, they need to pull Asn from the blood to make their proteins and to survive.

Doctors exploit this weakness by injecting L-asparaginase amidohydralase (L-ASNase) into patients which starves the cancer cell by depleting Asn levels in the blood. The cells around the cancer cells are fine because they can still make Asn.

Right now doctors use L-ASNase from two different bacterial sources: Escherichia coli and Erwinia chrysanthemi. But if a recent study by Costa and coworkers in Scientific Reports holds up, they might want to think about switching to using the Saccharomyces cerevisiae L-ASNase encoded by the ASP1 gene.

An older study had suggested that the yeast enzyme might be too weak to be useful. This new study finds that this is not the case.

The difference between the older study and this one was the purification protocol. The older study purified the native enzyme through multiple chromatography steps while this study used a single affinity chromatography step. The purified yeast and E. coli versions have comparable activity in this study.

They are also comparable in terms of being able to work with very low concentrations of Asn. This is important as Asn levels are very low in the blood.

What makes the yeast enzyme potentially better is that it is much worse at hydrolyzing a second amino acid, glutamine, than are the bacterial versions. This higher specificity for Asn is important because one of the major side effects of the current treatment is neurotoxicity caused by decreased levels of glutamine in the blood. Since the yeast version hydrolyzes glutamine at a lower rate, they predict patients may not suffer as badly from this side effect with the yeast version.

Of course this is all for naught if the yeast enzyme can’t kill cancer cells! Or if it kills cells indiscriminately.

The S. cerevisiae version was nearly as good as the E.coli version in tissue culture. After 72 hours of incubation, both versions had little effect on normal cells (HUVEC), and both were cytotoxic to the L-ASNase-sensitive cell line MOLT-4 with the E. coli version killing 95% of MOLT-4 cells and the yeast version killing 85% of them.

puppy

Move over dog, yeast is humanity’s best friend now. Image from pixabay.

Taken together these results suggest that the S. cerevisiae version may be an alternative to the bacterial versions. It may be able to kill cancer cells with fewer side effects.

But the yeast version is not the only alternative in town. Another group is engineering the E. coli version to lessen its propensity for hydrolyzing glutamine. Either way it looks like certain leukemia patients may be getting an effective cancer treatment with fewer side effects.

Beer, wine, bread, chocolate, and now maybe a treatment for a nasty form of leukemia. Yeast may be humanity’s best friend. #APOYG!

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

Categories: Research Spotlight Yeast and Human Disease

Tags: Recombinant protein therapy , Acute lymphocytic leukemia , Proteins , cancer

Turning to the Prion Expert: Yeast

February 15, 2017


You need an expert (not this guy!) for your plumbing problem. Just like you need yeast, and not some other organism, to study prions Image from flickr.

If you have a legal problem, you get a lawyer. A medical problem, a doctor. A leaky faucet, a plumber.

And if you are trying to find and figure out if a protein is a prion, you turn to the model organism where it is best understood. Yes, that is our old friend, the yeast Saccharomyces cerevisiae.

Prions became famous in the 1980’s when mad cow disease started to pop up in Britain. These are fascinating proteins that can cause an inheritable disease without affecting a cell’s DNA.

What happens is that prions can undergo a spontaneous conformational change. Now of course, this isn’t all that special. Lots of proteins can exist in different conformations.

But what makes a prion special is that the spontaneous change sets off a chain reaction where pretty much every copy of that prion protein is converted to that second conformation. And every translated prion protein thereafter both in the cell and any cells derived from that cell have that conformation too. So the new conformation along with the new traits it confers is passed on stably.

In mad cow disease, this spontaneous change causes neurological problems eventually resulting in death. But not every prion is so dangerous. Sometimes, as is the case in yeast, they can give new properties that allow survival in a new environment. Now these yeast have a new advantage in the absence of changed DNA.

Up until now prions have pretty much been confined to eukaryotes. That looks to change if a new study in Science by Yuan and Hochschild holds up.

They found that some bacteria have proteins that can and do behave as prions in a laboratory setting. The next step is to determine if they ever do so in the wild. If they do, then this would tell us that functioning prions may have evolved before the Bacteria/Eukaryota split and so be older than scientists previously thought.

The first step in finding a bacterial prion involved combing through a few bacterial genomes. Did I say a few? I meant something like 60,000 of them!

Of course you need the right tool for your search. This is where yeast’s incredibly well characterized set of prions comes in handy.

Yuan and Hochschild used an algorithm trained on known yeast prions to search through the bacterial genomes for proteins that have domains that would be predicted to be able to enter into a prion conformation. Among the proteins they found was the Rho protein in Clostridium botulinum E3 strain Alaska E43. Like the authors, I’ll call this protein Cb-Rho from here on out.

As you might remember, Rho is that famous transcription termination protein found in many different bacteria including E. coli. The E. coli version, however, does not look particularly like a prion nor did the authors find that it acts like one either.

A hallmark of prions is that one of the conformations involves their ability to form amyloid aggregates. These authors used a bacterial assay to show that this happened with Cb-Rho.

They next checked whether the prion portion of the protein behaved as a prion in a yeast cell. They substituted the prion domain from Cb-Rho with that of the one from Sup35p, a yeast prion. This chimeric protein behaved similarly to wild type Sup35p.

pipe

If you have a broken pipe, you find a plumber. But if you want to find and characterize a prion, you turn to yeast. Image from flickr.

They next tested their protein in E. coli. Often the prion conformation of the prion protein results in decreased activity of the protein. This is true in the Sup35p case, for example.

Sup35 acts as a translation release factor – it is an important factor in stopping translation at a stop codon. It is less active in its prion form meaning that there is now more read through of stop codons.

Rho is a transcription termination factor and so it would make sense that the prion conformation of Rho would result in lower levels of transcription termination. This is just what the authors found when they tested Cb-Rho in E. coli.

They used a reporter in which a transcription termination site was placed upstream of the lacZ gene. The idea is that if transcription termination is compromised, more lacZ will get made making the colonies a darker blue. And since termination is not 100% efficient, if Rho is doing its job, the colonies will be light blue.

When they plated out E. coli containing an engineered form of Cb-Rho, they got two classes of colonies, light blue and dark blue. And more importantly, this colony color was inheritable. In other words, light blue colonies gave more light blue colonies and dark blue colonies gave more dark blue colonies.

This isn’t to say that the colony color was a permanent state. It wasn’t. A bit under 1% of the colonies would spontaneously revert to the other form, as you’d expect from a prion protein.

So with the invaluable help of yeast, these authors were able to find and validate a protein from bacteria that can act as a prion. If they find a function for this in the wild, then yeast will have helped us better understand the evolution of life on Earth. Again. #APOYG!

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

Categories: Research Spotlight

Tags: amyloid , protein aggregate , lacZ , bacterial prion

Membrane Snorkeling with Arginine

February 06, 2017


If this swimmer’s snorkel were a glutamic acid and he was in a membrane, he couldn’t stay there for very long. His snorkeling tube would be too short! Image from pixabay.

Snorkeling is a blast. With a small tube stuck out into the air you can explore the wonders of the sea for much longer than you would be able to otherwise.

It is obviously important that the snorkel be long enough to reach out of the water. If it isn’t, you’ll be sucking down a lungful of water in no time.

Something similar can happen with membrane spanning proteins except that in this case, a snorkel is not essential to the protein swimming in the greasy confines of a membrane. Instead, positively charged amino acids like lysine or arginine can be tolerated much more often than you might think because of their structure.

Since membranes are hydrophobic, the amino acids in the part of the protein that span the membrane tend to be hydrophobic as well. Charged amino acids are usually trouble.

A proposed exception is amino acids like arginine and lysine. Their positive charges are each at the end of a long aliphatic chain:

Lysine & Arginine

What is thought to happen in that the aliphatic chain of the lysine or arginine snuggles up to the greasy part of the phospholipid and the positive part of these amino acids sticks out of the membrane to the more aqueous environment on the other side. The aliphatic side chain is long enough for the charged group to get out of the hydrophobic part. These amino acids may also work well because they can interact with the net negative charge that some phospholipid head groups have.

This is all very cool but, to date, there is very little in vivo work that supports this idea. Which of course means we need to turn to that workhorse model organism Saccharomyces cerevisiae to get some evidence!

In a new study out in GENETICS, Keskin and coworkers use yeast to provide some in vivo evidence that these positively charged amino acids are well tolerated in the membrane-anchoring domain of the yeast protein Fis1p. While I won’t have the space to go into some of the other fascinating experiments in this study, please read it over to learn more about how proteins are inserted into the mitochondrial outer membrane and the structure of this particular carboxy-terminal anchor.

The authors investigated the 27 amino acid carboxy-terminal anchor of this protein by first individually changing every one of its amino acids into every other amino acid (deep mutational scanning). Well, they didn’t get every possible combination. But 98.9% of them is pretty good!

Next they used a very clever screen to find which of these Fis1p mutants that could not properly insert into the mitochondrial membrane. Basically, they fused transcription activator Gal4p to their mutant library. If a mutant protein cannot insert into the membrane, then it is free to enter the nucleus and activate transcription of either a HIS3 or URA3 reporter.

When they did this they were surprised to see that the positively charged amino acids arginine and lysine were well tolerated in the membrane spanning portion of the protein. As expected, mutants with the negatively charged amino acids aspartic acid or glutamic acid in this region of the protein were not.

Here are these four amino acids:

LysArgAspGlu

The key difference here (besides the different charges) is the length of the aliphatic chain, the “snorkel” part of the amino acid. Both of the negatively charged amino acids are too short to be able to stick the charged part of the amino acid out of the membrane layer. They are like a snorkeler trying to snorkel with a tube that’s too short. It won’t work well.

So membrane spanning regions are not as hydrophilic fearing as many scientists and protein prediction programs may think.

It may be that the algorithms used to predict membrane spanning regions of proteins are missing some of them because they are too strict about the presence of charged amino acids like lysine and arginine. Perhaps these programs need to be modified to better allow for the presence of an errant arginine or lysine lurking in a long stretch of hydrophobic amino acids.

Of course we turned to yeast to help us better understand reality so we can come up with better algorithms. We may need to listen to yeast so that the programs can be adjusted to think about more than charge, and focus on the length of the snorkel too. #APOYG!

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

Categories: Research Spotlight

Tags: mitochondrial division , membrane insertion , amino acid snorkeling , deep mutational scanning , mitochondrial protein targeting

Not Recycling (RNA) Can be Bad for your Health

January 12, 2017


Improper disposal is bad for the environment and bad for cells. (Image from Wikimedia Commons)

Not too long ago, it was common to see people pouring used motor oil into street drains. Or to have people dumping old prescription drugs down their sinks.

Practices like these were (and are) terrible for the environment. Nature simply can’t deal with a buildup of this stuff (click here for some examples of the effects of pharmaceuticals on the environment).

Which is why it is so great that there are now ways to deal with waste like this. We can recycle it or at the very least dispose of it more carefully.

Turns out that things are similar in a cell. When its trash isn’t disposed of properly and/or recycled, the cell can suffer. And if cells suffer, so can the person made up of those cells.

One case where something like this is probably happening is in patients with the neurological disorder Pontocerebellar hypoplasia type 1B (PCH1B). These people have a mutated EXOSC3, an important gene for a cell’s RNA exosome. Presumably, this terrible disease is the result of certain cells not being able to properly clear some of their old RNAs.

In a new study out in GENETICS, Fasken and coworkers use good old Saccharomyces cerevisiae to begin to figure out what might be going on in the cells of these patients. They found that the most severe mutation seems to make it harder for the mutated protein to be part of the RNA exosome. As a result of being left out, the mutant protein is degraded more quickly leading to a buildup of some RNAs.

These sets of experiments were made a bit more complicated by the fact that human EXOSC3 cannot substitute for RRP40, the equivalent gene in yeast. This meant the researchers needed to focus on only those disease-causing mutations that hit the most highly conserved residues: EXOSC3-G31A, D132A and W238R.

Of these three, only the W238R yeast equivalent, rrp40-195R had much of an effect on the yeast. Fasken and coworkers propose that this is because this is the most deleterious of the three mutants.

Yeast harboring rrp40-195R grew more slowly at both 30 and 37 degrees C with the more pronounced effect at the higher temperature. At 37 degrees C, this mutant had higher levels of certain RNAs but not others. The RNA exosome was compromised for some but not all yeast RNAs.

And it wasn’t compromised everywhere. Although the RNA exosome works both in the nucleus and the cytoplasm, this mutant appeared to only be compromised in the nucleus. (Check the paper out for the cool way they figured this out.)

Next, the authors wanted to work out what this mutation did to the protein and the exosome. They were able to show that the mutant protein was more unstable than the wild type version and, interestingly, was even less stable when co-expressed with the wild type protein. They also showed that the mutant protein associated less well with the exosome complex and, again, this was exacerbated if the wild type protein was also present.

A reasonable model here is that RRP40 is more prone to degradation when it is not part of the RNA exosome. If true, then the mutant version of the protein is less stable because it is less often a part of the RNA exosome. And wild type RRP40 outcompetes the mutant protein making the mutant stay out of the complex for even more time.

Bad things can happen when you don’t recycle. (Image from Pixabay)

OK, so they have done some good work showing why this mutant of RRP40 affects the growth of a yeast cell. But what we really want to know is if these results explain what is going on in the cells of people with the disease.

Fasken and coworkers tackled this by looking at the effect of expressing the equivalent mouse Exosc3 mutant in the presence of wild type endogenous Exosc3 in mouse neuronal cells (the types of cells affected in PCH1B patients). They found that just like in yeast cells, the mutant was less stable in mouse neuronal cells.

So it looks like the recycling machinery for RNA is broken in these cells because of an unstable component and that this leads to a buildup of toxic RNAs. But if the yeast experiments hold up, not all RNAs are affected.

It is more like people still being able to recycle their cans and bottles but not their motor oil. Certain parts of the environment like waterways take a hit but other parts are left relatively unscathed.

This makes sense when you think about PCH1B. Only a few cell types are affected by the mutation in the EXOSC3 gene. In other words, most cells can deal with a slightly wonky RNA exosome.

Yeast has again helped researchers better understand a genetic disease. Awesome indeed. #APOYG

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

Categories: Research Spotlight

Tags: Pontocerebellar hypoplasia type 1B , EXOSC3 , RRP40

Budding Yeast, a Caffeine Wimp No More

December 16, 2016


Saccharomyces cerevisiae is an even bigger lightweight than this guy when it comes to caffeine. A little genetic engineering changed that. (Image from Ape Lad, flickr)

Some people get the jitters from a single espresso while others need a triple shot just to get started in the morning. Some of this is due to caffeine tolerance—a buildup of resistance to the marvelous effects of that wonderfully addictive substance, caffeine. But the rest has to do with genetic differences that affect how well each of us processes caffeine—our caffeine sensitivity.

Our best buddy Saccharomyces cerevisiae is a real wimp when it comes to caffeine. In fact, like a lot of other microorganisms, caffeine actually kills this yeast. S. cerevisiae is indeed a sensitive soul when it comes to caffeine.

In a new study in the Journal of Agricultural and Food Chemistry, Wang and coworkers were able to toughen up budding yeast against caffeine by adding bfr1, a gene from Schizosaccharomyces pombe that encodes the ABC transporter that shunts caffeine out of the cell. And then, using random mutagenesis, they were able to make bfr1 even better at its caffeine-exporting job. Although the yeast don’t get any of the pleasurable effects of caffeine, at least they can now happily grow in cultures that have more caffeine than a strong cup of coffee.

This new attribute could prove to be incredibly useful if caffeine producers ever want to start making caffeine biologically instead of synthetically. You can imagine adding the caffeine pathway from coffee to yeast and having the yeast merrily exporting caffeine to the culture medium where it can be harvested. And who knows, maybe they can have the yeast make caffeine and alcohol at the same time creating the equivalent of a vodka and Red Bull in a single step!

Previous research had shown that bfr1 was an important player in helping S. pombe deal with caffeine. When Wang and coworkers added the gene to S. cerevisiae, this newly engineered yeast could now better tolerate caffeine. For example, whereas wild type yeast barely grew with 8 mg/ml caffeine, the engineered yeast did OK.

These authors next turned to random mutagenesis of the bfr1 gene to screen for mutants that could tolerate even more caffeine. And boy did they win the lottery on this one! A mutant that they named bfr1-B did great even at concentrations of 25 mg/ml caffeine. Now they were getting somewhere.

Bfr1 doesn’t just export caffeine; it actually exports many different compounds. The authors found that bfr1-B was fairly specific for increased resistance to caffeine. For example, when they tested the bfr1-B mutant with theophylline, a structurally similar compound, and atropine, a structurally distinct compound, they found that S. cerevisiae expressing the mutant were, if anything, more sensitive to these compounds. They found what looked like a caffeine-specific mutant. 

When they looked at the mutant, Wang and coworkers found that there were 11 amino acid substitutions scattered across the protein. The next step was to figure out which ones mattered and which ones didn’t.

Maybe this genetic engineered yeast can make the equivalent of a Red Bull and vodka all in one step! (Image from Mark Hillary, flickr)

Using a bit of modeling with the 3-D structure of other ABC transporters, they settled on testing three mutations individually. Two of the mutations, S36 and D340 were in the nucleotide binding domain (NBD) and the third, Y497, was in the transmembrane domain (TMD). The NBD is where ATP binds to the transporter to supply the energy to move caffeine across the membrane. 

Of the three, only D340 in the nucleotide binding domain conferred caffeine resistance. While not as robust as bfr1-B, this mutant allowed yeast expressing it to tolerate caffeine concentrations up to 15 mg/ml, conditions under which cells with wild type bfr1 failed to grow. 

So while this mutation explains a lot of why bfr1-B is so good at dealing with caffeine, it is not the whole story. At least some of those other 10 mutations contribute to how well bfr1-B does with caffeine.

In the end we have a bullet-proof yeast when it comes to caffeine that should prove useful for anyone who wants yeast to synthesize caffeine for them. Of course unlike even the most grizzled 30 year coffee drinker with ideal genetics, the yeast almost certainly gets no joy from its morning Joe. But at least that cup of coffee won’t kill it!

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

Categories: Research Spotlight

Tags: BFR1 , Schizosaccharomyces pombe , ABC transporter , caffeine

Move Along, Transcription Factor

December 07, 2016


Sumo wrestlers throw each other out of the ring. Just like sumoylation throws Gcn4p off DNA. Image from Wikimedia Commons.

For an election to go smoothly, people cannot stay too long in the voting booth. If a lot of people stayed in the booth and answered emails, sent texts, etc., after they finished voting, then the whole process would grind to a halt.

There is some evidence that activating genes may work similarly. The transcription factors (TFs) that bind DNA and turn up the expression of nearby genes can’t stay too long. If they do, the activation starts to peter out.

What is thought to happen with these sorts of TFs is that they bind their preferred DNA, and then once they have attracted the cellular machinery needed to read the gene, they are targeted for destruction. Then a new TF can bind and repeat the process.

In a new study in GENETICS, Akhter and Rosonina set out to investigate the process by which the yeast transcription activator Gcn4p is removed after it has bound DNA and done its job. Gcn4p activates a number of genes in response to amino acid starvation.

They found that a key step in the process is the addition of SUMO proteins to DNA-bound Gcn4p, which gets the ball rolling on the destruction of Gcn4p. Imagine a sumo wrestler settling in next to a voter once he enters the booth and then throwing him out if he tarries too long.

Their model is that once Gcn4p binds DNA, it is sumoylated. Then the DNA-bound, sumoylated GCN4 is further modified by kinases like Cdk8p, a component of the mediator complex which acts as a bridge between TFs and the cellular machinery responsible for reading a gene. This modified TF is then sent off to the 26S proteasome where it is degraded making room for an unmodified Gcn4p.

Previous research had shown that sumoylation of GCN4 required DNA binding. The first thing these authors did in this study was to determine if Gcn4p had to bind to its target DNA sequence in order to be sumoylated. It did not.

When they fused a mutant Gcn4p that could not bind DNA to the DNA binding domain of Gal4p, they found that this molecule was sumoylated at the correct places on the Gcn4p part of the fusion protein, lysines 50 and 58, when bound to a Gal4p binding site. Therefore, Gcn4p does not need to occupy its own DNA binding site in order to be sumoylated.

Another set of experiments showed that while DNA binding was required for sumoylation, interaction with RNA polymerase II (RNAP II), the enzyme that reads the genes that Gcn4p activates, does not appear to be necessary. For one of these experiments they used a temperature sensitive mutant of the largest subunit of RNAP II, Rpb1p, and showed that even at higher temperatures when RNAP II is inactive in these cells, DNA-bound Gcn4p is still sumoylated. In the other experiment they showed that DNA-bound
Gcn4p was still sumoylated when they used the “anchor away” technique to drag Rpb1p out of the nucleus and into the cytoplasm.

So DNA binding is sufficient, and the specific site is not important. And Gcn4p doesn’t have to be activated in order to be sumoylated.

Of course, turnover like this is a delicate thing. If Gcn4p is pulled off too soon, then it can’t activate as much as it might otherwise be able to do. This might affect the cell’s response to starvation just as much as Gcn4p staying put too long. Sort of like the sumo wrestler throwing a voter out of the voting booth before they could finish their voting can muck up the election.

Akhter and Rosonina created a fusion protein of Gcn4p and the yeast SUMO peptide Smt3p. Unlike Gcn4p, this protein is sumoylated before it binds DNA.

They found that yeast expressing this fusion protein fared less well under starvation conditions compared to yeast cells that expressed the wild type version of GCN4. And using chromatin immunoprecipitation (ChIP) analysis they showed that at least at the ARG1 gene, this was because there was less of the fusion protein bound under activating conditions.

So cells need for TFs to stay at the right place for the right amount of time. If they are pulled off too early or stay too long, the levels of activation can fall below what is best for the cells.

Unfortunately, we don’t have time to go over other experiments that tease out which kinases are important and when, but I urge you to read about them for yourselves. They take full advantage of the genetic tools available in yeast to make this sort of study possible…#APOYG!

Integrating all of this gives the following model:

Gcn4p model

Gcn4p is only a dimer when bound to DNA and this dimerization may be the signal for sumoylation by Ubc9p. A preinitiation complex forms through its interaction with the DNA-bound, sumoylated Gcn4p which brings in the enzyme RNAP II to transcribe the gene. Once the polymerase has left the nest, the kinase Cdk8p comes in and phosphorylates Gcn4p which signals Cdc4p/Cdc34p to ubiquitinate Gcn4p. The ubiquitinated Gcn4p is then degraded by the 26S proteasome opening the upstream activator sequence (UAS) up to a fresh, new Gcn4p.

Here, with the help of our super hero Saccharomyces cerevisiae, Akhter and Rosonina have dissected out what happens to a transcription factor once it binds to DNA (at least ones that bind for short times). It will be fascinating to see if this translates to other TFs in other beasts. While I love yeast for all it can do for us for bread, wine, beer, human health, helping solve world problems like climate change, and so on, I think my favorite use is still that it allows us to better understand the basic biology of how our cells work.

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

Categories: Research Spotlight

Tags: Cdk8 , transcription , gene activation , Gcn4 , sumoylation

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