New & Noteworthy

A Factory Without Doors

April 29, 2015

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Imagine you have built a state-of-the-art factory to make a revolutionary product. The place is filled with gleaming assembly lines and you have hired the best talent in the world to run the place.

Unfortunately there was a glitch in the factory design—the builders forgot to put doors in! Now you can’t get the raw materials in to make that killer product that will change everything.

This may sound contrived or even silly, but it is sort of what is happening in attempts to use yeast to make biofuels from agricultural waste. Scientists have tweaked yeast cells to be able to turn xylose, a major sugar found in agricultural waste, into ethanol. But yeast has no transporter system for this sugar. A bit can get in through the windows, so to speak, but we need to put in a door so enough can get inside to make yeast a viable source for xylose-derived ethanol.

An important step was taken in this direction in a new study by Reznicek and coworkers. They used directed evolution to transform the Gal2 transporter of Saccharomyces cerevisiae into a better xylose transporter. And they succeeded.

After three successive rounds of mutagenesis, they transformed Gal2 from a transporter that prefers glucose into one that prefers xylose. When put in the right background, this mutant protein opens the door for getting yeast to turn agricultural waste into ethanol. Perhaps yeast can help us stave off cataclysmic climate change for just a bit longer. 

The first step was to find the right strain for assaying xylose utilization. They needed a strain lacking 8 hexose transporters, Hxt1-7 and Gal2, because these transporters can take up xylose (albeit at a very low efficiency). Deleting these genes “shuts the windows” and completely prevents the strain from utilizing xylose as a substrate (as well as impairing its ability to use glucose).

This strain was also engineered to be able to utilize xylose. It contained a xylose isomerase gene from an anaerobic fungus and also either overexpressed or lacked several S. cerevisiae genes involved in carbohydrate metabolism. With this strain in hand, the researchers were now ready to add a door to their closed off factory.  

The authors targeted amino acids 292 to 477 in Gal2. This region is thought to be critical for recognizing sugars, based on homology with other hexose transporters. They used mutagenic PCR conditions that generated an average of 4 point mutations in this region, and screened for mutants that grew better than others on plates containing 0.1% xylose.

In their initial screen they selected and replated the 80 colonies that grew best. They then chose the best 9 to analyze further. Of these 9, one mutant which they dubbed variant 1.1 grew better on xylose than a strain carrying wild-type GAL2. Variant 1.1 had a single amino acid change, L311R.

They repeated their assay using variant 1.1 as their starting source. Out of the 14,400 mutants assayed, they found four that did better than variant 1.1. These variants, dubbed 2.1-2.4, all shared the same M435T mutation.  Variant 2.1 had three additional mutations—L301R, K310R, and N314D.

These four new mutants showed better growth on 0.45% xylose, and after 62 hours, all the strains had pretty much used up the xylose in their media. Of the four, variant 2.1 appeared to be the best xylose utilizer: after 62 hours the authors could detect no xylose in the media at all. This variant also grew faster than the others in 0.1% xylose.

Reznicek and coworkers had definitely made Gal2 a better xylose transporter, but they weren’t done yet. They wanted to try to make a door that only let in the raw supplies (xylose) they wanted and not other sugars (glucose).

Up until now, the screens had been done with xylose as the sole carbon source. When they grew variant 2.1 in the presence of both 2% glucose and 2% xylose, they found that it preferentially used the glucose first. Their evolved transporter still preferred glucose over xylose!

Now in some ways this wasn’t surprising, as the mutations had not really affected the part of the protein thought to be involved in recognizing sugars. They next set out to evolve Gal2 so that it would transport xylose preferentially over glucose.

This time they used a slightly different background strain for their screen. This strain, which was deleted for hxk1, hxk2, glk1, and gal1, was unable to use glucose although it could transport it.

They repeated their mutagenesis and looked for mutants that grew best in 10% glucose and 2% xylose. We would predict that any growing mutants would have to transport xylose better than glucose. And this is just what they found.

When they analyzed the mutants, they found that the key mutation in making Gal2 prefer xylose over glucose in the variant 2.1 background was T386A. Based on homology with Hxt7, this mutation happens smack dab in the middle of the sugar recognition part of the protein. Most likely this mutation compromised the ability of Gal2 to recognize glucose, as opposed to improving recognition for xylose.

These experiments represent an important but by no means final step in engineering yeast to make fuel from biomass. We are on our way to a smaller carbon footprint and perhaps a world made somewhat safer from climate change.

First, beer, wine, and bread; next, keeping coral alive and saving countless species from extinction. Nice work, yeast.

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

Categories: Research Spotlight

Tags: biofuel, evolution, Saccharomyces cerevisiae

Sharing the Health

April 22, 2015

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A study published a few years ago made a big splash in the health news by showing that obesity is socially contagious. If one person gains weight, their friends tend to gain weight too—even if they don’t live in the same town! This works the opposite way too: thinner people are more likely to be socially connected with thinner people.

You might think this is because people tend to make friends with others of a similar size, but this doesn’t seem to be the case. The researchers concluded that there is actually a cause-and-effect relationship: we all influence the weight of our friends.

Well, S. cerevisiae cells are not so different. They may not have social lives, but since they can’t move on their own, they do tend to live together in colonies. And within these colonies, they influence each other: not in terms of weight, but in terms of the effect that calorie intake has on the length of their lives.

Turns out that like nematodes, fruit flies and even mice, living on a meager diet makes yeast live longer. And in a new study published in PLOS Biology, Mei and Brenner found that yeast cells actually share the life-extending benefits of calorie restriction with their neighbors, probably via a still-unidentified small molecule.  

Yeast are normally grown in the lab on medium containing 2% glucose. To a yeast cell, this is like an all-you-can eat buffet that goes on for its entire lifetime. Media with a glucose content of 0.5% or less represent a meager diet. But that deprivation comes with a benefit, in the form of an extended lifespan.

Mei and Brenner already had some hints from previous studies that yeast cells might excrete a substance that promoted lifespan extension. To study this systematically, they devised an experiment to test whether mother cells change the media surrounding them as they divide.

The researchers placed individual mother cells in specific spots on Petri plates containing an all-you-can-eat buffet (2% glucose), a restrictive diet (0.5% glucose), or a near-starvation diet (0.2% glucose). They watched as the cells budded, and removed each new daughter cell as it separated from the mother, counting the buds. The lifespan of a mother yeast cell, termed the replicative lifespan, is measured as the number of times she can bud during her lifetime.

After the mother cells had budded 15 times, half of them were physically moved to fresh parts of the same plate, while the other half were left in place. For the mothers on the 2% glucose plates where calories were abundant, the move didn’t change anything. The mothers that were moved had exactly the same replicative lifespan as those that stayed put.

On the plates where calories were restricted, it was a different story. The cells that stayed in place had extended lifespans, as expected under these low-calorie conditions. But the cells that were moved to new locations lost most or all of the life extension—even though calories were still restricted in their new locations. This suggested that the mother cells had secreted a “longevity factor” into the medium surrounding them, which then extended their lifespan when they got older.

There were a couple of metabolites that were prime candidates for the longevity factor: nicotinic acid (NA) and nicotinamide riboside (NR). NA and NR are precursors to nicotinamide adenine dinucleotide (NAD⁺), a compound that acts as an essential cofactor for many important enzymes. They had already been implicated in lifespan extension because mutating genes involved in their metabolism can affect how long various creatures live.

When the scientists tried supplementing calorie-restricted cells that had been moved to fresh medium with either NA or NR, they found that supplying these metabolites could restore the longevity benefit.  This finding strengthens the idea that NAD⁺ metabolism is involved.

But was the longevity factor actually NA or NR? To test this, Mei and Brenner grew yeast in liquid media with the different glucose concentrations and then tested for NA and NR in the medium using liquid chromatography-mass spectrometry analysis.  They found that under all the conditions, the amount of NA secreted by the cells didn’t change and secreted NR was undetectable, suggesting that neither was the factor induced by calorie restriction.

To ask directly whether there is a diffusible longevity factor, the researchers grew cells in liquid medium containing 2% or 0.2% glucose until all the glucose was used up, then separated out the cells and freeze-dried the remaining liquid. They suspended the dried “conditioned” medium in water and spread it on plates to repeat the cell-moving assay.

Just like before, cells grown in 2% glucose had the same lifespan after being moved to a fresh spot, and the addition of resuspended conditioned medium to the plate didn’t change that. However, the starved cells grown on 0.2% glucose not only kept their lifespan extension when moved to conditioned media, but actually lived 10% longer compared to starved cells on un-conditioned media that were not moved.

When the researchers dialyzed the conditioned medium so that molecules smaller than 3.5 kDa were lost, the longevity factor was lost too. So it looks to be a small molecule, and of course they are actively pursuing its identity. Intriguingly, this would explain why other scientists have been unable to detect calorie restriction-induced lifespan extension in yeast using microfluidic technology, where immobilized yeast cells are grown with a constant exchange of growth medium. Under these conditions, a small molecule that promotes longevity would be washed away.

So, even though they don’t have Facebook friends, yeast cells influence the health of their peers. Rather than spreading the influence through social interactions as we humans do, they broadcast a chemical that is the key to long life. 

It’s tempting to think that the identity of this chemical will tell us something about human aging. But if this mysterious molecule worked in humans the same way as it does in yeast, people would still have to eat just enough food to stay alive to get the benefits. Still, perhaps the molecule can point us towards finding a treatment that will let us live longer while enjoying lots of good food. We could have our cake and eat it too!

by Maria Costanzo, Ph.D., Senior Biocuration Scientist, SGD

Categories: Research Spotlight

Tags: aging, calorie restriction, NAD+, Saccharomyces cerevisiae

Harvesting Until the Last Minute

April 15, 2015

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In the science fiction novel Dune, the most precious thing in the universe is spice. It is harvested from the sands of the planet Dune under very dangerous conditions—every time people start to mine it, a gigantic worm comes to kill them. The spice is so valuable, though, that the miners harvest it until the very last second. As time runs out, a carryall whisks them away as the worm rises out of the desert.

While nothing quite so exciting happens in the nucleus of a yeast cell, one of the closest situations may be at the rDNA locus. Here the precious commodity is ribosomes and yeast cells need them to be made almost constantly. The only pauses in production are when this part of the genome needs to be replicated and when it needs to be segregated into a daughter cell. In both cases, as something akin to giant sandworms comes crashing onto the scene, harvesting stops and the machinery is removed with a cellular carryall.

Of course there aren’t harvesters extracting whole ribosomes from the yeast nucleus! Instead, it is RNA polymerase I (Pol I) and RNA polymerase III (Pol III) making the raw stuff of ribosomes, the 35S and 5S rRNAs, respectively.  These polymerases need to stop transcribing and clear the way for the rDNA replicating replisome during S phase and for condensins at the end of anaphase. If they don’t, the rDNA locus becomes unstable, resulting in the mother yeast cell living a shorter life and in its daughter not getting the rDNA locus (and the rest of the chromosome it is on) at all.

In a new study in Nature Communications, Iacovella and coworkers have identified the carryall that helps to remove Pol I from the rDNA locus. Surprisingly, it is a kinase, named Rio1, that was previously known to be involved in rRNA processing and building ribosomes in the cytoplasm.

Rio1 does not physically remove Pol I from the 35S gene. What this study suggests is that it phosphorylates one of the 14 subunits of Pol I, Rpa43, so that Pol I no longer interacts as strongly with the transcription factor Rrn3. The end result is the untethering of the polymerase and its release from the DNA. Now condensins can glom onto the rDNA locus at the end of anaphase and DNA polymerase can barrel through the region in S phase without wreaking genomic havoc.

The first key finding in this study was that Rio1 isn’t just active in the cytoplasm, but also in the nucleus. In fact, it was most active in the nucleolus, a small moon shaped section of the nucleus that is formed around the rDNA locus. 

The researchers went on to show that Rio1 is present in the nucleolus only at certain times in the cell cycle (S phase and anaphase). Using chromatin immunoprecipitation (ChIP) assays, they were able to show that Rio1 was enriched specifically at the rDNA’s 35S promoter and coding sequence.

They next created a conditional Rio1 mutant that could not enter the nucleus in the presence of galactose. When Rio1 was kept out of the nucleus, nucleoli became fragmented, there were no condensins on the rDNA locus at anaphase, and the mother yeast did not pass the replicated nucleolus to her daughter. Obviously Rio1 is a critical housekeeper for the rDNA locus!

They next used ChIP assays to show that when Rio1 was kept out of the nucleus, there was around 3-fold more Pol I at the 35S promoter and gene during anaphase than when Rio1 was allowed to go nuclear. This resulted in a 5-8 fold increase in 35S rRNA levels—implying that Pol I was still there cranking out rRNA.  

The most likely explanation is that all the hyperactive Pol I transcribing the rDNA locus prevents condensins from binding the DNA and that removal of Pol I by Rio1 allows the condensins to bind. The condensins then shrink the rDNA region such that it can move though the tiny bud opening into the daughter.

They got similar results in S phase, where lack of nuclear Rio1 caused an increase in Pol I occupancy at the rDNA and an increase in 35S transcription as well. Here the lack of Rio1 has more devastating consequences to the genome. Its absence most likely causes the replisome to collide head-on with the transcribing Pol I, resulting in double strand DNA breaks. Because the rDNA locus is such a repetitive region, the cell makes mistakes when it repairs the break using homologous recombination. The end result is nucleolar fragmentation and a shorter life span.  

The final set of experiments showed that Rio1 most likely affects Pol I occupancy by phosphorylating one of its subunits, Rpa43. First the authors used Western blot analysis to show that Rpa43 is less phosphorylated when Rio1 is kept out of the nucleus and when a mutant version of Rio1 lacking its kinase function is used. They also showed that Rio1 could phosphorylate Rpa43 in vitro.

They postulate that this phosphorylation causes the interaction between Pol I and the transcription factor Rrn3 to weaken, allowing Pol I release.  Alternatively, Rpa43 phosphorylation could lead to the disintegration of the Pol I enzyme itself. Confirmation of one or the other will require more research.

Taken together, these studies paint a fascinating picture of the rDNA locus. Here Pol I is frenetically transcribing as much 35S rRNA as it possibly can to keep up with the yeast cell’s unquenchable thirst for ribosomes. The only time it stops is when continuing could harm the cell, during DNA replication in S phase and DNA transmission in anaphase. And even then, like spice hunters on Dune, they stay until the very last minute. A spicy tale, indeed.

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

Rise, replisome, rise!

Categories: Research Spotlight

Once You Start Looking, Shu2 Homologs Are Everywhere

April 09, 2015

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Have you ever heard of the Baader-Meinhof phenomenon? (Don’t worry, we hadn’t either!) But you’ve surely experienced it.

The phenomenon describes the experience of suddenly seeing something everywhere, after you’ve noticed it for the first time. For those of us in Northern climates, it’s like like robins coming back in the springtime: one day, you see a single robin hopping on the grass; the next day, you look around and realize they’re all over.

Something similar happened to Godin and colleagues as they investigated the S. cerevisiae Shu2 protein. People knew about the protein and its SWIM domain but no one had looked to see just how conserved it was.

When the researchers looked for homologous proteins that contained the characteristic SWIM domain, they found homologs in everything from Archaea through primitive eukaryotes, fungi, plants, and animals. They were practically everywhere (in the evolutionary sense).

In a new paper in GENETICS, Godin and coworkers described their studies on this relatively little-studied, unsung protein. They found that it is an important player in the essential process of DNA double-strand break repair via homologous recombination, and its SWIM domain is critical to this function. Not only that, but being able to compare the SWIM domains from so many different homologs allowed them to refine its consensus sequence, identifying a previously unrecognized alanine within the domain was both highly conserved and very important.

Double-strand DNA breaks (DSBs) can happen because of exposure to DNA damaging agents, but they are also formed normally during meiotic recombination, in which a nuclease actually cuts chromosomes to start the process. Homologous recombination to repair DSBs is a key part of both mitosis and meiosis.

During homologous recombination, one strand of each broken DNA end is nibbled back to form a single-stranded region. This region is then coated with a DNA-binding protein or proteins, forming filaments that are necessary for those ends to find homologous regions and for the DSB to be repaired.

Shu2 is one of the proteins that participates in the formation of these filaments in S. cerevisiae. It was known that it was part of a complex called the Shu complex, and a human homolog, SWS1, had been identified. But the exact role of Shu2 and the significance of the SWIM (SWI2/SNF2 and MuDR) zinc finger-like domain that it contains were open questions.

One of the first questions the authors asked was whether Shu2 was widely conserved across the tree of life. Genes with similar sequences had been seen in fission yeast (Schizosaccharomyces pombe) and humans, but no one had searched systematically for orthologs. They used PSI-BLAST, a variation of the Basic Local Alignment Search Tool (BLAST) algorithm that that is very good at finding distantly related proteins, to search all available sequences.

Querying with both yeast Shu2 and human SWS1, the researchers found hits all across the tree of life—both in “lower” organisms such as Archaea, protozoa, algae, oomycetes, slime molds, and fungi, and in more complex organisms like fruit flies, nematode worms, and plants. The homologous proteins that they found across all these species also had the SWIM domain, suggesting that it might be important.

The sequence similarity was all well and good, but did these putative Shu2 orthologs actually do the same job in other organisms that Shu2 does in yeast? One way to test this is to do co-evolutionary analysis. Proteins that work together are subject to the same evolutionary pressures, so they tend to evolve at similar rates. Godin and colleagues found that evolutionary rates of the members of the Shu complex in fungi and fruit fly did generally correlate with those of other proteins involved in mitosis and meiosis.

The awesome power of yeast genetics offered Godin and coworkers a way to look at the function of Shu2. They first tested the phenotype of the shu2 null mutation, and found that it decreased the efficiency of forming filaments of the Rad51 DNA-binding protein on the single-stranded DNA ends that are created at DSBs. Formation of these filaments is a necessary step in repairing the DSBs by homologous recombination.

The comparison of SWIM domains from so many different proteins highlighted one particular alanine residue. This alanine hadn’t previously been considered part of the domain’s consensus sequence, but it was conserved in all the domains.

When the researchers changed the invariant alanine residue in yeast Shu2, the mutant protein bound less strongly to its interaction partner in the Shu complex, Psy3. When they mutated the analogous residue in the human Shu2 ortholog SWS1, this also decreased its binding to its partner, SWSAP1.

Other mutations within the SWIM domain of Shu2 also affected its interactions with other members of the Shu complex, and made the mutant cells especially sensitive to the DNA-damaging agent MMS. Diploid cells with a homozygous mutation in the Shu2 SWIM domain had very poor spore viability, suggesting that the SWIM domain is important for normal meiosis.

As one more indication of the SWIM domain’s importance, Godin and colleagues took a look in the COSMIC database, which collects the sequences of mutations found in cancer cells. Sure enough, a human cancer patient carried a mutation in that invariant alanine residue of the SWIM domain in the Shu2 ortholog, SWS1.

There’s still much more to be done to figure out exactly what Shu2 and the Shu complex are doing during homologous recombination. Yeast obviously provides a wonderful experimental system, and the discovery of Shu2 orthologs in two other model organisms that also have awesomely powerful genetics and happen to be multicellular, Drosophila melanogaster and Caenorhabditis elegans, expands the experimental possibilities even further.

There’s also a lot to be learned about the SWIM domain in particular. The discoveries that it affects the binding behavior of these proteins and that it is mutated in a cancer patient show that it’s very important, but just what does it do in Shu2? It will be fascinating to find out exactly how this domain works to help cells recover from the lethal danger of broken chromosomes. And it is amazing what you can see, once you start looking. 

by Maria Costanzo, Ph.D., Senior Biocurator, SGD

Categories: Research Spotlight

Tags: evolution, homologous recombination, model organism, Saccharomyces cerevisiae

Red Ink for the S. cerevisiae Genome

April 02, 2015

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Editing is an essential part of producing good writing. You might cringe when your masterpiece comes back covered in red ink, but in the end your paper is better for it.

These days, computers have made editing much, much faster and easier than using ink on paper. Some of us remember when cutting and pasting literally mean cutting the piece of paper on which your manuscript was typed and pasting the sections in a different order!

The same kind of revolution has happened when it comes to editing genomes. A seemingly obscure system used by bacteria to defend against invading phages, discovered in the 1950s, led to the development of restriction enzymes as the reagents that have enabled virtually all modern molecular biology. And now, an equally “obscure” system of bacterial immunity has opened the door to genome editing that is as precise and nearly as fast as your thesis advisor using Microsoft Word to edit your dissertation (maybe faster!).

This bacterial system is called CRISPR/Cas. Bacteria use it to defend against the foreign DNA—from infecting phages, bacteria that transfer plasmids via conjugation, or other sources—that constantly assaults them. And now scientists are using it to edit the genomes of most any beast they want, including our favorite yeast.

What makes this technique so powerful is that it is easily programmable. You can make virtually any sequence change that you wish, and even more impressively, make lots of changes all at once. CRISPR/Cas may be one of those technological leaps that changes everything.

The incredible potential of this approach, and the ethics of its use in humans, are hot topics right now. But while researchers and philosophers are hammering out guidelines for using the CRISPR/Cas system in larger organisms, yeast researchers are free to forge ahead and edit the S. cerevisiae genome to their hearts’ content. This has now been made much easier with the availability of a whole toolkit, created at the Delft University of Technology and described in a new paper by Mans et al. 

The yeast system has three essential components. The first is a plasmid that will express a specific guide RNA (gRNA). The gRNA leads a nuclease to the right place in the genome.  Mans and colleagues made a whole set of plasmids, with different nutritional and antibiotic resistance markers, that can express one or more gRNAs.

The second component of the system is the nuclease that binds to the gRNA and makes a double-stranded cut in the target DNA. The researchers used the Cas9 nuclease from Streptococcus pyogenes, and engineered a set of yeast strains that had the cas9 gene stably integrated into a chromosome and expressed from a strong yeast promoter.

The third component is one or more repair fragments: pieces of DNA that specify the modified sequence that the researcher wants to engineer into the genome.

So, for example, if a scientist wants to delete a gene precisely, she can create a gRNA that targets the gene, and then co-transform a Cas9-expressing yeast strain with both the plasmid expressing the gRNA and a repair fragment that corresponds to the gene’s upstream and downstream flanking sequences, fused together. When the gRNA is expressed in the transformant, it leads Cas9 to the gene, where it makes a double-stranded cut at a precise position in the DNA.

Now the researcher can let yeast do the rest of the work. S. cerevisiae has a powerful homologous recombination system, and it’s greatly stimulated by double-stranded breaks in DNA.

After Cas9 cuts the gene, yeast will repair the break, using as a template the repair fragment that the researcher designed. In this case, the upstream and downstream sequences will recombine with the homologous sequences in the chromosome, but since the coding sequence is missing from the repair fragment, the resulting strain will have a precise deletion of the gene of interest.

This example illustrates one of the simplest uses of the technique. Mans and colleagues tried successively more complicated tasks and were able to accomplish some amazing feats.

They were able to precisely delete six genes in one step by transforming with repair fragments for all six, along with three plasmids that each expressed two gRNAs. Also in one step, they replaced one yeast gene with six Enterococcus faecalis genes encoding subunits of the pyruvate dehydrogenase complex and other enzymes in the pathway. The E. faecalis genes were specified on six overlapping repair fragments that were cotransformed into the strain.

The CRISPR/Cas9 system designed by Mans and colleagues can do much more than gene deletions and replacements. By designing repair fragments specifying particular mutations, the method can also be used to create point mutations or other modifications.

For this technique to work, it’s important that there are no mismatches between the gRNA sequence and the chromosomal target sequence, and other sequence characteristics can influence the efficiency of the method.  So the authors created an online tool that helps researchers select optimal Cas9 targets in regions of interest and design gRNA sequences. Since they incorporated the sequences of 33 different S. cerevisiae strains into the tool, researchers can specify a strain and retrieve information on the best sequences for targets and gRNAs for their gene(s) of interest based on sequences found in that particular strain.

Importantly, the CRISPR/Cas9 technique allows researchers to make multiple changes in a single step. This is a big advantage, since transformation itself can be mutagenic. For example, a strain that has been commonly used to investigate the function of hexose transporters was engineered to carry multiple deletions in the conventional manner, using successive rounds of transformation and selection.  Its genomic sequence, which was recently determined, reveals that its genome is a complete mess, with many rearrangements and deletions. 

With the development of this toolkit, editing the S. cerevisiae genome is beginning to be almost as easy as editing a text document. And since Mans and colleagues have made all of the strains, plasmids, and online tool freely available to the world, everyone will be able to take advantage of them. Just think of the stories that yeast researchers will be able to write!

CRISPR/Cas in Bacteria

Way before this became a powerful tool for researchers, it was a very cool immune system for bacteria, allowing them to defend against assaults from foreign invaders. 

Bacteria collect foreign DNA sequences from invaders that threaten them, just like collecting mug shots of notorious criminals. They store these mug shots in a special place in their genome, integrated between blocks of a repeated sequence. (The CRISPR acronym refers to these repeats.) This region is transcribed, and the RNA is chopped into pieces containing individual mug shots. Because these mug shots, called crRNAs, are complementary to the DNA sequences of invaders, they can recognize and hybridize with those invading sequences.

Bacteria have an additional small RNA, the tacrRNA, that binds to both the crRNA and to a CRISPR-associated (Cas) nuclease. This forms a RNA-DNA-protein complex on the foreign DNA and allows the nuclease to do its work, cutting both strands of the DNA and neutralizing the invader.

To use this system for genome engineering, scientists have fused the two RNAs of the bacterial system into a single RNA, the guide RNA (gRNA).  It contains both the mug shot (with sequences complementary to the target) and the RNA sequence that recruits the nuclease. The gRNA, a Cas nuclease, and a repair fragment are the essential components of the system.

by Maria Costanzo, Ph.D., Senior Biocurator, SGD

Categories: Research Spotlight

Tags: CRISPR/Cas, genome engineering, Saccharomyces cerevisiae, synthetic biology

Conformity Preferred in Yeast

March 25, 2015

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In a classic Apple ad, the world is a gray, dreary place where everyone is the same. In charges a woman dressed in brightly colored clothing who hurls a hammer into a big black and white screen, smashing the old conformist world order. Individuality can now blossom.

This is a powerful story for people, but it turns out that if Mother Nature had her druthers, she would like that woman to either stay at home or dress and act like everyone else. At least this is true if we are talking about individuals that are genetically identical. In this case, alleles that cut down on cell to cell variation tend to be the ones that prosper.

This is confirmed in a new study in Nature in which Metzger and colleagues in the Wittkopp lab showed that there is a selection against mutations that cause increased variation between individuals in the S. cerevisiae TDH3 gene. In other words, mutations that cause more “noise” are selected against. The squeaky wheel is eliminated.

The TDH3 gene encodes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an important metabolic enzyme. Yeast cells can survive a deletion of this gene, but their fitness is greatly reduced. Overexpression of the gene also has noticeable effects. This suggested to the researchers that the level of TDH3 expression would be under selection pressure during evolution.

Metzger and coworkers compared the promoters of the TDH3 gene from 85 different strains of S. cerevisiae and found that the promoter had undergone selection out in the wild. The authors were interested in why certain polymorphisms were selected for and why others were selected against. To try to tease this out, they compared the activities of evolved changes to randomly selected ones.

The authors first used the sequences of the 27 haplotypes they saw in the 85 strains to predict what the original, ancestral promoter probably looked like. They then re-created this sequence and also sequences that represented the most likely intermediates on the way to the current promoters. They linked each of these promoter sequences to a yellow fluorescent protein (YFP) reporter and looked at mean activity and expression noise. In other words, they looked at how much promoter activity there was in aggregate and how much it varied between individual cells for the 10,000 cells in each culture.

They next set out to generate a pool of polymorphisms that didn’t make the cut during evolution, so they could compare these to the successful ones. To do this, they individually mutated 236 G:C to A:T transitions throughout the promoter region. They chose this transition because these are the most common spontaneous mutations seen in yeast and the most common SNP seen in this promoter out in the wild.

Now they were ready to do their experiment! Comparing the randomly created mutations to the evolved changes, they looked at both the overall level of expression and how much variation there was between each of the 10,000 individual cells in the tested culture.

What they found was that the effects on the mean level of activity were pretty comparable between the “selected” mutations and the random ones. But the same was not true for individual variation. The random mutations were much more likely to increase expression noise compared to the “selected” mutations.

From these results the authors conclude that there is a selection against mutations that increase the level of noise. In fact, they go a step further and conclude that at least for the TDH3 promoter, there was more of a selection against noise than there was a selection for a particular level of activity. It was more important that individuals had consistent activity than it was to have some mean level of activity.

This makes some sense, as a cell is a finely tuned machine where all the parts need to work in harmony together to succeed. If one part is erratic and shows different levels of activity in different individuals, then some of those individuals won’t do as well and so won’t survive.

This also means that certain paths to a more fit organism will be selected over others. And it could be that organisms miss out on some potential fitter states because they can’t survive the dangerous evolutionary journey that would be needed to get there.

So the cell prefers that all the parts work together in a predictable way. When you’re a population of individuals that are more or less genetically identical, nonconformists are dangerous.  The gray sameness of the Nineteen Eighty-Four world is preferable to a more bohemian atmosphere where diversity is celebrated.

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

Categories: Research Spotlight

Tags: evolution, promoter, Saccharomyces cerevisiae

From Sourdough Bread to Chemotherapeutic Drugs

March 18, 2015

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Microbes can achieve great things when they work together:

Microbiologists in the lab spend a lot of time and effort keeping each microbial strain and species separate. The conventional wisdom is that if you want to really understand an organism, you need to study it in isolation, as a pure culture. If contaminating colonies of some other bug appear on your Petri dishes, you’d better melt down those plates in the autoclave and trash them!

On the other hand, amateur microbiologists have known for centuries that mixtures of microbes can do great things. Sourdough bread, for example, is made using a culture of Lactobacilli and yeasts. They complement each other during the fermentation: the bacteria metabolize sugars that the yeast can’t use, and make new compounds that can be fermented by the yeast. The result is extremely tasty. 

A new study from Zhou and colleagues brings a microbial community into the lab to make a medicine called paclitaxel that is used to treat cancer. Yes, bread bowls for your clam chowder are cool, but this is obviously way more important for human health.

Paclitaxel is an incredibly successful drug for treating breast and ovarian cancer, but unfortunately there isn’t an easy way to make it.  It can be purified from the bark of the Pacific yew (killing the trees in the process), synthesized by plant cells cultured in vitro, or synthesized chemically. But all of these processes are expensive and complicated, which means this life saving drug is always in short supply.

The researchers wanted to produce it more cheaply and easily, and an obvious solution was to let microbes do most of the work. But neither of the most commonly used microbial workhorses, S. cerevisiae and E. coli, was exactly right for the job.

E. coli had already been engineered to overproduce the compound taxadiene. The taxadiene then needs to be oxidized to create oxygenated taxanes, which are paclitaxel precursors. This oxidation can be done by membrane-bound oxidoreductase enzymes called cytochrome P450s. But these enzymes are not found naturally in bacteria, and getting them expressed and functional in E. coli is challenging.

The researchers decided to see whether they could coax these two microbes into cooperating to produce oxygenated taxanes. After creating an E. coli strain that produced taxadiene and an S. cerevisiae strain that produced a P450 oxidoreductase, they grew them together in the same culture, with glucose as the carbon source. 

As planned, the E. coli pumped out taxadiene and it was able to diffuse into the yeast cells, where it became oxygenated. However, the two species weren’t as happy together as the researchers had hoped. 

One of the things that humans love about yeast is that when it grows on glucose, it produces ethanol. However, the E. coli cells didn’t love being bathed in ethanol: their yield of taxadiene went way down as the ethanol levels in the culture went up.

So Zhou and coworkers switched the carbon source to xylose. S. cerevisiae cannot consume xylose, but E. coli can. When growing on xylose, E. coli produces acetate, which the yeast can use—and they don’t produce ethanol under these conditions.

Growing the microbes in xylose doubled the yield of oxygenated taxanes over that of the glucose-grown culture. But still, only 8% of the taxadiene that was produced was getting oxygenated.

To be sure that the yeast cells were producing the P450 enzyme as efficiently as possible, the researchers tried driving transcription of the gene using several different promoters. Using the promoter that was strongest in the co-culture conditions made a significant difference in the proportion of taxadiene that was oxygenated.

The researchers guessed that another factor limiting the final yield was that the yeast cells were not growing as well as they could. Tweaking the ratio of the two species in culture and the contents of the media resulted in a three-fold increase in oxygenated taxanes. But Zhou and colleagues hoped to improve things even more.

Thinking that the limiting step in yeast growth might be the supply of acetate, the researchers tried to beef up the acetate synthesis pathway in E. coli. They engineered the bacteria to overproduce several of the enzymes in the acetate biosynthesis pathway, but this didn’t make a large difference. 

They reasoned that if they forced the E. coli to rely on the acetate biosynthesis pathway for energy, the cells might ramp up their acetate production. To do this they blocked oxidative phosphorylation by deleting the the atpFH gene that encodes a subunit of ATP synthase. Now more acetate was produced, the yeast cells grew better, and 75% of the taxadiene that was produced got oxygenated. They were in business!

Zhou and coworkers went on to show that the co-culture environment could be modified to generate several other isoprenoids. This class of naturally-occurring molecules includes some that are in use, or in development, as pharmaceuticals (paclitaxel, and others currently in clinical trials) and compounds that have other applications, from fragrances to fuels. 

There’s much more work to be done, and the potential of microbial communities is just beginning to be realized. Harnessing the power of multiple organisms means that different steps of pathways can be optimized separately and then mixed and matched for a desired result. This approach could turn out to be the best thing since sliced bread! But then again, sourdough bakers already knew that.

by Maria Costanzo, Ph.D., Senior Biocurator, SGD

Categories: Research Spotlight

Tags: paclitaxel, Saccharomyces cerevisiae, synthetic biology

Those Yeast Got Talent

March 11, 2015

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The winners of American Idol go through quite a selection process. They start out as one of tens of thousands of people who audition, and survive each cut until they are finally crowned.

At the first cuts, those with any sort of advantage are kept in the pool and the others dropped. As the cuts continue, contestants not only need to have had that stronger initial advantage (or a bit of luck), but they also need to have picked up some new skills from all of those off- and on-air performances.

Some of these contestants start with a lot of raw talent but then progress only a little, while others are able to hone their weaker initial talent with lots of practice. Once their numbers are winnowed down to a handful, it gets close to being anyone’s game because the remaining contestants are so talented.

A new study by Levy and coworkers paints a similar sort of picture for evolving populations of yeast. Very early on a whole lot of yeast stumble upon weak, beneficial mutations that keep them going in the population. These are the yeast that make the initial cut in the hurly-burly world of the Erlenmeyer flask.

At later times a few yeast end up with strongly beneficial mutations that allow them to start to dominate. These are the pool of yeast that are the finalists of the flask.

Of course a big difference (among many) between American Idol and the yeast in this experiment is that the pool of contestants in the flask hangs around—they are not thrown off the show. This means that some cell that didn’t do too well early on can suddenly gain a strongly beneficial mutation and begin to dominate. Until, of course, that cell is usurped by another more talented yeast, in which case that finalist will fade away unless it can adapt.

And this study isn’t just a fascinating dissection of evolutionary population dynamics either. It might also have implications for treating bacterial infections and even cancer.

Bacteria and cancer cells live in large populations with each cell trying to outcompete the others. By understanding the set of mutations that allow some cells to succeed against the others and become more harmful, researchers may be able to come up with new ways to treat these devastating diseases.

One of the trickiest parts of this experiment was figuring out how to follow lots of yeast lineages all at once in a growing culture. Levy and coworkers accomplished this by adding 500,000 unique DNA barcodes to a yeast population and using high-throughput DNA sequencing to follow the lineages in real time.

They set up two replicate cultures and followed them for around 168 generations. In both cultures the researchers saw that while most of the lineages became much less common, around 5% happened upon a beneficial mutation that allowed them to increase in number by generation 112.

In other words, around 25,000 lineages ended up with beneficial mutations that let them make the first cut in both cultures. This translates to a beneficial mutation rate of around 1 X 10^-6 per cell per generation and means that around 0.04% of the yeast genome (around 5000 base pairs) can change in a way that confers a growth advantage.

But of course not all mutations are the same. Weakly beneficial mutations are very common, which means both cultures have plenty of these early on. This is why the replicate cultures behave so similarly up to around generation 80.

Eventually, though, a few yeast stumble upon stronger, more beneficial mutations. Since these are rarer and harder to get, each replicate culture gets them at different generations. This is why the cultures begin to diverge as the 100 or so of the strongest beneficial mutations begin to dominate.

The experiment did not go on for long enough to see many double mutations. In other words, it was very rare in this experiment to see a yeast lineage succeed because it had developed additive beneficial mutations. This is because there simply wasn’t enough time for a yeast cell to get a beneficial mutation and establish itself and then have one of its lineage gain and establish a second beneficial mutation. There was no Jennifer Hudson who came in 7^th but then went on to win a Grammy and an Oscar.

When a cancerous tumor is developing, however, there is plenty of time for multiple “beneficial” mutations to be established. These mutations are only beneficial for the tumor; they are devastating for the person with cancer. This is why it is so critically important to understand not only which mutations are implicated in cancer, but also the dynamics of how they accumulate in the cancer cell population during progression of the disease. Talented yeast in the hands of talented researchers are helping us figure this out.

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

Categories: Research Spotlight

Tags: cancer, evolution, Saccharomyces cerevisiae

Sweet or Salty? It’s Hard to Tell Just By Looking

March 05, 2015

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If you have ever accidentally added salt to your coffee, you know that sugar and salt are very different things even though they look pretty much the same. Turns out that genes can sometimes be this way too. They can look similar at the DNA level but have very different functions.

A great example of this can be found in a new study in GENETICS by Varshney and coworkers. They found that a protein kinase in Candida albicans, Sch9, is important for ensuring that chromosomes end up in the right place when this yeast reproduces by budding.

Turns out that the same is not true for the Sch9 ortholog in our favorite yeast Saccharomyces cerevisiae. There is no evidence that Sch9 has anything to do with chromosome segregation there, even though the Sch9 sequences in these two yeasts look very similar.

C. albicans Sch9 is very important for keeping filamentous growth at bay under certain conditions (hypoxia and high levels of carbon dioxide). To understand better how Sch9 does this, Varshney and coworkers used chromatin immunoprecipitation (ChIP) to figure out where the protein binds in the genome. They were surprised when they found that it bound mostly to centromeres.

Despite this binding, the authors saw no evidence that Sch9 was involved in stabilizing the kinetochore, the protein structure that forms at the spindle of sister chromatids. When a kinetochore is destabilized, a cell’s nuclear morphology changes, its centromeres decluster during the cell cycle, and the centromeric histone Cse4 delocalizes away from its centromeres. The authors saw none of these things in a C. albicans strain in which the SCH9 gene was deleted.

They did, however, find that C. albicans cells lacking Sch9 had anywhere from a 150 to a 750-fold increase in chromosome loss. They found this by using a strain of C. albicans that had an arginine marker on one copy of its chromosome 7 and a histidine marker on the other, and looking for how often cells lost one of the two markers. From this the authors concluded that like many other kinetochore associated proteins, Sch9 is involved in chromosome segregation.

As a final experiment, Varshney and coworkers used ChIP to see if the Sch9 protein bound to centromeres in S. cerevisiae. It did not. While the authors did not directly test whether Sch9 had any effect on chromosome segregation in S. cerevisiae, the presumption is that it didn’t, as it doesn’t appear to interact with centromeres and no such effect has been seen previously.

But Sch9 isn’t completely different in the two yeasts. A close look at the ChIP data showed that Sch9 bound the rDNA locus in both C. albicans and S. cerevisiae.

How did orthologous proteins in two budding yeasts end up with such different functions? One idea is that the ancestral gene to Sch9 was important for rDNA regulation and that it later gained a function in chromosome segregation in C. albicans. Another possibility is that the ancestral gene had both functions and that centromere binding was lost in S. cerevisiae. More work will need to be done to tell the difference.

Whichever explanation is correct, this study reminds us that, just like sugar and salt, even if two genes look similar they may have quite different functions. Assuming that similar appearance means identical function may lead to an experimental result that is just as unpleasant as salty coffee!

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

Categories: Research Spotlight

Tags: Candida albicans, chromosome segregation, ortholog, Saccharomyces cerevisiae

Mannan From Heaven

February 25, 2015

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Thousands of years ago, humans encountered very little yeast in their diet. This all changed with the invention of beer and bread.

Now of course we didn’t include the yeast as a carbon source…we just liked fluffy bread and getting hammered. But it was a different story for the bacteria in our gut. They now found the mannans in yeast cell walls raining down on them like manna from heaven.

Unlike the Israelites, who could eat manna, most of our microbiota probably couldn’t use this newfound carbon source. But at some point, the polysaccharide utilization loci (PULs) of a few species changed so that they could now digest the mannans found in yeast cell walls. And, as a new study in Nature by Cuskin and coworkers shows, it was almost certainly a great boon for them.

They found that at least one gram negative bacterium, Bacteroides thetaiotamicron, can live on just the mannans found in the yeast cell walls. This isn’t just a cool story of coevolution either. It might actually help sick people get better one day.

Glycans, like the mannans found in yeast cell walls, have been implicated in autoimmune diseases like Crohn’s disease. One day doctors may be able to treat and/or prevent diseases like this by providing bacteria that can eliminate these potentially harmful mannans: a probiotic treatment for a terrible disease.

Cuskin and coworkers identified three loci in B. thetaiotamicron, MAN-PUL1, MAN-PUL2, and MAN-PUL3, that were activated in the presence of α-mannan. Deletion experiments showed that MAN-PUL2 was absolutely required for the bacteria to utilize these mannans.

The authors next wanted to determine whether the ability to use yeast cell walls as a food source provided an advantage to these bacteria. For this work they turned to a strain of mice that lacked any of their own gut bacteria.

The authors colonized these gnotobiotic mice with 50:50 mixtures of two different strains of B. thetaiotamicron—wild type and a mutant deleted for all three PULs. They found that in the presence of mannans, the wild type strain won out over the mutant strain and that in the absence of mannans, the opposite was true.

So as we might expect, if you feed mice mannans, bacteria that can break them down do best in the mouse gut. The second result, that mutating the PULs might be an advantage in the absence of glycans, wasn’t expected. This result suggests some energetic cost of having active PULs in the absence of usable mannans.

To better mimic the real world, Cuskin and coworkers also looked at the gut bacteria of these mice when they were fed a high bread diet. In this case, the mutant still won out over wild type but not by as much. Instead of being reduced to 10% as was true in the glycan-free diet, the final proportion of wild type bacteria in guts of mice on the high bread diet was 20%. Modest but potentially significant.

We do not have the space to discuss it here, but the authors next dissected the mannan degradation pathway in fine detail. If you are interested please read it over. It is fascinating.

The authors also analyzed 250 human metagenomic samples and found that 62% of them had PULs similar to the ones found in B. thetaiotamicron. So the majority of the people sampled, but not all, had gut microbiota that could deal with the mannans in yeast cell walls.

Human microbiota have adapted to use the energy from the bits of yeast left in bread and alcoholic beverages. Given how little there is, it might be better to think of it as the filth the peasants collected in Monty Python and the Holy Grail instead of manna. Even though it isn’t much, it has given these bacteria a niche that no one else has (or probably wants).

Still, it has allowed them to at least survive. And if these bacteria can one be repurposed as a treatment for disease like Crohn’s disease, thank goodness they adapted to using these mannans.  

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

Categories: Research Spotlight

Tags: evolution, glycans, gut microbiome

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