New & Noteworthy

Studying Aging in Yeast Just Got Easier

April 02, 2012

Watching a yeast cell age can be a real pain.  In budding yeast like Saccharomyces cerevisiae, the buds quickly outnumber the mom.  Which means scientists need to remove the buds as they appear.

Up until now, scientists have had to use a 50-year-old method that involves removing the buds by hand.  Not only is this labor intensive, but the field is held back by the inability to use high resolution microscopy to investigate the aging process. 

These technical limitations may soon be swept aside with a new microfluidic dissection technique described by Lee and coworkers in a recent study out in PNAS. These researchers were able to monitor 50 aging yeast at once with a variety of microscopic techniques without having to remove the buds by hand.  And unlike the older technique, they were able to keep a constant environment for the yeast cells (i.e. no decrease in nutrients and/or build up in wastes).

Basically Lee and coworkers tucked the yeast mother cells under a micropad which they then washed with a constant flow of nutrients.  Because the daughter cells are smaller than the mother, they are washed away as they emerge.  So no manual bud removal is required.

Sounds convenient but the researchers needed to show that this new technique gave similar results as compared to the old one.  And they did.

They showed that mutant strains behaved similarly with both techniques.  So a SIR2 deletion mutant still had a shorter lifespan and a FOB1 deletion mutant still lived longer with microfluidic dissection.  Not only that, but the number of divisions in an average yeast’s lifetime was comparable with both techniques.  At first blush the techniques do seem comparable.

Now they were ready to take their new technique out for a spin to see what it could do.  First they were able to show heterogeneity in how yeast cells age.  Some cells died as spheres around their 12th division while others died as ellipsoids after their 25th division.  The shape of the yeast later in life correlated with how long that yeast lived.

The researchers were also able to use GFP to explore the vacuoles of aging yeast.  They found three classes of vacuoles: tubular, fused, and fragmented.  The tubular vacuoles were only found in the longer-lived ellipsoid yeast.

Researchers could not have discovered these properties of aging yeast without the new microfluidic dissection technique.  And these findings are really just the tip of the iceberg of what can now be learned about aging by studying yeast.  It will be exciting to see what else scientists will be able to learn about the twilight of a yeast cell’s life.

Life and Death of a Single Yeast

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

Categories: Research Spotlight

Tags: aging, microfluidic, vacuole, yeast

Sopping up Uranium with Yeast

March 23, 2012

Yeast may be good for more than making bread and beer or understanding how eukaryotes like humans work.  They may also be useful for cleaning up high volume, low concentration waste uranium (think uranium waste water).

The idea would be to add yeast to the contaminated area, have the yeast take the uranium up, put the yeast into radioactive waste and repeat with new yeast.  This would be a relatively cheap, simple way to detoxify this form of radioactive waste.

An obvious way to improve on this idea is to identify yeast strains that can accumulate more uranium than the wild type strain.  In a new study out in Geomicrobiology Journal, Sakamoto and coworkers have started down this path by identifying genes that allow yeast to grow in the presence of uranium and those involved in uranium accumulation.

They did this with two different screens using a set of 4,098 non-essential gene deletion strains.  In the first they identified 13 strains that grew more poorly than wild type at 0.5 mM uranium.  And in the second, they identified 17 strains that accumulated less uranium than wild type.

There was very little overlap between the two sets of strains suggesting different pathways (or sets of pathways) may be involved in accumulation and growth.  However, there were two deletion strains that showed up in both screens.  Both of the identified genes, PHO86 and PHO2, are involved in phosphate metabolism.

These genes definitely make sense.  A number of previous studies had hinted strongly that uranium accumulates on the surface of yeast in the form of insoluble uranium-phosphate complexes. 

The idea behind the importance of these genes is that yeast deals with higher uranium levels by scavenging more phosphate.  When genes involved in this process are knocked out, the yeast can’t get the extra phosphate it needs to form the insoluble uranium phosphate complexes.  Now it grows poorly and has less uranium on its surface. 

It will be interesting to see how the other genes are involved in uranium survival or accumulation.  Perhaps one day researchers will be able to turn yeast into a grade A uranium sponge.  Here’s hoping they can!

For those really interested, here is a list of the genes identified in each screen:

Uranium sensitive: PHO2, PHO84, PHO86, PHO87, VPS74, ENT5, CPR1, GLO2, OPI1, ATG15, PTC6, SLC1, and uncharacterized ORF, YPR116W.

Uranium accumulation: OPI1, PHO86, APL4, PEX10, VPS74, PHO2, SPT20, GAL11, SWP82, IVY1, FLO1, DIT2, RPL2A, and uncharacterized ORFs, YGL214W, YJR098C, YNL035C, and YPR116W.       

A nice lecture on bioremediation (using biology to clean up toxic waste)

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

Categories: Research Spotlight

Tags: bioremediation, nuclear waste, Saccharomyces cerevisiae, uranium, yeast

Not any Cyclin Will Do

March 16, 2012

Getting through a cell cycle is a complicated process. All sorts of proteins need to work and stop working at the right times in the right places to get the DNA copied, get the cells growing larger, have the cells divide and so on.

Key regulators in this process are the cyclins and their dependent kinases. Different cyclins are expressed at different points in the cell cycle, at which time they direct their cyclin-dependent kinase (CDK) to the appropriate subset of proteins to be phosphorylated. A big part of cell cycle regulation, then, comes from when a cyclin is expressed. But this is not the whole story.

In a study out in this month’s issue of GENETICS, DeCesare and Stuart showed that at least for the B-type cyclin Clb5 in the yeast S. cerevisiae, timing isn’t everything. And even more unexpectedly, they found that a key part of this cyclin’s specificity comes from its N terminus.

In yeast, Clb5 is involved in premeiotic DNA synthesis. Many researchers had previously argued that any B-type cyclin expressed at the right time would be sufficient to promote this function. DeCesare and Stuart were able to show that this was not the case by putting two different cyclins, Clb1 and Clb3, under the control of the CLB5 promoter. These cyclins were now expressed at the right time but neither could substitute for Clb5.

The authors next set out to discover what part of Clb5 conferred this specificity by creating chimeric versions of Clb3 and Clb5. They identified two regions in Clb5 important for premeiotic DNA synthesis — a hydrophobic patch and the N terminus.

The hydrophobic patch was expected; this region is highly conserved in all cyclins and has previously been shown in to be involved in interacting with protein substrates. But the N terminus was a surprise. It was thought to be involved primarily in cyclin stability and/or subcellular localization and not protein-protein interactions.

The authors were not able to identify which specific part of the N terminus of Clb5 was involved in conferring specificity. In their experiments, there was a gradual decline in the ability of the Clb3-Clb5 chimera to promote premeiotic DNA synthesis as more and more of Clb5 was replaced with Clb3. It is as if the whole region is involved in determining specificity.

And the decreasing ability of the Clb3-Clb5 chimera to induce premeiotic DNA synthesis was not due to the loss of kinase activity. When paired with Cdc28 (also known as Cdk1), all of the chimeras in the experiment were equal or even more active than the wild type Clb5/Cdc28 pair.

What it looks like is happening is that Clb5 uses both its hydrophobic patch and its N terminus to bring appropriate proteins to Cdk1 for phosphorylation. Different parts of each region are used to interact with different subsets of proteins involved in premeiotic DNA synthesis. At least for Clb5 and premeiotic DNA synthesis, it looks like not any cyclin will do.

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

Categories: Research Spotlight

Stellar Strain Yields Increased SAM Production

March 10, 2012

Yeast geneticists often go the extra mile to get their mutant. But Huang and coworkers went hundreds of extra miles to get theirs. Hundreds of miles straight up, that is.

In a recent study published online in the Journal of Applied Microbiology, Huang and coworkers identified and improved upon a strain of Saccharmoyces cerevisiae that makes increased amounts of S-adenosyl-L-methionine or SAM. This is an important chemical in many pharmacological and medical uses and is primarily made via microbiological synthesis. Increased production would be an obvious boon to researchers and the pharmaceutical industry.

What makes this study interesting is that the researchers obtained their initial strain from outer space. They shot cultures up into space on a satellite where the poor yeast had to endure the harsh environment there for 18 days. Researchers then collected the samples when the satellite returned to Earth.

Out of six hundred random clones from the flight, researchers found 43 that made at least 10% more SAM than their wild type counterpart. A second round of selection yielded strain H5M147 which made 84% more SAM than the wild type strain.

Unfortunately the researchers were not able to (or did not report in this study) why the strain made extra SAM. They used a technique called AFLP that allowed them to see that there were differences in the new and host strain’s genome, but it did not allow them to pinpoint what those differences were nor which ones were significant. That will have to wait for a future study.

They did manage to ramp up SAM production even further in this strain though. First they added an extra copy of a key player in SAM production, the MAT2 gene. Researchers have tried to coax other strains of yeast to make more SAM by adding MAT2 but to no avail. This space strain apparently has genetic mutations that allow extra MAT2 to increase SAM production. The new strain with the integrated version of MAT2 was called H5MR38.

Finally the researchers tinkered with culture conditions to optimize SAM production in H5MR38 further. They found that using sucrose as the carbon source and adding peptone to yeast extract and urea yielded the most SAM.

In the end, Huang and coworkers managed to get 7.76 grams of SAM per liter of culture after 84 hours. This compares to the previous high in Sacchromyces cerevisiae of 5.7 gram per liter after 120 hours and 3.6 grams per liter from a strain of Pichia pastoris in 100 hours. Clearly their space-derived yeast strain is an improvement over anything else identified so far.

Huang and coworkers aren’t the only ones putting yeast into space

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

Categories: Research Spotlight

Engineering Magnetic Yeast

March 02, 2012

With a bit of genetic manipulation and a hearty diet of iron, Nishida and Silver report in the latest issue of PLOS Biology that they have caused yeast cells to become magnetic. And this isn’t just a parlor trick. Their research could one day help other scientists create new therapies for the sick and new applications for research and industry.

The first step in the process was to load the yeast up with magnetic iron. The authors took a couple of different approaches.

The simplest was to grow the yeast in lots of iron. Surprisingly, without any other manipulation, this was enough to make the yeast a bit magnetic. But the authors wanted more magnetism.

To accomplish this goal, they needed to keep yeast from transporting excess iron to their vacuole where it is nonmagnetic. They did this by knocking out the gene encoding the vacuolar iron transporter, CCC1.

When grown in lots of ferric citrate, the ccc1Δ strain was about 1.8 times more magnetic than wild type. Nice, but to get even more magnetic yeast, Nishida and Silver added back the three human genes necessary to reconstitute human ferritin. This new strain was now about 2.8 times more magnetic than wild type.

None of this was really earth-shattering yet. Scientists knew that iron was needed to make a cell magnetic and that ferritin-iron complexes were a bit magnetic. What made these initial studies important was that they gave Nishida and Silver the tools to study the underlying mechanisms of magnetism.

The authors took a directed approach to study this problem and knocked out genes known to be involved in iron homeostasis or oxidative stress. Of the 60 knockout strains tested, tco89Δ was the only one to consistently be less magnetic than the wild type strain. On average it was about two fold less magnetic.

Tco89p is a nonessential part of TORC1, a complex involved in the regulation of cell growth in response to nutrients, stress, and redox states. As might be predicted from TORC1 function, the authors determined that nutrients and the redox state of the medium affected the yeast’s magnetism. They then expanded their screen to look for genes involved in carbon metabolism and mitochondrial redox that might affect magnetism and discovered several (POS5, YFH1, SNF1, and ZWF1).

The current model is that the redox state within the cell and in particular, within the mitochondria, impacts the amount of iron precipitation and hence magnetism in yeast. This is consistent with the iron deposits the authors saw in electron micrographs of the mitochondrial membrane of the magnetic yeast.

These findings should help point researchers in productive directions for engineering magnetic cells in other systems but it is only a first step. Science has a long way to go before therapies based on cell magnetism are helping patients.

More details on these magnetic yeast

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

Categories: Research Spotlight

Tags: ferritin, magnetic, MRI, yeast

Symposium in remembrance of Jon Widom, 1955 – 2011

February 22, 2012

As many of you know, Jonathan Widom died suddenly last summer.  He was a Professor in the Department of Molecular Biosciences at Northwestern University.  The beauty of Jon’s scientific research was more than matched by his surpassing intellectual brilliance and personal warmth, and he is deeply missed by those who knew and loved him.  Jon’s family and colleagues have organized a symposium celebrating his life and work, which will be held at Northwestern’s Evanston campus on March 16, 2012.  Information about this gathering, called “Unraveling the Mysteries of Life:  Recognizing the Life and Work of Jon Widom”, can be found online here.  All are welcome. Additionally, tributes to Jon that will be shared at the meeting (and afterward) can be contributed here.

Categories: News and Views

Finally Great Tasting, Low Alcohol Beer

February 17, 2012

Let’s face it: low alcohol beer just doesn’t taste that great.  This is because the alcohol is either diluted or removed chemically after fermentation.  Both methods wreak havoc with a beer’s flavor.

Dr. John Morrissey of University College Cork is trying to change this.  His lab is working to generate a strain of yeast that turns some but not all of its sugar into alcohol.  That way the beer process is the same, just with less alcohol at the end.

This is different from stopping fermentation early.  In that case there are still sugars in the final product which ruin a beer’s taste even more than removing the alcohol!  Here the same amount of sugars are used up, it is just that only part of that energy has gone into making the alcohol.  Same sugar content, less alcohol.

Although we don’t have all the details because of intellectual property issues, what we do know is that he compared the genomes of yeast species that make a lot of alcohol and those that don’t.  In an email he stated that he focused on genes that would affect carbon metabolism without perturbing redox balance in a significant way.  Presumably he then swapped the appropriate genes between strains and created his low alcohol strain.

This is not only a godsend for low alcohol beer, but it may be useful for other fermentation processes as well.  For example, maybe something similar can be done for low or no alcohol wines which, apparently, are even less tasty than low alcohol beer.  Designated drivers everywhere will be thanking Dr. Morrissey profusely if he can make decent tasting, low alcohol drinks a reality.

And apparently it isn’t just designated drivers that want this stuff.  Judging by recent upticks in sales of the relatively low quality low alcohol beers currently on the market, there is definitely a market out there for such beverages.  A cool science project, decent low alcohol beer and nice profits to boot!  Who could ask for more? 

How beer is made, from Modern Marvels, http://www.history.com

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

Categories: Research Spotlight

Tags: carbon metabolism, fermentation, low alcohol beer, redox, yeast

Expression Data and LiftOver Files Available for Download

February 14, 2012

RNA expression data that are included in SGD’s SPELL expression analysis tool are now available for download in the expression directory. Datasets have been grouped by publication and are in PCL format.

LiftOver files that allow conversion of chromosomal coordinates between different S. cerevisiae genome versions are also now available for download via the genome_releases link in the sequence directory.

Categories: New Data, Website changes

Multicellularity a Snap? Maybe so…

February 10, 2012

Some people might think that the transition from single-celled creatures to multi-cellular ones must have been tough.  After all, single celled organisms ruled the world for the first one or two billion years of life here on Earth. 

And yet, all multi-celled beasts didn’t evolve from the same ancestor.  Current theories are that multicellularity evolved dozens of times over the ages.  In fact, all of the transitional stages of multicellular life can be seen in the volvocine green algae species around today.  So maybe it isn’t so tricky after all.

Using a very clever screen in yeast, Ratcliff and coworkers have shown that they can get crude multicellular life to evolve in the lab.  Basically they only let the yeast that settled easily to the bottom of a shaking flask go on to reproduce.  Within 60 or so days, they had the beautiful, snowflake-like, multicellular beasts made up of multiple yeast cells shown in the image to the right.

Of course multicellular is more than having a bunch of cells stuck together.  Heck, yeast do that now in something called flocs.  No, to be multicellular, these yeast need to reproduce in a way that generates new multicellular yeast and to have specialized cells.  The snowflake yeast from this experiment did both.

These yeast did not reproduce by creating sperm and eggs that combine to generate progeny.  Instead they reproduced more like a lot of plants do.  They produced smaller versions of themselves which then went on to grow to “adulthood.”  Multicellular life gave birth to more multicellular life.

Cells within these snowflakes were also willing to die for the common good.  For example, the cell where the juvenile snowflake was attached would undergo apoptosis so the juvenile could be released.  No single-celled organism would willingly take that kind of hit for other cells.

So it looks like these researchers managed to evolve multicellular organisms from single-celled ones in just a few months.  Pretty amazing what can be learned from yeast!

Of course some care is needed here.  Yeast actually evolved from a multicellular ancestor so some sort of memory of multicellular life may still be lurking in its genes.  If true, this might make the transition from one to many simpler in yeast than in other single-celled organisms. 

This is why the researchers plan to try similar experiments with single celled organisms that have been single cells throughout their evolutionary life.  Then they’ll have an even better idea about how easy the “one to many cells” transition is.

Multicellular yeast having babies.

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

Categories: Research Spotlight

Tags: evolution, multicellular, Saccharomyces cerevisiae, yeast

A Simpler Way to Evolve

February 06, 2012

As life evolves there is a tendency for increased complexity.  Up until now, scientists have mostly focused on gain of function mutations as the motor for this change.  This has proven fertile ground for evolution deniers who have claimed that life’s complexity could not have arisen from these rare, gain of function mutations alone.

A new study by Finnigan and coworkers provides an important counterpunch to this argument.  These authors resurrected ancient proteins and showed that an increase in complexity can come from much more common, loss of function mutations.  Time for the deniers to find a new argument…

Making Molecular Machines

Finnigan and coworkers focused on the evolution of a protein called vacuolar H+ -ATPase or V-ATPase for short.  Like other molecular machines, this proton pump consists of many different proteins all working together in a coordinated fashion.  One key part of this machine is a rotary ring called V₀.  (This would be the ring of C proteins in the image to the right.)

In most eukaryotes, V₀ is made up of five identical subunits (called Vma3) and one subunit called Vma16.  In fungi, a third protein, Vma11, has replaced one of the Vma3 subunits.  In other words, the fungal version is a bit more complex than other eukaryotic versions.

Current theories are that these three proteins all arose through gene duplication.  Duplication of the Vma3 gene first led to the Vma16 gene and then later in fungi, Vma3 duplicated again this time becoming Vma11.  Yeast V-ATPase absolutely requires Vma11 to function and other eukaryotic Vma3 family members cannot replace Vma11.

Using the 139 family members of the Vma family available in GenBank, members of the Thornton and Stevens lab recreated the ancestral proteins that existed before and after the Vma11 gene duplication event.  Before the arrival of Vma11, there were only two proteins which the authors have named Anc.3-11 and Anc.16.  Anc.3-11 presumably has functions of both Vma3 and Vma11.  After the gene duplication event, there were three ancient proteins: Anc.3, Anc.11, and Anc.16.

Using these ancient proteins, the authors first showed that Anc.3-11 could substitute for either Vma3 or Vma11 in yeast. It could even partially rescue a yeast strain that lacked both of the other genes.  They then showed Anc.16 could replace Vma16 and most importantly, that the two ancient proteins could replace the three modern ones.  They reconstructed an ancient molecular machine that works.

The next step was to figure out what happened after Anc.3-11 duplicated again and the two genes began to evolve into the separate proteins, Anc.3 and Anc.11.  Again using the GenBank sequences, the authors predicted that two single mutations were an initial step on the way to the separation of Anc.3-11 activities into the Anc3 and the Anc11 proteins. 

The authors engineered each mutation independently into the Anc.3-11 protein and found that one mutation made Anc.3-11 more like Anc.3 and the other made Anc.3-11 more like Anc.11.  The complex now required all three Anc proteins instead of just the two for maximal activity.  The authors had recapitulated the first evolutionary steps that led to the formation of the three subunit V₀ rotary ring.

Finally the authors showed that each of these mutations were loss of function mutations.  The Anc.3-11 protein has two different interfaces that interact with Anc.16.  The first mutation weakened one interface on Anc.3 and the second mutation weakened the other interface on Anc.11 causing both proteins to now be required to reconstitute the ring.  The added complexity arose from a combination of gene duplication and relatively common loss of function mutations.

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

Categories: Research Spotlight

Tags: ATPase, evolution, gain of function, gene duplication, loss of function, mutation, proton pump

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