New & Noteworthy
October 23, 2014
In case there were still any doubters, the world is definitely heating up. April to September 2014 were the warmest these months have ever been since we started keeping records in 1880. And about 35,000 walruses were forced ashore this summer in Alaska because there wasn’t enough room left for them on the sea ice where they normally hang out. Unfortunately, the trend looks to be more record breaking heat for as long as we can see in the future.
This is bad news for us, and even worse news for nature. But all is not lost! Our hero yeast may be able to swoop in and make us biofuels to replace gasoline. This won’t stop global warming, but it might at the very least slow it down.
But yeast in its current form probably couldn’t make enough biofuels to make a difference without using too much precious food in the process. Like the X-men, it will need a helpful mutation or two to increase its efficiency to the point where it can make a real dent in slowing down the relentless rise in global temperatures. And ironically, one new trait yeast needs to pull this off is to be able to survive better in the heat.
To efficiently turn the parts of the plants we can’t use (mostly anything to do with cellulose) into biofuels, we need for yeast to grow in cultures where enzymes are chopping cellulose into bite sized pieces at the same time. These enzymes work best at temperatures that are a real struggle for wild type yeast.
In a new study in Science, Caspeta and coworkers isolated mutants better able to tolerate high temperatures by simply growing Saccharomyces cerevisiae at around 39.5° C for over 300 generations. They did this in triplicate and ended up with strains that grew on average about 1.9 times faster than wild type yeast at these temperatures.
The next step was to use genome sequencing and whole-genome transcription profiling to figure out why these mutant strains were heat tolerant. This is where things got really interesting.
They found many genes that were affected, but identified ERG3 as the most important player. All of the thermotolerant strains that they selected contained a nonsense mutation in ERG3. And in fact, when they introduced an erg3 nonsense mutation into wild type yeast, they found that this engineered strain grew 86% as well as the original mutant strain at high temperatures. In other words, most of the heat tolerance of these strains came from mutations in the ERG3 gene.
This makes sense, as membrane fluidity is a key factor in dealing with higher temperatures, and ERG3 codes for a C-5 sterol desaturase important for membrane composition. When Caspeta and coworkers looked at the membranes of the mutant strains, they found a buildup of the “bended” sterol fecosterol. Since “bended” sterols have been shown to protect the membranes of plants and Archaea from temperature swings, this could be the reason that the mutant strains dealt with the heat so well.
So as we might predict, changing the composition of the cell membrane affects how the yeast respond to temperature. What we couldn’t predict is that in order to get to this point via artificial selection, the yeast had to have a second mutation in either the ATP2 or the ATP3 genes that actually make yeast less able to grow at higher temperatures.
Because ATP2 and ATP3 are needed for yeast to grow on nonfermentable carbon sources, the authors hypothesized that perhaps thermotolerance could not evolve while oxidative respiration worked at full speed. Consistent with this, Caspeta and coworkers found that their evolved thermotolerant strains were more susceptible to oxidative stress and could not grow on nonfermentable carbon sources. This was not true of the strain they engineered to only have the mutation in the ERG3 gene—it grew well at 40° C and could use nonfermentable carbon sources.
So not only have these authors found a mutant yeast with the superpower of growing at higher temperatures, but they also showed that sometimes engineering works better than natural selection at creating the mutant they want. Evolving sometimes requires passing through an intermediate that makes the final product less useful than it could have been. In this case it worked best to use a combination of artificial selection and engineering to build a better mutant.
Even this super engineered yeast mutant won’t stop global warming in its tracks, but it might help us to slow it down enough so that natural systems have a chance to adapt. A superhero indeed…
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
October 21, 2014
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October 16, 2014
A train without working brakes can cause a lot of destruction if it careens off the tracks. And it turns out that a runaway RNA polymerase II (pol II) can cause a lot of damage too. But it doesn’t cause destruction, so much as disease.
Unlike a train, which has its brakes built right in, pol II has to count on outside factors to stop it in its tracks. And one of these brakes in both humans and yeast is a helicase: Sen1 in yeast and Senataxin, the product of the SETX gene, in humans.
Mutations in SETX are associated with two devastating neurological diseases: amyotrophic lateral sclerosis type 4 (ALS4) and ataxia oculomotor apraxia type 2 (AOA2), both of which strike children and adolescents. One idea is that these mutations may short circuit the brakes on pol II, causing it to keep on transcribing after it shouldn’t. And this is just what Chen and colleagues found in a new paper in GENETICS.
The researchers used the simple yet informative yeast model system to look at some of these mutations, and found that they disrupted the helicase function of Sen1 and caused abnormal read-through of some transcriptional terminators. Looks like bad brakes may indeed have a role in causing these devastating diseases.
Some human proteins can function perfectly well in yeast. Unfortunately, Senataxin isn’t one of those; it could not rescue a sen1 null mutant yeast, so Chen and coworkers couldn’t study Senataxin function directly in yeast. But because Senataxin and Sen1 share significant homology, they could instead study the yeast protein and make inferences about Senataxin from it.
First, they sliced and diced the SEN1 gene to see which regions were essential to its function. They found that the most important part, needed to keep yeast cells alive, was the helicase domain. But this wasn’t the only key region.
Some flanking residues on either side were also important, but either the N-terminal flanking region or the C-terminal flanking region was sufficient. Looking into those flanking regions more closely, the researchers found that each contained a nuclear localization sequence (NLS) that directed Sen1 into the nucleus. This makes perfect sense of course…the brakes need to go where the train is! If we don’t put the brakes on the train, it won’t matter how well they work, the train still won’t stop.
These flanking sequences appeared to do more than direct the protein to the nuclear pol II, though. When the authors tried to use an NLS derived from the SV40 virus instead, they found that it couldn’t completely replace the function of these flanking regions even though it did efficiently direct Sen1 to the nucleus.
Next the researchers set out to study the disease mutations found in patients affected with the neurological disease AOA2. They re-created the equivalents of 13 AOA2-associated SETX mutations, all within the helicase domain, at the homologous codons of yeast SEN1.
Six of the 13 mutations completely destroyed the function of Sen1; yeast cells could not survive when carrying only the mutant gene. When these mutant proteins were expressed from a plasmid in otherwise wild-type cells, five of them had a dominant negative effect, interfering with transcription termination at a reporter gene. This lends support to the idea that Sen1 is important for transcription termination and that the disease mutations affected this function.
The remaining 7 of the 13 mutant genes could support life as the only copy of SEN1 in yeast. However, 5 of the mutant strains displayed heat-sensitive growth, and 4 of these showed increased transcriptional readthrough.
Taken together, these results show that the helicase domains of Senataxin and Sen1 are extremely important for their function. They also show that Sen1 can be used as a model to discover the effects of individual disease mutations in SETX, as long as those mutations are within regions that are homologous between the two proteins.
It still isn’t clear exactly how helicase activity can put the brakes on that RNA polymerase train, nor why runaway RNA polymerase can have such specific effects on the human nervous system. These questions need more investigation, and the yeast model system is now in place to help with that.
So, although it might not be obvious to the lay person (or politician) that brainless yeast cells could tell us anything about neurological diseases, in fact they can. Yeast may not have brains, but they definitely have RNA polymerase. And once we learn how the brakes work for pol II in yeast cells, we may have a clue how to repair them in humans.
by Maria Costanzo, Ph.D., Senior Biocurator, SGD
October 15, 2014
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