New & Noteworthy
April 17, 2017
With the construction of a global genetic interaction network in S. cerevisiae, it’s not hard to see why yeast genetic interactions remain a treasure trove for biological discovery. When combined with tools for visualization and analysis, these data can be used to draw powerful functional maps of the cell and infer potential functions for uncharacterized genes.
In a recent paper published in G3: Genes|Genomes|Genetics, Usaj et al. describe a web-based resource for exploring the global yeast genetic interaction network: TheCellMap.org. TheCellMap.org is an online database and visualization tool for quantitative yeast genetic interaction data. It provides an interactive version of the global yeast genetic interaction similarity network described by Costanzo et al., enabling users to scroll through and zoom in on different clusters of functionally related genes within the network. Users can search for specific genes or alleles, extract and re-organize sub-networks for genes of interest, functionally annotate genetic interactions, and more. Further, if more details about a gene are needed, users can even double-click on the gene to be taken to its respective locus summary page at SGD!
April 4, 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.
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
March 23, 2017
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.
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
March 17, 2017
For almost 50 years, the legendary Yeast Genetics & Genomics course has been taught each summer at Cold Spring Harbor Laboratory. (OK, the name didn’t include “Genomics” in the beginning…). The list of people who have taken the course reads like a Who’s Who of yeast research, including Nobel laureates and many of today’s leading scientists.
The application deadline is April 15th, so don’t miss your chance! Find all the details and application form here.
This year’s instructors – Grant Brown, Maitreya Dunham, and Elçin Ünal – have designed a course (July 25 – August 14) that provides a comprehensive education in all things yeast, from classical genetics through up-to-the-minute genomics. Students will perform and interpret experiments, learning about things like:
- How to Find and Analyze Yeast Information Using SGD
- Isolation and Characterization of Mutants
- Transformation of Plasmids & Integrating DNAs
- Meiosis & Tetrad Dissection as well as mitotic recombination
- Synthetic Genetic Array Analysis
- Next-Gen. whole-genome and multiplexed DNA barcode sequencing
- Genome-based methods of analysis
- Visualization of yeast using light and fluorescence microscopy
- Exploring synthetic biology with CRISPR/CAS9-directed engineering of biosynthetic pathways
Scientists who aren’t part of large, well-known yeast labs are especially encouraged to apply – for example, professors and instructors who want to incorporate yeast into their undergraduate genetics classrooms; scientists who want to transition from mathematical, computational, or engineering disciplines into bench science; and researchers from small labs or institutions where it would otherwise be difficult to learn the fundamentals of yeast genetics and genomics. Significant stipends (in the 30-50% range of total fees) are available to individuals expressing a need for financial support and who are selected into the course.
Besides its scientific content, the fun and camaraderie at the course is also legendary. In between all the hard work there are late-night chats at the bar and swimming at the beach. There’s a fierce competition between students at the various CSHL courses in the Plate Race, which is a relay in which teams have to carry stacks of 40 Petri dishes (used, of course). There’s also a sailboat trip, a microscopy contest, and a mysterious “Dr. Evil” lab!
The Yeast Genetics & Genomics Course is loads of fun – don’t miss out!