New & Noteworthy
May 26, 2016
If you’re not already using JBrowse to view all your favorite S. cerevisiae genes…you should be! SGD’s JBrowse is a quick and easy way to browse through the information-rich yeast genome. Using JBrowse, you can visualize spatial relationships between genes, locate SGD annotations throughout the yeast genome, and align chromosomal features to hundreds of experimental data sets.
Our upcoming webinar on June 1st will provide a short 15-minute tutorial on the basics of JBrowse. We will demonstrate how to navigate the genome with JBrowse, locate your favorite genes or chromosomal features, and visualize experimental data with data tracks. Whether you’re an experienced GBrowse user looking to try JBrowse for the first time, or someone new to genome browsing as a whole, this webinar is sure to help you get started.
If you are interested in attending this event, please register using this online form: http://bit.ly/SGDwebinar3
This is the third episode of the SGD Webinar Series. For more information on the SGD Webinar Series, please visit our wiki page: SGD Webinar Series.
May 18, 2016
A long time ago, through the mists of time, finding a phrase or a passage in a document was hard. If you didn’t have the foresight to highlight it, you were stuck.
You could reread the whole document but that might take way too much time. Or you could narrow down where to look by remembering the chapter it was in and rereading just that.
Nowadays of course, finding that phrase or passage is trivial in a Google or Word doc. You just use the find function!
Finding the genetic variant responsible for a given trait is still, in many ways, in the age of the typewriter. A genetic association study can find a part of the DNA responsible for that trait but homing in on the exact variant is terribly time consuming and labor-intensive.
Enter that wonder of a gene editing tool, CRISPR.
In a new study in Science, Sadhu and coworkers have essentially turned CRISPR into a find function for genetic association studies. They use CRISPR’s double stranded DNA cutting ability and mitotic recombination in a heterozygote yeast strain to blanket a region of DNA with crossover events.
Sifting through the results let them find the actual amino acid change responsible for manganese sensitivity with much less effort than would have been required with the old method. They only needed to use 358 lines instead of the 7,500 or so they predict they would have had to go through without good old CRISPR.
And think how much time might be saved with a more complicated beast! Given its short generation time and relatively small genome, yeast is like a magazine article. A human is more like War and Peace. This new system might make linking human traits/diseases to the causative SNP much simpler.
The usual way to link a trait to the DNA that causes it is to see what known traits tend to get passed down with it. If you know trait A is at position X on chromosome Y and the trait you are interested in, trait B, is usually passed down with A, then trait A and trait B are close to each other (this is called linkage mapping). This is the easy part.
The hard part is getting from A to B. To get finer mapping, you need to rely on the recombination that happens during meiosis. This is the step where this new study can help.
Instead of relying on the slow, natural process of meiotic recombination, these authors use the CRISPR-Cas9 system to jump start mitotic recombination.
Once Cas9 cuts the DNA, the cell uses homologous recombination to repair the cut using the DNA on the other chromosome in the pair as the template. This results in a loss of heterozygosity (LOH) which can reveal recessive traits.
They picked 384 lines, approximately 4 from each cutting event and found that 95% of these had undergone LOH with hardly any off-target effects.
They next measured the growth of these strains in 12 different conditions and found that one trait, growth on 10 mM manganese sulfate could work for their purposes. Using the more traditional approach with 768 segregants obtained through meiotic recombination, they were able to narrow it down to an overlapping piece of DNA of 3,900 bases. However, using CRISPR-Cas9, they were able to narrow down the location of the variant to a 2,900 base pair stretch of DNA. Score one for the new method!
Next they did finer mapping by targeting the 2,900 bases they had identified with CRISPR-Cas9. Even though they only used three guide RNAs that targeted three locations, they were able to look at many more places on the DNA than this because the length of DNA repaired around the cut varies a bit between strains.
They isolated 358 lines of which 46 or 13.1% had a recombination event in the 2,900 bases they were interested in. Only 0.7% of segregants obtained the old way were in the right place. Score another one for the new method!
After measuring how well these 46 lines grew in manganese sulfate, the researchers were able to narrow the search to a single polymorphism that resulted in an amino acid change in the PMR1 gene. A perfectly reasonable result, given that Pmr1p is a manganese transporter. They then showed that this variant, pmr1-F548L, did indeed make a strain sensitive to manganese all on its own.
By targeting recombination events to regions of interest, these authors have provided proof of principle for a technique that should make connecting traits (phenotype) with DNA variants (genotype) much easier than it is currently. Behold yet again the awesome power of yeast genetics (#APOYG)!
by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
May 10, 2016
SGD’s new JBrowse genome browser allows quick and easy browsing of the information-rich yeast genome.
Take a look at our newest video tutorial to learn how to download or upload JBrowse data tracks. Let us know if you have any questions or suggestions.
For more SGD Help Videos, visit our YouTube channel, and be sure to subscribe so you don’t miss anything!
May 4, 2016
As HBO’s Silicon Valley scathingly relates, the mapping app from Apple was truly terrible when it was first launched. There are all kinds of funny (scary?) stories in which people following the directions ended up in the wrong place. (Click here for a few more of the epic fails.)
And sometimes it would show impossible ways to get from one location to the other. For example, to get to a certain place, my iPhone would recommend two different routes. After choosing the seemingly easiest route I quickly realized that it would take me through a building. Sometimes, even though it seems there are a couple of different ways to get somewhere, there is actually only one.
It turns out that this can be true in gene expression too. While you might increase expression by either mutating the promoter or duplicating the whole gene, sometimes only duplication is enough. There is just one route from here to there.
This point is driven home in a new study out in GENETICS by Rich and coworkers. Here they show that, under sulfate-limiting conditions, the only way that yeast can boost the expression of the SUL1 high affinity sulfate transporter enough to thrive is by duplicating it.
In many previous experiments, whenever yeast is starved for sulfate, after 100 generations or so the population almost always ends up selecting for a SUL1 duplication rather than increasing expression with a promoter mutation. This duplication results in a fitness advantage of 35% or more, which makes sense – when there isn’t much sulfate available, those cells that can get more of what’s there will outcompete their neighbors.
There are a couple of possible reasons things go down like this. It could be that the duplication is simply the most likely way to increase expression enough to survive, meaning that promoter mutations are possible but rare. Alternatively there may be no way to mutate the promoter enough to adequately increase the activity in such a short window of time. You simply can’t get to enough increased fitness by this route.
The first step in figuring out how to get somewhere is to map the roads in the area. This is also what Rich and coworkers needed to do – they needed to figure out how SUL1 is regulated. They did this in the standard way by nibbling away bits of the sequences upstream of the gene until there was a significant impact of gene expression. This is how they identified the 493 base pairs upstream of SUL1 as its promoter.
The next step was to generate a library of mostly single point mutations in this promoter using error-prone PCR. As might be expected, most of the mutants had no effect or decreased gene expression but a few did increase activity. However, none of these last set increased expression as much as duplicating the gene.
They found 8 mutants that gave a 5% or better increase in fitness with the best being a 9.4% increase. Even when they combined these point mutations they could not increase the fitness much beyond 11%, not even half of the increase in fitness that an extra SUL1 gene gives. It seems that to get the most bang for its buck, yeast needs to duplicate SUL1. At least in the time frame of the experiment, that is.
Using what is essentially the scanning mutagenesis of this promoter, they were able to identify three sites that were important for SUL1 regulation. One site at -465 to -448 corresponded to a Cbf1-Met28-Met4 regulatory site, the second site at around -407 was most similar to either a Met31 or Met32 site and the third site at around -350 matched a Met32 site.
Mutations that resulted in increased activity tended to bring one of these three sites closer to the consensus transcription factor binding site. For example, the strongest point mutation, -353T>G, did this with the Met32 site at -350.
Even with these stronger consensus sequences for sulfate-regulatory transcription factors, none of these point mutations could get the yeast to where it needed to be in the fitness landscape in order to be able to thrive under sulfur limitation. Point mutations just can’t hold a candle to simply duplicating the SUL1 gene. Sometimes there really is just one way to get from point A to point B.
by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics