New & Noteworthy

Support Model Organism Databases!

June 22, 2016


Image from http://biology.uiowa.edu/.

Model Organisms such as yeast, worm, fly, fish, and mouse are key drivers of biological research, providing experimental systems that yield insights into human biology and health. Model Organism Databases (MODs) enable researchers all over the world to uncover basic, conserved biological mechanisms relevant to new medical therapies. These discoveries have been recognized by many Nobel Prizes over the last decades.

NHGRI/NIH has recently advanced a plan in which the MODs will be integrated into a single combined database, along with a 30% reduction in funding for each MOD (see also these Nature and Science news stories). While integration presents advantages, the funding cut will cripple core functions such as high quality literature curation and genome annotation, degrading the utility of the MODs.

Leaders of several Model Organism communities, working with the Genetics Society of America (GSA), have come together to write a Statement of Support for the MODs, and to urge the NIH to revise its proposal. We ask all scientists who value the community-specific nature of the MODs to sign this ‘open letter’. The letter, along with all signatures, will be presented to NIH Director Francis Collins at a GSA-organized meeting on July 14, 2016 during The Allied Genetics Conference in Orlando. We urge you to add your name, and to spread the word to all researchers who value the MODs.

In other words, sign this letter!

Look Before You Leap…Into DNA Repair

June 9, 2016


It is worth a pause before leaping into action. This is true at the edge of a cliff, making a life or death situation, and before DNA repair. Cliff jumping image from Evan Bench on flickr, emergency technician image from Wikimedia Commons

Sometimes in an emergency, it can be useful to take a pause before trying to fix a problem. If the ambulance is on the way you may not want to start removing someone’s appendix right then and there. Yes you may save them, but the ambulance and EMTs will do less damage to the patient once they get there.

The same sort of logic applies to cells too. For example, a double stranded break is a deadly emergency that can wreak havoc with a cell’s genome.

The cell can deal with this in a few ways; the big two being non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ is the simplest in that the broken ends are simply stuck back together. However, it often results in a bit of DNA damage as a few bases are added or lost at the ligation point.

The other option, homologous recombination (HR), involves using a homologous DNA region as a template to essentially resynthesize the broken area. There are two possible ways this can happen in a cell: gene conversion and break induced replication (BIR).

If both ends are available without too much of a gap and in the right orientation, the cell opts for gene conversion, the safer of the two options. Only a small section of DNA is resynthesized, keeping most of the original DNA intact.

It is a different story for BIR, the second approach. Here, a large swath of DNA gets resynthesized, meaning a big loss of heterozygosity (LOH)—two sections of DNA are now identical over a large region. BIR only happens when there is only one end available or when there is a big gap of missing DNA.

Of the two HR possibilities, gene conversion is preferable over BIR. This is why the cell wisely waits to make sure there is no other option before starting down this path. This pause goes by the name of recombination execution checkpoint (REC).

A new study by Jain and coworkers out in GENETICS has identified two of the key players involved in this checkpoint—SGS1 and MPH1. Both are highly conserved 3’ to 5’ helicases.

These researchers found that when both are deleted, the REC disappears. And that isn’t all. In situations where the wild type cell would choose to repair its DNA by BIR, the double deletion strain no longer does. Instead, it now uses a process that is more similar to gene conversion.

Jain and coworkers found this using a reporter that contained an HO endonuclease site in the middle of the LEU2 gene. Once the HO endonuclease is activated, it makes a double strand DNA break, cutting the LEU2 gene in half.

The cell was provided with a variety of templates to be used in HR. One of these only provides the part of the LEU2 gene to the right of the HO endonuclease site. As only one of the ends has homology to the cleaved LEU2 gene, with this template, the cell can only use BIR to repair the break.

The researchers saw a huge increase in how fast the DNA was repaired using the BIR-only template in the strain deleted for both sgs1 and mph1 compared to the wild type strain. The double mutant repaired the DNA using BIR nearly as quickly as a wild type cell could using gene conversion. The pause before repair was essentially lost in the double mutant.

The double deletion strain wasn’t just faster with the BIR-specific template either. It repaired DNA via this pathway about 4 times better than the wild type strain did.

There are at least a couple of different reasons why the double mutant could be so much better at repairing DNA in BIR situations. In the first, the cell is just better at BIR—it can initiate the BIR pathway much more quickly than wild type. The second possibility is that this double mutant isn’t using the BIR pathway anymore and is using something closer to gene conversion.

Jain and coworkers found that the second option is the more likely of the two. The way they figured this out was to make a mutation in the POL30 gene, a gene required for DNA repair by BIR but not gene conversion. So, they tested what happens when POL30 is mutated in the wild type and the sgs1 mph1 double deletion strains.

They found that while a dominant negative mutant of pol30 had the predicted effect of severely compromising repair in wild type cells using their BIR-specific reporter, it had no effect in the double deletion mutant. Since Pol30p is needed for the BIR pathway, the strain deleted for sgs1 and mph1 must be using a different pathway to fix the DNA damage. So the pause is eliminated because the cell isn’t really using the BIR pathway anymore.

car going off a cliff

Image from The Petrick on vimeo

We don’t have time to go into it here, but there is a lot more research in this paper that looks at why the REC might be lost and that probes the differences in how MPH1 and SGS1 influence the BIR pathway that I encourage you to read. For example, deleting SGS1 is not identical to deleting MPH1 in terms of the BIR DNA repair pathway. And each single mutant still uses the BIR DNA pathway as the pol30 mutation severely compromises the ability of each to repair the BIR-specific reporter.

There is a lot more fascinating stuff like this in the study. The bottom line, though, is that MPH1 and SGS1 are the level-headed people urging folks not to panic and to wait for the EMTs to get to the accident scene to help. When MPH1 and SGS1 are gone, the cell dives right in and starts repairing breaks without waiting for the safest option—gene conversion. Who knows what havoc is wreaked in their absence!

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

New High-throughput GO Annotations Added to SGD

June 6, 2016


We’ve added 1,400 high-throughput (HTP) cellular component GO annotations from a new paper published by Maya Schuldiner’s lab. In this paper, Yofe et al., 2016 devised and implemented a methodology, called SWAT (short for SWAp-Tag), creating a parental library containing 1,800 strains, all known or predicted to localize to the yeast endomembrane system. Once created, this novel acceptor library serves as a template that can be ’swapped’ into other libraries, thus facilitating the rapid interconversion to new libraries by simply replacing the acceptor module with a new tag or sequence of choice. As proof of principle, this paper describes the parental library (N’ SWAT-GFP), and its utility as a gateway to the construction of two additional libraries (N’ mCherry and N’ seamless GFP). A high-content screening platform was used to generate images that were then manually reviewed and used to assign subcellular locations for proteins in these collections. Based on these results, SGD has incorporated GO annotations for proteins when at least two of three tags gave the same cellular localization. In addition, Locus Summary page descriptions for genes within this collection that did not have a known cellular location prior to this study have been updated. Finally, this study also provides access to a list of proteins predicted to contain signal peptides using three different algorithms. We would like to thank Maya Schuldiner and members of her lab for help with the integration of this information into SGD.

Sometimes You Need Half a Trillion

June 1, 2016


Image courtesy of The Internet Speculative Fiction Database.

In the Foundation series by Isaac Asimov, Hari Seldon invented a field of study called psychohistory which was able to “make general predictions” about human history. It only worked because the population of the Galactic Empire was in the quadrillions. You need huge numbers to get useful predictions.

Sometimes real life biologists need lots of individuals to see what they are looking for too. In the case of a recent paper in PNAS by Lee and Stevens, they needed to sift through half a trillion yeast to find what they were looking for. And boy was it worth it!

They saw an intron jump from one gene to another. Twice. In 500 billion tries.

This is the first example where we have caught an intron in the act of moving from one place to another. This has important implications for the study of evolution where over time introns spread or recede. It might even help us better understand cancer where intron loss can play a role.

Seeing something as rare as this means making a very specific, complicated reporter. You don’t want to manually sort those 500 billion colonies…or at least I wouldn’t want to.

Here is a “simplified” schematic of the reporter they came up with:

It is as complicated as it looks but it got the job done! Let me walk you through what everything is and how it works.

The GU and AG sequences are splice site junctions. Sequences between a GU and an AG can be spliced out.

The red gene is the S. pombe his5+ gene that has a promoter driving its expression shown with the red arrow. It is in the reverse orientation compared to the eGFP gene which is under the control of the promoter represented by the blue arrow.

The S. pombe his5+ gene works fine in S. cerevisiae his3 mutants to make up for histidine auxotrophy. But it won’t work from this construct.

See, the his5+ gene has an intron that keeps it from being translated correctly unless the intron is spliced out. But this intron can’t be spliced out when the gene is transcribed using the red promoter because the splice junctions GU2 and AG1 are in the wrong orientation. Any transcripts from the red promoter will not work because the intron cannot be spliced out.

You also can’t get His5 from the blue promoter. Even with splicing (which will work in that orientation), the gene can’t be translated because it is in the wrong orientation.

To get any his5+ transcript, the yeast needs to take the spliced RNA from between GU1 and AG2 and get it into DNA. It also needs to get rid of the intron in the middle of his5+ gene before it happens.

So what they are looking for are yeast that glow green and can survive in the absence of histidine. There are a couple of ways this can happen.

The more common way results in the his5+ gene recombining back into the plasmid such that the intron is missing from its middle. Basically the RNA between GU2 and AG1 is spliced out and the resulting RNA is reverse transcribed into DNA. This DNA then undergoes homologous recombination with the plasmid DNA resulting in a working his5+ gene. They got over 10,000 of these in their experiment.

The much less common way involves the intron being inserted into a gene within the genome. This way uses some of the same steps with one extra one—a reverse splicing event.

As I said, they got two of these with one ending up in the RPL8B gene and the other in the ADH2 gene.

Here’s how they think this happened…

The spliced out RNA from the plasmid was left for a brief time in the spliceosome (or what remained of it after the splicing reaction). During this time, a second mRNA arrived for splicing while the intron from the reporter was still there.

Then something called reverse splicing happened which basically replaced the native intron of the RPL8B or the ADH2 gene with the spliced out intron from the reporter. Next this was turned into DNA with reverse transcriptases and then this construct ended up in the genome through homologous recombination.

find

Only with yeast can we sift through 500,000,000 cells to find the two introns that have moved to new genes. Image from Needle in a Haystack, on Tumblr.

No wonder this was so rare! Reverse splicing is thought to be really uncommon in vivo as is reverse transcription of DNA. Add in a loitering intron on the remnants of a spliceosome and you can see why this was a 1 in 250 billion shot.

So there you have it, one way a transposon can hop. It is no transposon, but occasionally an intron can move to a new gene.

And of course we turned to the awesome power of yeast genetics to help us figure this out. Only with yeast can we sift through half a trillion cells to find the two that show us how intronogenesis, the introduction of an intron to a new site, might happen. #APOYG!

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

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