New & Noteworthy

Can’t Get There Like That

May 04, 2016


Sometimes when there should be two ways to get someplace, there is only one. What is true for mapping apps can also be true for mutations that increase fitness. Image from flickr.

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.

In the meantime, they also managed to develop a broadly applicable methodology for investigating any promoter – saturation mutagenesis, chemostat selection, and DNA sequencing to track variants.

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.

candles

Point mutations just can’t hold a candle to simply duplicating the SUL1 gene. Image from flickr.

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

Categories: Research Spotlight

Tags: gene duplication , saturation mutagenesis , fitness , adaptation , chemostat selection