The best geneticists are smart but lazy; they use genetics to avoid laborious and tedious analyses, and in the process produce very elegant experiments, generating the maximum information with the minimum work. Our biochemical analysis of how sxy mRNA folds has been a lot of work but I don't feel that we've learned much more than we already knew (from genetics), and I'm thinking it's time to give genetics another chance.
We discovered (and named) sxy as the site of a mutation that causes cells to be competent when normal cells aren't. We now have five such mutations, but when we started we didn't even know if such mutations could arise. But it was relatively easy to find out, using a genetic selection experiment.
We took normal cells, gave them a small dose of a mutagen to increase the number of cells containing new mutations, and grew them under conditions where normal cells wouldn't become competent. Then we gave them some DNA with an antibiotic-resistance gene, and put them on agar plates containing that antibiotic. Normal cells die on these plates because, not being competent, they can't take up the antibiotic-resistance gene. So cells that do survive this selection are likely to have a mutation that changes how competence is regulated.
Note that we didn't have to test all the cells we started with, to see if they might have the kind of mutation we were looking for. We just gave them DNA, put them on the antibiotic plates, and let them find out for themselves whether they were the lucky one in a billion that had a mutation that kept them alive. That's what I mean by letting the bacteria do the work.
We've had these five 'hypercompetence' mutations for a long time (more than 10 years!). Because four of them wouldn't change the Sxy protein, but each of them would prevent a base pair from forming when two separate parts of the sxy mRNA came together, we hypothesized that RNA base pairing limits expression of the sxy gene.
A former grad student did the definitive test by making 'compensatory mutations'. These are mutations that restore the base pairing without restoring the wildtype DNA sequence. The most elegant one she made combined two independent hypercompetence mutations that affected the two partners in the same hypothesized base pair. The original mutations each prevented the base pair from forming, but the double mutant allowed the mutant bases to pair with each other. If base pairing wasn't important the double mutant should be even MORE hypercompetent that the single mutants, but if the single mutants caused hypercompetence by preventing base pairing, the double mutant should be much LESS hypercompetent, and that's what she found.
We now know more about the consequences of these mutations. They affect both the amount of sxy mRNA and the amount of Sxy protein made from each sxy mRNA, and they reduce the ability of purine nucleotide pools to limit sxy expression. But we don't know anything more about how the mutations do this, despite a lot of work trying to directly examine the folding of sxy mRNA.
So I think it's time to go back to genetics, and look for more mutations in sxy. We still have some of the original mutagenized cells in the freezer, and we could repeat the original selection for hypercompetent mutants on these, or on a fresh batch of mutagenized cells. We could also limit our mutagenesis to the sxy gene, by randomly mutagenizing a cloned or PCR-amplified sxy gene and then transforming it into normal cells and selecting for hypercompetence mutants. The results of such a mutant hunt could identify other parts of the mRNA that participate in regulatory base pairing, or that otherwise limit transcription or translation (perhaps by interacting with a purine).
Unfortunately selection for hypercompetent mutants won't give us the other kind of regulatory mutations, those that reduce sxy expression. In principle we could set up the reverse selection, a treatment that would kill all the cells that did take up DNA, but this would require having some sort of 'killer gene'. Such genes exist in other systems, but using the same principles to select for cells that can't become competent is likely to be very difficult or impossible.
But given that we are only interested in mutations in sxy (for this specific problem, not all our research), we can invest the time to do a genetic screen instead of a selection. That is, we'll have to do more of the work ourselves. We don't want to make random mutations throughout the genome, as many genes other than sxy are needed for DNA uptake. Techniques are available (kits!) to create specific mutations at any desired positions, and we can use these to create a set of many different mutations in sxy. We can then screen each mutant separately for the ability to take up DNA. Luckily the sxy gene is small, and we can use the results of our selection for hypercompetent mutants to guide this screen.
August 2015 Desktop Calendar
5 hours ago in Moss Plants and More