The experimental work of this thesis was designed to provide the lab with tools for quantifying rates of recombination in males and females of two Drosophila species for a broader selection project. The mei-P22 gene is known to play a major role in the meiotic recombination process in Drosophila females, through initiation of DSB formation. It was selected as a marker for recombination. Among the many species of Drosophila only one, D. ananassae, has been known to show recombination in males. Using this particular species of flies and comparing it with the well-studied D. melanogaster could provide the lab with a way to study the effects of sexual selection on rates of recombination and help test the hypothesis that sexual selection plays a role in lowering rates of recombination in males.
It is also worth noting that not all strains of D. ananassae have shown male recombination. Consequently, we contacted a Dr. Muneo Matsuda in Japan, who possesses strains of D. ananassae known to have shown recombination in males, and asked him to send samples for us to use. However, due to difficulties in communicating between the labs, strict customs rules and regulations, and now the earthquake, we have been unable to obtain strains with male recombination. For the purposes of working out the techniques described here, we have used strains of D. ananassae that we obtained from the U.S. stock center. It is not known whether they have male meiotic recombination.
As a necessary first step of the experiment the sequence of mei-P22 in D. melanogaster and that of its putative homolog in the D. ananassae species were identified. In order to design primers, these sequences were compared to each other to find a region of greatest homology between the two. The last 252 bp region was identified as having the highest homology between the two species and was used to design primers. Following a DNA extraction and a PCR reaction using the designed primers, the products of the PCR reaction were run on a gel, the samples were purified from the gel and sent off for sequencing. The results of DNA sequencing confirmed that the sequences of mei-P22 in both species and within all strains had a high degree of homology with those available on databases. These results allowed us to verify that the correct sequence of mei-P22 was being studied and that the designed primers were functional and ready to be used in the RT-PCR reaction.
As the second main step of the experiment RNA was extracted from males and females of each species. In general, even though the RNA yields from females typically fell within an acceptable concentration range, those obtained from males were significantly lower. In order to eliminate this problem, flies were raised on yeast for a period of 4 to 6 days. Even after feeding flies on the yeast diet, the RNA yields, especially for males, did not improve substantially. As a next step, we used an alternative RNA extraction protocol that eliminated the use of QIAshredder spin columns, which were used in initial attempts. This change could potentially prevent loss of significant amounts of RNA through the use of the shredders. Despite our efforts to increase male RNA yields, even the alternative RNA extraction protocol did not seem to increase male yields significantly, although female concentrations of RNA more than doubled in some cases. Another significant step of the RNA extraction was to add a DNase treatment to the extraction procedure in order to remove any traces of DNA and thus to ensure that primers were using RNA as their template and not DNA during the following RT-PCR reactions. Unfortunately, this step does not seem to have had the expected effect, and further work is necessary to obtain DNA-free RNA extracts.
In order to test whether the mei-P22 gene was expressed in males and females, the RNA samples were used in an RT-PCR reaction along with our designed primers. A crucial step of the RT-PCR experiment was to add a set of controls to the reaction to test for DNA and other sources of contamination, as well as to ensure that the reaction setup and the procedure functioned properly. After visualization of the RT-PCR results, bands were observed in both DNA contamination and in some of the negative control lanes. This was an indication that our samples and possibly some reagents were most likely contaminated. The lanes corresponding to both mei-P22 and Adh positive controls also contained secondary bands of 200 bp size that could not be explained. We suspect that this last result might have been cross contamination between primers. Even though we had verified functionality of the mei-P22 primers and that the correct sequence of the gene was being used, the results indicate the possibility of several technical errors. Given the challenges posed by the molecular biology techniques throughout the experiment and after running out of time, we were not ultimately able to test the expression patterns of males and females of the two species.