Identifying the Molecular Basis of the fs(1)234 Phenotype
Christa Ventresca, Vassar College ’17 and Prof. Nancy Pokrywka
The fs(1)234 mutation causes sterility in female Drosophila melanogaster (Swan et al., 2001), because actin cell membranes within the germline of egg chambers break down during oogenesis between stages 2 and 9. Previous deletion mapping reduced the portion of the DNA containing this mutation to several candidate genes. The genes examined in this project included Rab18, Sirt4, CG4119, Tre1, and RpL35, which each code for proteins, and one noncoding region, CR45520. Each of these genes are located within the boundaries of the deletion mapping, were examined for mutations within the DNA, and were examined for mutations within the RNA levels as well. We amplified candidate regions by PCR and obtained the nucleotide sequence of the genes, comparing the fs(1)234 sequence with a control fs(1)186 sequence, as well as a reference sequence from FlyBase. Differences between the sequences would indicate a genetic mutation causing a change in Drosophila oogenesis. In addition to this, quantitative polymerase chain reaction (qPCR) technique was used to investigate differences in the RNA production of the genes as well. qPCR technique involves isolating the RNA from fs(1)234 and fs(1)186 flies, reverse transcribing the RNA to obtain cDNA, and then amplifying the sequence while measuring its output. This indicates the levels of RNA within the flies, and substantial differences in output would indicate a change in transcription levels altering the process of oogenesis. By the end of this project, the majority of the DNA within this region was sequenced, but no nucleotide changes or mutations were revealed. Similarly, the qPCR data found did not show significant changes in the expression of the genes.