Characterisation of genes involved in oogenesis in Drosophila melanogaster

• Main Author: MacIver, Ian Bryce
• Published: University of Edinburgh 1997
• Subjects: 591.35
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.654407

We isolated a cDNA with an in situ hybridisation expression pattern that showed gene expression very early in oogenesis with rapid localisation of the transcript to the developing oocyte. Egg chambers at mid-stages of oogenesis exhibited localisation of the transcript to the anterior region of the developing oocyte while late stages of oogenesis showed strong expression in the nurse cells and loss of localisation in the oocyte. A number of localised transcripts are known to have crucial roles in axis determination in the developing oocyte and embryo. Work presented in this thesis shows that this gene encodes a class V conventional myosin. Examples of this class have been described in mouse and yeast and there is evidence that they are involved in vesicle trafficking. Additional cDNAs were isolated and sequenced although no full length cDNAs were found. A PCR and genomic DNA cloning strategy was used to obtain most of the remaining coding sequence. Hybridisation to polytene chromosomes from the salivary glands of third instar larvae located the gene to the 43BC region on the right arm of the second chromosome. Results mapping the class V myosin to one of three genes in this region will be presented. A region from the carboxyl terminus of the gene was expressed in Escherichia coli with the recombinant protein used to raise antibodies in rabbits. Preliminary results show these antibodies detecting protein from ovary tissue. A cDNA encoding a putative sodium-dependent inorganic phosphate co-transporter was isolated and sequenced in full. A PCR approach designed to amplify from the class V myosin ATP binding site identified sequence from a previously uncharacterised gene showing similarity to an ATPase required for meiotic spindle formation in <I>Caenorhadbitis elegans. </I>The implications of these findings and the future directions this work can take are discussed.