The structure and sequence of the single calmodulin gene of Drosophila melanogaster
Doyle, Kevin Edward
Beckingham, Kathleen M.
Doctor of Philosophy
The work described involves the isolation and characterization of the gene encoding the calmodulin protein in the dipteran species, Drosophila melanogaster. The two major protein coding exons, designated 2 and 3, had been previously isolated. Together they encode 139 of a total of 148 amino acids. These exons were used to screen a D. melanogaster embryonic cDNA library in order to isolate clones containing sequences derived from the remaining exons. The clones obtained in this manner eventually led to the identification of two additional exons. Exon 4 contains the remaining protein coding sequences as well as the 3$\sp\prime$ untranslated trailer. Two different cDNA size classes were discovered, which, after sequencing, were found to result from the use of alternative polyadenylation signals. Northern blot analysis of polyadenylated transcripts isolated from 0 to 12 hour-old embryos confirmed the existence of two mRNAs of the approximate sizes 1.7 and 1.9 kilobases. Northern blot analysis of poly-A+ RNA isolated from developmentally staged animals revealed that the relative levels of these two transcripts vary in a stage-specific manner. Exon 1 was originally presumed to be the exon situated furthest 5$\sp\prime$, containing the translation initiation codon as well as all of the 5$\sp\prime$ untranslated leader. Primer extension and S1 nuclease analysis to determine the 5$\sp\prime$ limit of the transcription unit revealed the presence of an additional small 5$\sp\prime$ exon. Several cDNA libraries, both stage- and tissue-specific, were screened using sequences derived from exon 1 in order to isolate a clone complete at its 5$\sp\prime$ end. The sequence of the 50 residue exon 0 was therefore determined and used to locate the exon in genomic DNA. The genomic region containing the 5$\sp\prime$ flank of the transcription start site, exon 0, and the first intron of the gene was sequenced. Sequence comparisons of the 5$\sp\prime$ flanking region with those of other calmodulin genes were performed.
Molecular biology; Genetics; Entomology