Developmental regulation of alternative exon usage in the house fly Vssc1 sodium channel gene.

Abstract

Sequence analysis of cDNA clones amplified by PCR from house fly ( Musca domestica L.) Vssc1 voltage-sensitive sodium channel alpha subunit transcript templates identified 11 putative alternatively spliced exons. Nine of these corresponded to the 7 optional exons (designated a, b, e, f, h, i, and j) and 2 mutually exclusive exons (designated c/d) identified previously in the orthologous para sodium channel alpha subunit genes of Drosophila melanogaster and Drosophila virilis, whereas two segments represented new mutually exclusive exons in Vssc1 (designated k/l) located in a region not previously identified as a site of alternative splicing in para. Diagnostic PCR assays on individual Vssc1 cDNA templates detected the presence or absence of each putative alternative exon in multiple partial cDNAs (42-96 individual clones per cDNA pool) from newly emerged first instar larvae, pupae, day 1 adult heads, and day 1 adult bodies. Exons h and i were present in all cDNA clones from all developmental stages. Exon d was also present in all clones from all developmental stages that encoded full-length amino acid sequences; however, 1 of 42 clones from adult head contained an exon c-like segment in which the coding sequence was terminated by a premature stop codon. In contrast, the frequencies of exons a, b, e, f, j, k, and l differed between developmental stages and adult anatomical regions. Analysis of the Vssc1 region containing alternative exons a, b, c/d, e, f, h, and i as a single amplified cDNA segment identified nine Vssc1 splice variants involving these exons. The splice variant containing exons a, d, h, and i was the most abundant form in all cDNA pools examined, but the observed patterns of splice variant expression were specific to each developmental stage and adult anatomical region. Our results document the strong conservation of alternative exon location and structure between the Vssc1 gene of the house fly and the para gene of D. melanogaster but identify marked differences in exon usage between these species.