PCR-Based homology probing reveals a family of GABA receptor-like genes in Drosophila melanogaster

Abstract

A polymerase chain reaction (PCR)-based homology probing strategy was employed to screen Drosophila melanogaster genomic DNA for sequences encoding a conserved amino acid ‘signature motif’ known to be present in vertebrate GABA receptor and glycine receptor subunit genes. This approach yielded three discrete amplified sequence elements (designated LCCH1, LCCH2, and LCCH3) that contained open reading frames and > 40% amino acid sequence identity to the corresponding regions of vertebrate ligand-gated chloride channel genes. Genomic DNA clones corresponding to each element were isolated and sequenced, and predicted amino acid sequences corresponding to the second (M2) and third (M3) transmembrane domains of vetebrate genes were analyzed for identity or similarity to known sequences. LCCH1 was identical to the Rdl gene, a known GABA receptor subunit gene from D. melanogaster, whereas LCCH2 and LCCH3 were novel D. melanogaster sequences that exhibited structural similarity to other members of the ligand-gated chloride channel gene family. LCCH2 was equally divergent in M2 and M3 (46–49% amino acid identity) from all other known members of this family and may therefore represent a new subunit or receptor class within this family. LCCH2 was localized by in situ hybridization to cytogenetic region 75A on the left arm of chromosome 3. LCCH3 was closely related to mammalian (79% amino acid identity) and snail (96% amino acid identity) GABA receptor β subunits and may therefore be the homologue in D. melanogaster of this subunit class. LCCH3 was localized by in situ hybridization to cytogenetic region 13F on the X chromosome. Sequence analysis of genomic DNA clones corresponding to LCCH1, LCCH2, and LCCH3 showed that each transcription unit lacked at least one of the intron-exon splice junctions found to be conserved between mammalian and snail GABA receptor subunit genes. These results provide evidence for the existence of a diverse family of genes in D. melanogaster that are structurally related to the ligand-gated chloride channel gene family of vertebrates.