PCR-based phylogenetic walking: Isolation of para-homologous sodium channel gene sequences from seven insect species and an arachnid

Abstract

The voltage-sensitive sodium channel is the site of action of two important classes of insecticides, DDT and pyrethroids. We recently used the polymerase chain reaction (PCR) to amplify sodium channel gene sequences in the house fly genome and showed the direct use of the amplification product as a conspecific hybridization probe. This report describes the use of this method to isolate sodium channel gene sequences from seven insect species (representing four orders) and an arachnid, thereby demonstrating its general utility for quickly and efficaciously isolating homologous sequences from distantly related species. DNA sequence analysis of the amplified products revealed that all but a few were homologous to the IS5-6 region of the para gene of Drosophila melanogaster, the region upon which the design of the target primers was based. Although unique nucleotide sequences were obtained for each species (with some species having more than one sequence variant), the inferred amino acid sequences of the 15 residue stretch between the amino acid target sequences were found to be completely conserved or to contain a single conservative replacement of serine with threonine. We suggest that this methodology now permits specific knowledge obtained from molecular genetic analysis of D. melanogaster to be applied straightforwardly to the characterization of many genes and the primary products of their expression in other insect specs.