Accelerated evolution of snake venom phospholipase A₂ isozymes for acquisition of diverse physiological functions

The nucleotide sequences of two cDNAs and four genes encoding Trimeresurus gramineus venom gland phospholipase A₂ (PLA₂) isozymes were determined and compared internally and externally with those encoding Trimeresurus flavoviridis venom gland PLA₂ isozymes. It was reveald that the protein-coding regions are much more diversified than the 5' and 3' untranslated regions (UTRs) and the introns except for the signal peptide domain. The numbers of nucleotide substitutions per site (K(N)) for the UTRs and the introns were approximately one-quarter of the numbers of nucleotide substitutions per synonymous site (K(s)) for the protein-coding regions and were at the same level as the K(N) value of T. gramineus and T. flavoviridis TATA box-binding protein (TBP) genes, indicating that the protein-coding regions of PLA₂ isozyme genes are unusually variable and that the UTRs including the introns of venom gland PLA₂ isozyme genes have evolved at similar rate to those of non-venomous genes. The numbers of nucleotide substitutions per non-synonymous genes. The numbers of nucleotide substitutions per non-synonymous site (K(A)) values were close to or larger than the K(s) values for the protein-coding regions in venom gland PLA₂ isozyme genes, indicating that the protein-coding regions of snake venom gland PLA₂ isozyme genes have evolved via accelerated evolution. Furthermore, the evolutionary trees derived from the combined sequences of the 5' and 3' UTRs and the signal peptide domain of cDNAs were in accord with the consequences from taxonomy. In contrast, the evolutionary trees from the mature protein-coding region sequences of cDNAs and from the amino acid sequences showed random patterns. Estimations of nucleotide divergence of genes and the phylogenetic analysis reveal that snake venom group II PLA₂ isozyme genes have been evolving under adaptive pressure to acquire new physiological activities.