Regional evolution of venom-gland phospholipase A(2) isoenzymes of Trimeresurus flavoviridis snakes in the southwestern islands of Japan

Conventional chromatographic analysis showed that phospholipase A(2) (PLA(2 )) isoenzymes of the venom of Trimeresurus flavoiridis (Habu snake) of Okin awa island are profoundly different in composition from those of T. flavovi ridis of Amami-Oshima and Tokunoshima islands. The most striking feature wa s that myotoxic [Lys(49)]PLA(2) isoenzymes, called BPI and BPII, which are expressed abundantly in the venoms of Amami-Oshima and Tokunoshima T. flavo viridis, are missing from the venom of Okinawa T. flavoviridis. Northern bl ot analysis of Okinawa T. flavoviridis venom-gland mRNA species showed the absence of BPI and BPII mRNA species. Analysis by single-stranded conformat ional polymorphism-PCR of venom-gland mRNA species of T. flavoviridis from three islands, with reference to five DNA species each encoding different P LA(2) isoenzymes from Tokunoshima T. flavoviridis venom gland, also suggest ed that BPI and BPII mRNA species are not expressed in Okinawa T. flavoviri dis venom gland. In contrast, genomic Southern blot analysis with a variety of probes showed that only the bands corresponding to the upstream and dow nstream regions of the genes for BPI and/or BPII can be detected in Okinawa T. flavoviridis. These results suggested that the genes for BPI and BPII i n Okinawa T. flavoviridis genome had been inactivated to form pseudogenes. Differently from Amami-Oshima and Tokunoshima T. flavovirdis genomic DNAs, PCR amplification of the segments of BPI and BPII genes between the 5' moie ty of second exon and the middle portion of second intron failed for Okinaw a T. flavoviridis genomic DNAs. In sequence analysis of the two segments in volving polymorphism between BPI and BPII genes, which are located in first exon and third exon, respectively, only one base was detected at the polym orphic positions for pseudogene in Okinawa T. flavoviridis genome. Based on these facts, it became evident for pseudogene that the upstream region of BPI gene down to the 5' moiety of second exon and the downstream region of BPII gene starting from the middle portion of second intron are in a linked form with a possible insertion. Such observations suggest that venom-gland genes for PLA(2) isoenzymes in T. flavoviridis snakes isolated for one to two million years have evolved independently. Their evolution is regional a nd seems, from several lines of consideration and observation, to be adapti ve to the environment.