Unique structural characteristics and evolution of a cluster of venom phospholipase A2 isozyme genes of Protobothrops flavoviridis snake

Protobothrops flavoviridis (Crotalinae) venom gland phospholipase A2 (PLA2) isozyme genes have evolved in an accelerated manner to acquire diverse physiological activities in their products. For elucidation of the multiplication mechanism of PLA2 genes, a 25,026 bp genome segment harboring five PLA2 isozyme genes was obtained from Amami-Oshima P. flavoviridis liver and sequenced. The gene PfPLA 2 encoded [Lys49]PLA2 called BPII, the gene PfPLA 4 neurotoxic [Asp49]PLA2 called PLA-N, the gene PfPLA 5 basic [Asp49]PLA2 called PLA-B, and PfPLA 1(ψ) and PfPLA 3(ψ) were the inactivated genes. The 5′ truncated reverse transcriptase (RT) elements, whose intact forms constitute long interspersed nuclear elements (LINEs), were found in close proximity to the 3′ end of PLA2 genes and named PLA2 gene-coupled RT fragments (PcRTFs). The facts that PcRTFs have the stem–loop and repetitive sequence in the 3′ untranslated region (UTR) which is characteristic of CR1 LINEs suggest that PcRTFs are the debris of P. flavoviridis ancestral CR1 LINEs, denoted as PfCR1s. Since the associated pairs of PLA2 genes and PcRTFs are arranged in tandem in the 25,026 bp segment, it is thought that an ancestral PLA2 gene-PfCR1 unit (PfPLA-PfCR1) which was produced by retrotransposition of PfCR1 by itself to the 3′ end of PLA2 gene duplicated several times to form a multimer of PfPLA-PfCR1, a cluster of PLA2 genes, in the period after Crotalinae and Viperinae snakes branched off. Recombinational hot spot of a 37 bp segment, named Scomb, was found in the region 548 bp upstream from the TATA box of PLA2 genes. Thus, it could be assumed that multiplication of PfPLA-PfCR1 occurred by unequal crossing over of the segment, -Scomb-PfPLA-PfCR1-Scomb-. The PfCR1 moieties were afterward disrupted in the 5′ portion to PcRTFs. The detection of two types of PcRTFs different in length which were produced by elimination of two definitive sequences in PfCR1 moiety possibly by gene conversion clearly supports such process but not multiplication of the PLA2 gene-PcRTF unit.