Analysis of evolution and phylogeny of snakes of Trimeresurus genus in the southwestern islands of Japan based on structural information of venom-gland isozymes

1.竹叶青蛇（Trimeresurusflavoviridis）蛇毒磷脂酶A_2（PLA_2）同工酶的基因突变，产于奄美大岛、德之岛和冲绳的竹叶青蛇蛇毒含有碱性[Asp^<49>] PLA_2，分别称为PLA-B '、PLA-B和PL-Y。它们具有1-4个氨基酸取代，被认为是由于在隔离环境下发生了突变。从奄美大岛黄绿胸苷蛇毒中分离到一种神经毒素PLA_2，命名为PLA-N。Tokunoshima T.flavoviridis毒液也含有这种同工酶，但冲绳T.flavoviridis毒液含有一种氨基酸取代的同系物，称为PLA-N（O）。这也是一个基因突变的例子。据推测，冲川岛首先从原来的岛屿分裂出来，然后其余的岛屿分裂成奄美大岛和德之岛。从日本西南诸岛竹叶青蛇（Trimeresurus）蛇毒中分离到两种新的PLA_2同工酶（[Arg^<49>]PLA_2），分别命名为TeBP（R）-I和TeBP（R）-II。它们在位置3处是多态的，TeBP（R）-I的瓦尔和TeBP（R）-II的Ile。从其毒腺中克隆了编码[Asp 49]PLA_2的cDNA，命名为TePLA_2。从tokarensis蛇毒中纯化出中性的[Asp 49]PLA_2。然而，它的毒腺cDNA文库的生产失败。本文对日本西南诸岛竹叶青蛇属（T.tokarensis除外）的蛇毒PLA_2同工酶进行了系统发育树的构建。在[Lys 49]PLA_2型的分支中，线虫的TeBP（R）-I和TeBP（R）-II与黄病毒线虫的TfBPI和TfBPI相比，更接近于TmK 49 PLA_2。在PLA_2型中，TePLA_2比TfPLA_2更接近TmPLA_2。因此，秀丽隐杆线虫是比黄病毒隐杆线虫更接近黑鳞隐杆线虫的物种。此外，明确指出，冲绳锥虫和禾谷锥虫是与黄绿锥虫、秀丽锥虫和粘鳞锥虫相当远的物种。

1. Interisland mutation of Trimeresurus flavoviridis venom phospholipase A_2 (PLA_2) isozymes.The venoms of T.flavoviridis snakes from Amami-Oshima, Tokunoshima and Okinawa islands contain basic [Asp^<49>]PLA_2s called PLA-B', PLA-B and PL-Y, respectively. They have 1-4 amino acid substitutions that are considered due to occurrence of interisland mutation under isolated environments. A neurotoxic PLA_2 isolated from Amami-Oshima T.flavoviridis venom was named PLA-N. Tokunoshima T.flavoviridis venom also contained this isozyme but Okinawa T.flavoviridis venom contains one amino acid-substituted homolog, called PLA-N(O). This is also a example of interisland mutation. It is speculated that Okiawa island first splitted from the original island and then the rest was disunited into Amami-Oshima and Tokunoshima.2. Evolution and phylogeny of snakes of Trimeresurus genus in the southwestern islands of JapanTwo novel PLA_2 isozymes with Arg at position 49 ([Arg^<49>]PLA_2) were isolated from T.elegans venom and named TeBP(R)-I and TeBP(R)-II. They are polymorphic at position 3, Val for TeBP(R)-I and lle for TeBP(R)-II. A cDNA encoding [Asp49]PLA_2 called TePLA_2 was cloned from its venom gland. A neutral [Asp49]PLA_2 was purified to homogeneity from T.tokarensis venom. However, production of its venom gland cDNA library failed. A phylogenetic tree was constructed for venom PLA_2 isozymes of Trimeresurus genus, except for T.tokarensis, in the southwestern islands of Japan. In the branch of [Lys49]PLA_2-type group, TeBP(R)-I and TeBP(R)-II of T.elegans are much closer to TmK49PLA_2 of T.mucrosquamatus than TfBPI and TfBPI of T.flavoviridis. In PLA2-type group, TePLA_2 is closer to TmPLA_2 than TfPLA2. Thus, T.elegans is the species closer to T.mucrosquamatus than T.flavoviridis. In addition, it is clearly noted that T.okinavensis and T.gramineus are the species fairly distant from T.flavoviridis, T.elegans and T.mucrosquamatus.