Elsevier

Gene

Volume 313, 14 August 2003, Pages 111-118
Gene

Molecular evolution and diversification of snake toxin genes, revealed by analysis of intron sequences

https://doi.org/10.1016/S0378-1119(03)00637-1Get rights and content

Abstract

The genes encoding erabutoxin (short chain neurotoxin) isoforms (Ea, Eb, and Ec), LsIII (long chain neurotoxin) and a novel long chain neurotoxin pseudogene were cloned from a Laticauda semifasciata genomic library. Short and long chain neurotoxin genes were also cloned from the genome of Laticauda laticaudata, a closely related species of L. semifasciata, by PCR. A putative matrix attached region (MAR) sequence was found in the intron I of the LsIII gene. Comparative analysis of 11 structurally relevant snake toxin genes (three-finger-structure toxins) revealed the molecular evolution of these toxins. Three-finger-structure toxin genes diverged from a common ancestor through two types of evolutionary pathways (long and short types), early in the course of evolution. At a later stage of evolution in each gene, the accumulation of mutations in the exons, especially exon II, by accelerated evolution may have caused the increased diversification in their functions. It was also revealed that the putative MAR sequence found in the LsIII gene was integrated into the gene after the species-level divergence.

Introduction

Snake venom is a mixture of various toxic proteins with different functions. Among them, α-neurotoxins (postsynaptic neurotoxins) have been investigated more than any other snake toxins. They are the major components of the Elapidae family snake venom and have characteristic three-dimensional (3D) structure called the three-finger-structure, that look like a duck's foot (Low et al., 1976, Tsernoglou and Petsko, 1976, Inagaki et al., 1978). They are classified into two groups, long and short chain neurotoxins, based on the number of amino acid residues. Cardiotoxins, κ-neurotoxins, and fasiculins are also famous toxic proteins isolated from Elapidae snakes, having the same 3D structures as α-neurotoxins. These toxins and α-neurotoxins are structurally relevant toxins, and are thought to have evolved from a common ancestor. Erabutoxins are short chain α-neurotoxins and are the main toxic components of Laticauda semifasciata (sea snake) venom. Three isoforms (Ea, Eb, and Ec) have been identified from the venom, and the cDNAs corresponding to these isoforms have been cloned (Tamiya and Abe, 1972, Tamiya et al., 1985, Obara et al., 1989). LsIII is a long chain α-neurotoxin isolated from L. semifasciata venom, and the cDNA corresponding to this neurotoxin and its isoform have been also cloned (Tamiya et al., 1999). The first information on the snake toxin gene (Ec) was reported by Fuse et al. (1990). This gene has three exons and two introns, and the length of the gene is 1.2 kbp. Subsequent studies have revealed the gene structures of various three-finger-structure toxin genes from land snakes, including α-neurotoxins (Chang et al., 1999, Afifiyan et al., 1999), κ-neurotoxins (accession nos. Y11768 and Y17699) and cardiotoxins (Chang et al., 1997a). However, the sizes of the genes are quite different from that of the Ec gene, the exon-intron organizations of the genes are the same as that of the Ec gene. The intron length of the Ec gene (197 bp) is the shortest among the known three-finger-structure genes (more than 1000 bp) (Chang et al., 1997a, Chang et al., 1997b, Chang et al., 1999). Comparing the homology of intron I sequences of 11 different snake toxin genes, Afifiyan et al. (1999) discussed that all of the absent regions in intron I of the Ec gene have been evolutionarily acquired in land snake toxin genes in the course of evolution.

In this report, the sequences and gene organizations of short and long chain neurotoxin genes from two species of genus Laticauda are reported. Based on the analysis of these genes, the evolution of three-finger-structure toxin genes is discussed and a detailed model is presented.

Section snippets

Cloning of Laticauda semifasciata three-finger-structure genes

Total genomic DNA was prepared from the liver of Laticauda semifasciata as described previously (Fujimi et al., 2002), and partially digested with Sau3AI. A L. semifasciata genomic library was constructed with a Predigested Lambda FIXII Vector cloning Kit (Stratagene, USA), according to the manufacture's protocol. Sense (LsIIIspeF: 5′-AAGGATCCTGAATCCTCATGACACTC-3′) and antisense (LsIIIspeR: 5′-AAGTCGACATTTCTGTTACGGATATGTG-3′) oligonucleotide primers were synthesized according to the nucleotide

Structures of α-neurotoxin genes from Laticauda semifasciata

Ten gene clones were isolated from genus Laticauda (Table 1). These clones have not been isolated from this genus previously. Five gene clones, encoding three isoforms (two clones of Ea, two Eb and one Ec) of erabutoxin (short chain α-neurotoxin), were isolated from L. semifasciata genomic library. The nucleotide sequences of these new five Etx gene clones and a Ec gene previously reported (accession no. X51410) (Fuse et al., 1990) were highly conserved with each other. There was more than 97%

Sequence analysis of Intron II

A comparison of the 11 three-finger-structure toxin genes from five snake species (four kinds of toxins) revealed that the nucleotide sequences of intron II were highly conserved. Among the 11 genes, sequence homologies of any two intron IIs were more than 82%. In contrast to the homology of intron IIs, many mutations were found in the exon II sequences, and their mutation rate clearly increased in exon II (Fig. 3A). Chang et al. (1999) pointed out that neurotoxins might have evolved via

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The nucleotide sequence data of the genes reported here have been submitted to the DDBJ sequence data bank. Erabutoxin a, AB098526 and AB098527; Erabutoxin b, AB098528 and AB098529; Erabutoxin c, AB098530; LsIII, AB098531; NLNTP, AB098532; Laticauda laticaudata long chain neurotoxin, AB098533; LlOKI-01, AB098534; LlOKI-10, AB098535.

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