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Crotamine is a small basic myotoxin peptide of Crotalus durissus venom, with beta-defensin scafold and variable concentration in individual venoms. The crotamine gene was mapped to the end of chromosome 2 and the signal intensity differed significantly between the two homologues. In contrast to crotamine, the paralogous crotasin gene is scarcely expressed in the venom glands. In this study, we analyzed the crotamine concentrations in the venoms of a total of 23 rattlesnakes from diverse Brazilian localities by ELISA as well as the copy number of both crotamine and crotasin genes by real-time PCR. Crotamine was found to constitute 5-29% of venom proteins varying greatly among individual animals. The crotamine gene exists from 1 to 32 copies per haploid genome, whereas the crotasin gene is present from 1 to 7 copies. Furthermore, we observed that the crotamine concentration and crotamine gene copy number are positively correlated (r(2)=0.68), implying the variation of crotamine in venom results from the variation of the gene copy number. Sequencing of 50 independent copies of crotamine and crotasin genes from four different rattlesnakes revealed the presence of six crotasin isoforms with a single amino acid difference from the original crotasin sequence, whereas only two additional crotamine isoforms were observed. Taken together, our results suggested that after duplication from a common ancestor gene, crotamine and crotasin may have diverged in such a way that the crotamine gene underwent repetitive duplication to increase its copy number, whereas the crotasin gene diversified its sequence.

Snakes are among the most successful groups of reptiles, numbering about 3,000 extant species. In spite of centuries of comparative anatomical and morphological studies, many aspects of snake systematics remain unsolved. To better understand the evolution and diversity of genomic characteristics in Serpentes, we analyzed online sequence data of mitochondrial and nuclear genes, as well as cytogenetic data and reviewed other genomic characteristics such as toxin genes. After the analysis of the whole-genome and chromosomal organization, we find that: (1) cytogenetic comparisons could provide a useful tool to investigate intergeneric and tribal relationships within the extremely diverse neotropical xenodontine snakes; (2) toxin genes could also help to understand snake evolution if special care is taken to choose the sequences because of the difficulty in avoiding paralogs; (3) snake phylogeny based on mitochondrial genome sequences is largely consistent with the relationship obtained using nuclear genes., (Copyright 2010 S. Karger AG, Basel.)