Cloning and nucleotide sequences of crotamine genes

Abstract

A cDNA library containing snake toxin genes was constructed in bacteriophage lambda by using mRNA isolated from the glands of the South American rattlesnake, Crotalus durissus terrificus. The first high-density screening of 400,000 plaques for crotamine-containing genes yielded over 800 positives when a labeled cDNA probe with sequence homology to crotamine was used. Four of these clones with insert sizes from 270 to 400 base pairs were chosen and their inserts subcloned into pGEM-3Z and sequenced. Nucleotide sequence analysis of the cloned cDNAs predicted the existence of multiple variants of the crotamine toxin. The different forms, identified from the DNA sequences, displayed discrepancies in amino acid sequence for crotamine when compared with previously published reports. Direct amino acid sequencing of commercially purified crotamine and CNBr fragments thereof confirmed the structures predicted by the nucleic acid sequences.