Direct cloning of a target gene from a pool of homologous sequences: Complete cDNA sequence of a weak neurotoxin from cobra Naja kaouthia
Selective cloning of the cDNA coding for a weak neurotoxin (WTX) from cobra N. kaouthia including the 5′- and 3′-non-translated regions (NTR) is described. The known amino acid sequence of WTX was used together with the nucleotide sequence of a weak neurotoxin NNAM2 from cobra Naja atra, to design WTX-specific primers for direct amplification of an internal WTX cDNA fragment by RT - PCR. The sequence of the complete WTX cDNA was determined in sequencing runs on internal PCR products, cloned 3′- and 5′-RACE-fragments and several full-length cDNA clones. The cDNA coding sequence is in excellent agreement with the previously determined WTX amino acid sequence, has a high homology with other known weak toxin cDNAs, whereas even higher homology (up to 96%) with several classes of 3-finger toxins was detected in the 59 bp 3′-NTR consensus sequence. A possible function of the highly conserved nucleotide sequence elements is discussed.