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.
Oustitch
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Eppendorf 5 Prime, Inc., 6531 Gunbarrel Av., Boulder, CO 80301, USA.
Peters
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Utkin
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Tsetlin
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004546:Elapid Venoms / Q000737:chemistry / Q000235:genetics*; D017815:Elapidae / Q000235:genetics* / Q000378:metabolism; D015723:Gene Library; D008969:Molecular Sequence Data; D009498:Neurotoxins / Q000235:genetics* / Q000378:metabolism
D004546:Elapid Venoms; D009498:Neurotoxins