Gene, 128 (1993) 165-171 0 1993 Elsevier Science Publishers B.V. All rights reserved. 0378-l 119/93/$06.00 GENE 07136 Cloning and characterization of cDNAs that code for Na+-channel- blocking toxins of the scorpion zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Centruroides noxius Hoffmann (Recombinant DNA; hgtl 1 cDNA library; Southern blot genomic analysis; nucleotide sequence; polymerase chain reaction; toxin gene evolution; introns) Baltazar Becerril, Alejandra Vhzquez, Consuelo Garcia, Miguel Corona, Francisco Bolivar and Lourival D. Possani zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA lnstituto de Biotecnologia. Vniversidad National Autdnoma de Mkico, Cuernavaca, Morelas 62271, Mexico Received by L.K. Miller: 23 September 1992; Revised/Accepted: 19 January/20 January 1993; Received at publishers: 1 March 1993 165 SUMMARY With the purpose of studying the organization and characteristics of the genes that code for toxins present in the venom of the Mexican scorpion, Centruroides noxius Hoffmann (CnH), we prepared a hgt 11 cDNA library from the venom glands. Using specific oligodeoxyribonucleotides (oligos) designed according to known amino acid (aa) sequences of CnH toxins (STox), we detected several positive clones, determined their nucleotide (nt) sequences and deduced their aa sequences. A comparative analysis of these sequences with previously reported STox revealed that CnH cDNAs code for a family of very similar STox. The cDNA coding for a known STox, 11-10, was cloned. Additionally, three other complete (new) nt sequences were obtained for cDNAs encoding peptides similar to STox 1 from CnH or variants 2 and 3 from Centruroides sculpturatus Ewing. Southern blot genomic DNA analysis showed a minimum size of approxi- mately 600 bp as EcoRI fragments for elements of this family. PCR amplifications of CnH genomic DNA and hybridiza- tion of PCR products with specific probes indicated that the genomic structural regions that code for these genes do not contain introns, or at least not large introns. INTRODUCTION Of the 134 species and sub-species of Mexican scorpi- ons, only eight are dangerous to man (Dehesa-DBvila, 1989). Species which cause the most frequent cases of scorpion poisoning in Mexico belong to the Centruroides genus. They represent a serious health problem with more than 200000 people stung per year and a mortality rate Correspondence to: Dr. L.D. Possani, Instituto de Biotecnologia, UNAM, Av. Universidad 2001, Apartado Postal 510-3, Cuernavaca, Morelos 62271, Mexico. Tel. (52-73) 172799; Fax (52-73) 172388; e-mail: possani@pbr322.ceingebi.unam.mx Abbreviations: aa, amino acid(s); bp, base pair(s); cDNA, DNA comple- mentary to RNA, Clt, Centruroides limpidus tecomanus; Cngt, cDNA from CnH cloned into kgtl 1; CnH, Centruroides noxius Hoffmann; CsE, Centruroides sculpturatus Ewing; Css, Centruroides suffisus suffusus; of 700-800 people per year (D. Lbpez- Acuiia and A. Alagbn, communicated during the technical session of the Sociedad Mexicana de Salud Ptiblica, Mexico City, March 1979). Scorpion venoms constitute a rich source of low-M, peptides toxic to a variety of organisms including man (Miranda et al., 1970; reviews by Zlotkin et al., 1978; Possani, 1984). The most dangerous scorpion venoms so ctsd DNA, calf thymus sonicated and denatured DNA; Denhardt’s solu- tion, 0.02% each of bovine serum albumin/ficoll/polyvinylpyrrolidone; EtdBr, ethidium bromide; kb, kilobase or 1000 bp; N, any nucleoside; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCl/O.OlS M Na,citrate pH 7.6; SP, signal peptide( STox, scorpion toxin(s); T,, melting temperature (temperature at which 50% of the hybrids formed between two nucleic acids are dissociated).