LOCALIZATION OF CROTAMINE GENE OF Crotalus durissus terrificus BY FLUORESCENT IN SITU HYBRIDIZATION (FISH)

Oguiura, N.(1), Svartman, M.(2), Batistic, R.F.(1), Almeida, T.M.B.(4), Rádis-Baptista, G.(3), Yamane, T.(3), Vianna-Morgante, A.(2)

(1)Laboratório de Herpetologia do Instituto Butantan, (2)Departamento de Biologia do Instituto de Biociências da USP, (3)Laboratório de Toxinologia Molecular e (4)Laboratório de Genética do Instituto Butantan.

Crotamine belongs to a closely related group of crotamine-like proteins, sharing up to 98% of similarity, present in most rattlesnake venoms. However, its presence and amount can vary according to the subspecies or the geographic locality of a given population. The quantity of this toxin ranges from 10 to 40% of dried venom. The crotamine gene of crotamine-plus Crotalus durissus terrificus consists of approximately 1800bp: three exons separated by a long phase-1 (900bp) and a short phase-2 (140bp) introns. Exon1 codifies the 5’-untranslated region and the first 19 amino acids of signal peptide, exon 2 codifies a total of 42 amino acids, 3 belonging to the signal peptide and 39 to the crotamine. Exon 3 codifies the last 3 amino acids of the mature toxin, the terminal lysine which is removed after post-translation processing and the 3’-untranslated region. In this work, we mapped the crotamine gene to metaphase chromosomes of C. d. terrificus. Chromosome preparations were obtained from bone marrow. The probe, MR20 phage DNA containing the crotamine gene, was labeled with biotin by nick translation. FISH was performed as described by Viegas-Péquignot (1992) with minor modifications and immunodetection was performed with avidin conjugated with FITC. The crotamine gene mapped to the telomeric region of the long arm of chromosome 2 in C. d. terrificus crotamine-plus karyotype. The intensity of the signal differed between the two homologues in all cells analyzed, what could be due to different number of copies of the crotamine gene. The variable amounts of crotamine found in the venom of crotamine-plus C. d. terrificus could reflect differences in gene copy numbers.

Financial support: FAPESP 99/02675-6

CORRESPONDENCE TO:

Nancy Oguiura, Rua Rumaica, 332 casa 6, São Paulo, SP, CEP: 05057-020, Brasil, Email: naniogui@terra.com.br