Regulation of foreign ADC gen expression in T. cruzi

SERRA MP; CARRILLO C; GONZALEZ NS; ALGRANATI ID

Cataratas del Iguazú, Misiones, Argentina

Congreso; XL Reunión Anual de la SAIB; 2004

SAIB

FATE OF THE EXOGENOUS ORNITHINE DECARBOXYLASE GENE IN TRANSFECTED T. cruzi. Carrillo, C., Zadikian, C, González, NS. and Algranati, ID. Fundación – Instituto Leloir, Instituto de Investigaciones Bioquímicas – FCEyN, UBA y CONICET. P. Argentinas 435, Cap. Fed. (1405), Argentina. E-mail: ccarrillo@leloir.org.ar Polyamines, a group of aliphatic cations, play essential roles in cellular proliferation and differentiation in T. cruzi and other eukaryotic cells. The biosynthesis of polyamines in most of the organisms begins with the conversion of ornithine into putrescine, catalysed by ornithine decarboxylase (ODC). Previously, we have demonstrated that T. cruzi epimastigotes are auxotrophic for polyamines because they lack ODC gene. Then, we have transformed T. cruzi epimastigotes with the ODC gene form C. fasciculata, cloned into the vector p-Ribotex. The transgenic T. cruzi expressed ODC activity and becomes autotrophic for polyamines and sensitive to difluoro-methylornithine (DFMO). The presence of DFMO in transfected cultures elicited the selection of resistant parasites that showed ODC gene amplification, increased levels of specific mRNA and higher enzymatic activity. We analyzed the fate of the exogenous ODC gene through the time post-transfection in both sensitive and resistant parasites by PCR and PFGE. We have found that ODC gene was initially free as an episome into the cell (1-3 hs post-transfection), partially integrated into the parasite genome at 48 hs, and mostly or totally integrated after a week of transfection (this last point was also suggested by miniprep and hybridization with a specific probe of ODC). Restriction analyses of DNA from either sensitive or resistant T. cruzi cells indicated that one copy of the recombinant plasmid was integrated at each recombination event.