tailieunhanh - Báo cáo khoa học: Modulation of oat arginine decarboxylase gene expression and genome organization in transgenic Trypanosoma cruzi epimastigotes

We have previously demonstrated that wild-typeTrypanosoma cruziepi-mastigotes lack arginine decarboxylase (ADC) enzymatic activity as well as its encoding gene. A foreign ADC has recently been expressed in T. cruzi after transformation with a recombinant plasmid containing the complete coding region of the oatADCgene. | iFEBS Journal Modulation of oat arginine decarboxylase gene expression and genome organization in transgenic Trypanosoma cruzi epimastigotes Maria P. Serra Carolina Carrillo Nélida S. Gonzalez and Israel D. Algranati Fundacion Institute Leloir Buenos Aires Argentina Keywords free episome genome organization heterologous ADC gene expression plasmid integration Trypanosoma cruzi transformation Correspondence I. D. Algranati Fundacion Instituto Leloir Avenue Patricias Argentinas 435 1405 Buenos Aires Argentina Fax 5411 5238 7501 Tel 5411 5238 7500 E-mail ialgranati@ Received 24 November 2005 accepted 12 December 2005 doi We have previously demonstrated that wild-type Trypanosoma cruzi epi-mastigotes lack arginine decarboxylase ADC enzymatic activity as well as its encoding gene. A foreign ADC has recently been expressed in T. cruzi after transformation with a recombinant plasmid containing the complete coding region of the oat ADC gene. In the present study upon modulation of exogenous ADC expression we found that ADC activity was detected early after transfection subsequently it decreased to negligible levels between 2 and 3 weeks after electroporation and was again detected w 4 weeks after electroporation. After this period the ADC activity increased markedly and became expressed permanently. These changes of enzymatic activity showed a close correlation with the corresponding levels of ADC transcripts. To investigate whether the genome organization of the transgenic T. cruzi underwent any modification related to the expression of the heterologous gene we performed PCR amplification assays restriction mapping and pulse-field gel electrophoresis with DNA samples or chromosomes obtained from parasites collected at different time-points after transfection. The results indicated that the transforming plasmid remained as free episomes during the transient expression of the foreign gene. Afterwards the free plasmid disappeared