Trypanosoma cruzi as a model system to study the expression of exogenous genes coding for polyamine-biosynthetic enzymes

ALGRANATI ID; CARRILLO C; GONZALEZ NS

Waterville Valley, NH

Conferencia; Gordon Research Conference on Polyamines; 2007

Gordon Research

<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:EN-US;} @page Section1 {size:595.3pt 841.9pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:35.4pt; mso-footer-margin:35.4pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> Trypanosoma cruzi as a model system to study the expression of exogenous genes coding for polyamine-biosynthetic enzymes.   Trypanosoma cruzi is the only eukaryotic cell unable to syntesize polyamines de novo because its genome contains neither ornithine decarboxilase (ODC) nor arginine decarboxylase (ADC) genes, presumably lost during evolution. Since T. cruzi behaves as a natural deletion mutant for ODC and ADC genes, we have used this parasite as a model system to study the regulation of a heterologous ODC gene expression. Transgenic T. cruzi obtained after transformation of wild type parasites with a recombinanat plasmid containing the ODC-coding region from Crithidia fasciculata became autotrophic for putrescine and at the same time susceptible to DFMO, an irreversible inhibitor of ODC. We have studied the emergence of DFMO-resistant T. cruzi after one.step selection of ODC-transformed parasites cultivated in the presence of high levels of the drug. Our results have indicated a duplication of the ODC-gene copy number in the drug-resistant cell line. The ODC transcripts and the corresponding translation products showed very significant increases (about 7 and 25-fold, respectively) in DFMO- resistant parasites, while the ODC enzymatic activity was only 5 times higher than in drug-sensitive T. cruzi. The unequal increases of ODC-protein and enzymatic activity in DFMO-resistant  protozoa strongly suggest that in addition to gene amplification and enhaced transcription and translation, the assembly of ODC-polypeptide chains into dimeric active enzyme molecules might also contribute to regulate the ODC-gene expression and the level of DFMO-resistance.