Modulation of oat arginine decarboxylase gene expression and genome organization in transgenic Trypanosoma cruzi epimastigotes.
We have previously demonstrated that wild-type Trypanosoma cruzi epimastigotes 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 approximately 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 almost completely for several weeks and, finally, when the expression of the ADC gene became stable, two or more copies of the transforming plasmid arranged in tandem were integrated into a parasite chromosome (1.4 Mbp) bearing a ribosomal RNA locus. The sensitivity of transcription to alpha-amanitin strongly suggests involvement of the protozoan RNA polymerase I in the transcription of the exogenous ADC gene.
Serra
María P
MP
Fundación Instituto Leloir, Buenos Aires, Argentina.
Carrillo
Carolina
C
González
Nélida S
NS
Algranati
Israel D
ID
D000546:Amanitins / Q000494:pharmacology; D000818:Animals; D018554:Avena / Q000201:enzymology* / Q000235:genetics; D002262:Carboxy-Lyases / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D016678:Genome; D030781:Organisms, Genetically Modified; D012318:RNA Polymerase I / Q000037:antagonists & inhibitors / Q000378:metabolism; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D013045:Species Specificity; D013997:Time Factors; D014158:Transcription, Genetic / Q000502:physiology; D014349:Trypanosoma cruzi / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism
D000546:Amanitins; D012333:RNA, Messenger; D012318:RNA Polymerase I; D002262:Carboxy-Lyases; C019722:arginine decarboxylase