Tài liệu Báo cáo khoa học: Modulation of oat arginine decarboxylase gene expression and genome

Modulation of oat arginine decarboxylase gene expression

and genome organization in transgenic Trypanosoma cruzi

epimastigotes

Marı´a P. Serra, Carolina Carrillo, Ne´lida S. Gonza´lez and Israel D. Algranati

Fundacio´n Instituto Leloir, Buenos Aires, Argentina

Trypanosoma cruzi, the etiological agent of Chagas’

disease, is a parasitic protozoan with a digenetic life

cycle involving an insect vector and a mammalian

host. The parasite undergoes major morphological

and biochemical changes during the different stages of

its life cycle. The epimastigote form is noninfective and

proliferates extracellularly in the insect gut where it

differentiates into metacyclic trypomastigotes, which

can then infect the mammalian host cells and replicate

intracellularly after transforming into amastigotes [1–4].

Epimastigotes from different wild-type strains of

T. cruzi are able to grow continuously in vitro in a rich

culture medium [5], but proliferation stops after a few

passages in a semidefined medium, which contains only

traces of polyamines [6,7]. T. cruzi remain viable for

several weeks in the defined medium and are able to

resume normal growth only upon the addition of exo￾genous polyamines to the culture [7]. These results

confirm previous reports from our and other laborat￾ories indicating that T. cruzi epimastigotes are unable

Keywords

free episome; genome

organization; heterologous ADC gene

expression; plasmid integration;

Trypanosoma cruzi transformation

Correspondence

I. D. Algranati, Fundacio´n Instituto Leloir,

Avenue Patricias Argentinas 435 (1405),

Buenos Aires, Argentina

Fax: +5411 5238 7501

Tel: +5411 5238 7500

E-mail: [email protected]

(Received 24 November 2005, accepted

12 December 2005)

doi:10.1111/j.1742-4658.2005.05098.x

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

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 chromo￾somes obtained from parasites collected at different time-points after trans￾fection. The results indicated that the transforming plasmid remained as

free episomes during the transient expression of the foreign gene. After￾wards, 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 integra￾ted into a parasite chromosome (1.4 Mbp) bearing a ribosomal RNA locus.

The sensitivity of transcription to a-amanitin strongly suggests involvement

of the protozoan RNA polymerase I in the transcription of the exogenous

ADC gene.

Abbreviations

ADC, arginine decarboxylase; G418, geneticin; ODC, ornithine decarboxylase; PFGE, pulse-field gel electrophoresis.

628 FEBS Journal 273 (2006) 628–637 ª 2006 The Authors Journal compilation ª 2006 FEBS