Shuttle mutagenesis and targeted disruption of a telomere-located essential gene of Leishmania

Autor(es)

Squina‚ F. M. ; Pedrosa‚ A. L. ; Nunes‚ V. S. ; Cruz‚ A. K. ; Tosi‚ L. R. O.

• Tipo de documento: Artigo Científico
• Fonte: Parasitology; v. 134, n. 04, p. 511-522, Apr. 2007.
• Área do conhecimento: Ciências Biológicas - Bioquímica
• Assunto(s): Protozoa Leishmania braziliensis Estruturas cromossômicas Telômero

Resumo

Leishmania mutants have contributed greatly to extend our knowledge of this parasite's biology. Here we report the use of the mariner in vitro transposition system as a source of reagents for shuttle mutagenesis and targeted disruption of Leishmania genes. The locus-specific integration was achieved by the disruption of the subtelomeric gene encoding a DNA-directed RNA polymerase III subunit (RPC2). Further inactivation of RPC2 alleles required the complementation of the intact gene, which was transfected in an episomal context. However, attempts to generate a RPC2 chromosomal null mutant resulted in genomic rearrangements that maintained copies of the intact locus in the genome. The maintenance of the RPC2 chromosomal locus in complemented mutants was not mediated by an increase in the number of copies and did not involve chromosomal translocations, which are the typical characteristics of the genomic plasticity of this parasite. Unlike the endogenous locus, the selectable marker used to disrupt RPC2 did not display a tendency to remain in its chromosomal location but was targeted into supernumerary episomal molecules. (AU)

• Processo FAPESP: 01/02527-9 - Nocaute sistematico de genes em (leishmania major). utilizacao de sistema "mariner mos1" de transposicao "in vitro".
• Beneficiário: Fábio Márcio Squina
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 99/12403-3 - Do genoma à biologia de Leishmania: uma abordagem compreensiva
• Beneficiário: Angela Kaysel Cruz
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 98/09805-0 - Análise genômica funcional e comparativa em Leishmania ssp
• Beneficiário: Luiz Ricardo Orsini Tosi
• Modalidade de apoio: Auxílio à Pesquisa - Jovens Pesquisadores