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

Published online by Cambridge University Press:  15 December 2006

F. M. SQUINA ,
A. L. PEDROSA ,
V. S. NUNES ,
A. K. CRUZ  and
L. R. O. TOSI
F. M. SQUINA
Affiliation:
Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brasil
A. L. PEDROSA
Affiliation:
Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brasil Current address: Departamento de Ciências Biológicas, Universidade Federal do Triângulo Mineiro, Avenida Frei Paulino, 30, 38025-5045, Uberaba – MG, Brasil.
V. S. NUNES
Affiliation:
Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brasil
A. K. CRUZ
Affiliation:
Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brasil
L. R. O. TOSI
Affiliation:
Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brasil
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Abstract

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.

Keywords

Leishmaniain vitro transpositiontargeted mutagenesistelomere
Type
Research Article
Information
Parasitology , Volume 134 , Issue 4 , April 2007 , pp. 511 - 522
Copyright
© 2006 Cambridge University Press

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