Mapping of a Leishmania major gene/locus that confers pentamidine resistance by deletion and insertion of transposable element

Revista do Instituto de Medicina Tropical de São Paulo

Adriano C. Coelho²  Luiz R. O. Tosi¹  Paulo C. Cotrim² 

[1] ,Universidade de São Paulo Instituto de Medicina Tropical São Paulo SP ,Brasil

关键词: Leishmania major;    Pentamidine;    Drug resistance;    Gene transfection;    Overexpression;    Gene mapping;    Transposable element;   

DOI  :  10.1590/S0036-46652004000200010

来源: SciELO

【 摘 要 】

Pentamidine (PEN) is an alternative compound to treat antimony-resistant leishmaniasis patients, which cellular target remains unclear. One approach to the identification of prospective targets is to identify genes able to mediate PEN resistance following overexpression. Starting from a genomic library of transfected parasites bearing a multicopy episomal cosmid vector containing wild-type Leishmania major DNA, we isolated one locus capable to render PEN resistance to wild type cells after DNA transfection. In order to map this Leishmania locus, cosmid insert was deleted by two successive sets of partial digestion with restriction enzymes, followed by transfection into wild type cells, overexpression, induction and functional tests in the presence of PEN. To determine the Leishmania gene related to PEN resistance, nucleotide sequencing experiments were done through insertion of the transposon Mariner element of Drosophila melanogaster (mosK) into the deleted insert to work as primer island. Using general molecular techniques, we described here this method that permits a quickly identification of a functional gene facilitating nucleotide sequence experiments from large DNA fragments. Followed experiments revealed the presence of a P-Glycoprotein gene in this locus which role in Leishmania metabolism has now been analyzed.

【 授权许可】

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