Identification by extrachromosomal amplification and overexpression of a zeta-crystallin/NADPH-oxidoreductase homologue constitutively expressed in Leishmania spp.

A gene which overexpresses a 36-kDa protein (p36) in tunicamycin-resistant Leishmania was mapped by transfection and overexpression to the upstream region of the drug maker in the extrachromosomal amplicon. Complete sequencing of this region revealed a single open reading frame of about 1 kb. Authenticity of the cloned gene is verified by immunologic specificity of its recombinant products and sequence identity with a p36 peptide. The gene shares an overall sequence similarity of about 50% with members of the eukaryote alcohol dehydrogenase family at the amino acid level, including essentially all 13 evolutionarily conserved residues and a nucleotide-binding domain. The binding ligands for both structurally and catalytically important zinc atoms are absent, similar to the zeta-crystallin/NADPH:quinone oxidoreductase gene. Consistent with hydrophilicity of its primary sequence and the presence of a nucleotide binding site, p36 is a soluble molecule non-sedimentable at 105,000 x g and binds Blue Sepharose, elutable only with NADPH. The p36 gene is expressed constitutively in both stages of the wild-type and is conserved among all Leishmania species examined, suggestive of its functional significance different from evolutionarily related homologues.