IDENTIFICATION BY EXTRACHROMOSOMAL AMPLIFICATION AND OVEREXPRESSION OF A ZETA-CRYSTALLIN NADPH-OXIDOREDUCTASE HOMOLOG CONSTITUTIVELY EXPRESSED IN LEISHMANIA SPP

A gene which overexpresses a 36-kDa protein (p36) in tunicamycin-resis tant Leishmania was mapped by transfection and overexpression to the u pstream region of the drug marker in the extrachromosomal amplicon. Co mplete sequencing of this region revealed a single open reading frame of about 1 kb. Authenticity of the cloned gene is verified by immunolo gic specificity of its recombinant products and sequence identity with a p36 peptide. The gene shares an overall sequence similarity of abou t 50% with members of the eukaryote alcohol dehydrogenase family at th e amino acid level, including essentially all 13 evolutionarily conser ved residues and a nucleotide-binding domain. The binding ligands for both structurally and catalytically important zinc atoms are absent, s imilar to the zeta-crystallin/NADPH:quinone oxidoreductase gene. Consi stent 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 homo logues.