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.
Liu
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Department of Microbiology/Immunology, University of Health Sciences/Chicago Medical School, IL 60064.
Chang
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KP
D000426:Alcohol Dehydrogenase / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D003459:Crystallins / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D005784:Gene Amplification; D017125:Genes, Protozoan; D007891:Leishmania / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics*; D016366:Open Reading Frames; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
D003459:Crystallins; D017931:DNA Primers; D014415:Tunicamycin; D000426:Alcohol Dehydrogenase; D016660:NAD(P)H Dehydrogenase (Quinone)