IDENTIFICATION BY EXTRACHROMOSOMAL AMPLIFICATION AND OVEREXPRESSION OF A ZETA-CRYSTALLIN NADPH-OXIDOREDUCTASE HOMOLOG CONSTITUTIVELY EXPRESSED IN LEISHMANIA SPP
IDENTIFICATION BY EXTRACHROMOSOMAL AMPLIFICATION AND OVEREXPRESSION OF A ZETA-CRYSTALLIN NADPH-OXIDOREDUCTASE HOMOLOG CONSTITUTIVELY EXPRESSED IN LEISHMANIA SPP
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DOI：
10.1016/0166-6851(94)90147-3
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发表时间：
1994-08-01
影响因子：
1.5
通讯作者：
CHANG, KP
中科院分区：
文献类型：
--
作者：
LIU, X;CHANG, KP
在衣霉素耐药利什曼原虫中，一个过表达36 kDa蛋白(P36)的基因通过转染和过度表达被定位在染色体外扩增子药物标记的上游区域。对该区域的完整测序显示了一个约1kb的单一开放阅读框。通过重组产物的免疫学特异性和与p36肽序列的同源性来验证克隆基因的真实性。该基因与真核细胞酒精脱氢酶家族成员在氨基酸水平上的序列相似性约为50%，基本上包括所有13个进化上保守的残基和一个核苷酸结合区。结构上和催化上重要的锌原子的结合配体都不存在，类似于Zeta-Crystal allin/NADPH：Quone氧化还原酶基因。与其一级序列的亲水性和核苷酸结合位点的存在一致，p36是一种可溶性分子，在105000×g时不沉淀，并与蓝色琼脂糖结合，只能用NADPH洗脱。P36基因在野生型的两个阶段都有结构性表达，并在所研究的所有利什曼原虫物种中保守，这表明其功能意义不同于进化上相关的同源物。
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 marker 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.