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标题：Cloning and expression of human deoxycytidine kinase cDNA.
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作者：E G Chottiner ; D S Shewach ; N S Datta 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1991
卷号：88
期号：4
页码：1531-1535
DOI：10.1073/pnas.88.4.1531
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Deoxycytidine (dCyd) kinase is required for the phosphorylation of several deoxyribonucleosides and certain nucleoside analogs widely employed as antiviral and chemotherapeutic agents. Detailed analysis of this enzyme has been limited, however, by its low abundance and instability. Using oligonucleotides based on primary amino acid sequence derived from purified dCyd kinase, we have screened T-lymphoblast cDNA libraries and identified a cDNA sequence that encodes a 30.5-kDa protein corresponding to the subunit molecular mass of the purified protein. Expression of the cDNA in Escherichia coli results in a 40-fold increase in dCyd kinase activity over control levels. In dCyd kinase-deficient murine L cells, transfection with dCyd kinase cDNA in a mammalian expression vector produces a 400-fold increase over control in dCyd phosphorylating activity. The expressed enzyme has an apparent Km of 1.0 microM for dCyd and is also capable of phosphorylating dAdo and dGuo. Northern blot analysis reveals a single 2.8-kilobase mRNA expressed in T lymphoblasts at 5- to 10-fold higher levels than in B lymphoblasts, and decreased dCyd kinase mRNA levels are present in T-lymphoblast cell lines resistant to arabinofuranosylcytosine and dideoxycytidine. These findings document that this cDNA encodes the T-lymphoblast dCyd kinase responsible for the phosphorylation of dAdo and dGuo as well as dCyd and arabinofuranosylcytosine.