Affiliation:
1. Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, The Biomedical Center, S-751 23 Uppsala, Sweden,1 and
2. Center for Enzyme Research, Institute of Molecular Biology, University of Copenhagen, Copenhagen, Denmark2
Abstract
ABSTRACT
Deoxyribonucleoside salvage in animal cells is mainly dependent on two cytosolic enzymes, thymidine kinase (TK1) and deoxycytidine kinase (dCK), while
Escherichia coli
expresses only one type of deoxynucleoside kinase, i.e., TK. A bacterial whole-cell system based on genetically modified
E. coli
was developed in which the relevant bacterial deoxypyrimidine metabolic enzymes were mutated, and the cDNA for human dCK or TK1 under the control of the
lac
promoter was introduced. The TK level in extract from induced bacteria with cDNA for human TK1 was found to be 20,000-fold higher than that in the parental strain, and for the strain with human dCK, the enzyme activity was 160-fold higher. The in vivo incorporation of deoxythymidine (Thd) and deoxycytidine (dCyd) into bacterial DNA by the two recombinant strains was 20 and 40 times higher, respectively, than that of the parental cells. A number of nucleoside analogs, including cytosine arabinoside, 5-fluoro-dCyd, difluoro-dCyd, and several 5-halogenated deoxyuridine analogs, were tested with the bacterial system, as well as with human T-lymphoblast CEM cells. The results showed a close correlation between the inhibitory effects of several important cytostatic and antiviral analogs on the recombinant bacteria and the cellular system. Thus,
E. coli
expressing human salvage kinases is a rapid and convenient model system which may complement other screening methods in drug discovery projects.
Publisher
American Society for Microbiology
Subject
Infectious Diseases,Pharmacology (medical),Pharmacology
Cited by
11 articles.
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