Molecular Cloning and Cell Cycle-Specific Regulation of a Functional Human Thymidine Kinase Gene

• Published in: Proceedings of the National Academy of Sciences - PNAS 1983-09, Vol.80 (18), p.5588-5591
• Main Author: Bradshaw, Harvey D.
• Format: Article
• Language: English
• Subjects: Bacteriophages; Base Sequence; Cell Cycle; Cell lines; Cells; Cloning, Molecular; DNA; DNA - analysis; DNA - metabolism; DNA probes; gene expression; Gene Expression Regulation; Genes; HeLa Cells; Humans; L cells; man; restriction endonuclease mapping; thymidine kinase; Thymidine Kinase - genetics; transduction; Transformed cell line
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.80.18.5588

A functional thymidine kinase (TK; ATP:thymidine TK+EC 2.7.1.21) gene has been molecularly cloned from human DNA. The gene was rescued from a genomic library of TK-deficient mouse L cells transformed to the LTK-phenotype with total HeLa cell DNA. Of 14 overlapping clones, only one contained the intact human TK gene. The cloned recombinant bacteriophage carries a 16-kilobase insert derived entirely from human DNA and is capable of transforming TK+cells to 10 TK+with an efficiency of 106colonies per ng of DNA per LTK-cells. Restriction endonuclease mapping shows that the functional human TK gene is at least twice as long as that reported for chicken. A 1.6-kilobase Xho I/EcoRI fragment was subcloned and found to hybridize to a human mRNA of 1.5 kilobases. When introduced into LTK-cells, the cloned human TK gene is regulated in the cell cycle-specific manner characteristic of TK+mammalian cells. That is, TK activity in synchronized cells increases markedly with the onset of DNA synthesis. The signals governing the S-phase induction of TK activity reside within 16 kilobases of human DNA and are correctly interpreted by mouse cells.