Direct isolation of the functional human thymidine kinase gene with a cosmid shuttle vector.

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We have developed a new recombinant DNA cloning system to isolate directly the functional unit of the human thymidine kinase (TK) gene. The system utilizes a cosmid vector that can shuttle cloned DNA sequences between bacteria and mammalian cells. A complete human cosmid library was constructed and DNA from the total library was transfected to mouse L cells deficient in TK (LTK-) by calcium phosphate precipitation. The transfected cells were then selected with hypoxanthine/aminopterin/thymidine (HAT) medium, and one HAT-resistant cell clone was isolated. This cell line became resistant to HAT selection by acquiring the TK gene derived from the human cosmid library. As the cosmid vector contains the cohesive ends of the bacteriophage, we could directly retrieve the human DNA sequences from the transformed mouse L cells. Total DNA from the transformed TK+ L cells was packaged in vitro with lysogenic bacterial extracts and used to infect Escherichia coli. One of the two recombinant cosmids isolated contained a 43.8-kilobase human DNA insert and was capable of converting TK- L cells to the TK+ phenotype in both acute and stable transformation assays. Thus, we have isolated the functional human TK gene in this recombinant cosmid. The gene was further localized on a 14.5-kilobase BamHI DNA fragment, and it transcribed a mature mRNA of about 1,500 nucleotides. This method of gene isolation has several special features: (i) an intact structural gene can be cloned directly based on its function without knowledge of its amino acid or nucleotide sequence; (ii) the functional gene sequences can be recovered faster and more efficiently than with the usual DNA transfection method; and (iii) in conjunction with cell-sorting techniques, this method can be used to clone genes encoding cell surface markers.

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