We previously isolated and characterized the structure of murine thymidine kinase (tk) genomic and cDNA sequences to begin a study designed to identify regions of the tk gene important for regulated expression during the transition of cells from Go to a proliferating state. In this report, we describe the stable transfection of the cloned gene into L-M(TK-) cells and show that both thymidine kinase (TK) enzyme activity and DNA synthesis increase in parallel when transfectants in Go arrest are stimulated by serum. To define promoter and regulatory regions more precisely, we have constructed a series of tk minigenes and have examined their expression in stable transfectants after serum stimulation. We have identified a 291-base-pair DNA fragment at the 5' end of the tk gene that has promoter function, and we have determined its sequence. In addition, we have found that DNA sequences which mediate serum-induced expression of TK are transcribed, since expression of the murine tk cDNA, fused to a promoter from either the murine tk gene, the simian virus 40 early region, or the herpes simplex virus tk gene, is stimulated by serum. Our constructs also reveal that the murine tk polyadenylation signal is not required for regulation, nor is most of the 3' untranslated region. RNA dot blot analysis indicates that murine cytoplasmic tk mRNA levels always parallel TK enzyme activity. Nuclear runon transcription assays show less than a 2-fold increase in transcription from the cloned tk gene in serumstimulated transfectants, but an 11-fold increase in mouse L929 cells, which are inherently TK+. These results taken together suggest that the murine tk gene is controlled in serum-stimulated cells by a transcriptional mechanism influenced by DNA sequences that flank tk and also by a posttranscriptional system linked to gene sequences that are transcribed.Thymidine kinase (TK; EC 2.7.1.21) participates in the salvage pathway for pyrimidine nucleotide biosynthesis, catalyzing the phosphorylation of thymidine (TdR) to form thymidine 5'-monophosphate. Two major versions of the enzyme have been characterized in mammalian cells; one is found in the cytoplasm, and the other is found in the mitochondria (8, 27). They are encoded by genes located on different autosomes (15,30,37,63), and although they catalyze the same basic phosphorylation reaction, the biochemical characteristics of each enzyme and of each reaction are unique (27). The cytosolic form of TK is of special interest, since its activity is regulated in association with several cellular metabolic activities and events. Enzyme activity is high in cells infected with certain oncogenic viruses (for a review, see reference 29), in many rapidly proliferating tumor cell populations (14), and in cells during passage through the S phase of the cell cycle (3, 35). In contrast, nonproliferating cells, including those that have terminally differentiated (42), and also cells that are not in the S phase (3, 35), express little or no TK activity.We previously described the cloning and phys...

Two functional cytosolic thymidine kinase (tk) cDNA clones were isolated from a mouse L-cell library. An RNA blot analysis indicated that one of these clones contains a nearly full-length tk sequence and that LTKcells contain little or no TK message. The nucleotide sequences of both clones were determined, and the functional mouse tk cDNA contains 1,156 base pairs. An analysis of the sequence implied that there is an untranslated 32-nucleotide region at the 5' end of the mRNA, followed by an open reading frame of 699 nucleotides. The 3' untranslated region is 422 nucleotides long. Thus, the gene codes for a protein containing 233 amino acids, with a molecular weight of 25,873. A comparison of the coding sequences of the mouse tk cDNA with the human and chicken tk genes revealed about 86 and 70% homology, respectively. We also isolated the tk gene from a mouse C57BL/10J cosmid library. The structural organization was deternmined by restriction mapping, Southern blotting, and heteroduplex analysis of the cloned sequences, in combination with a mouse tk cDNA. The tk gene spans approximately 11 kilobases and contains at least five introns. Southern blot analysis revealed that this gene is deleted in mouse LTK-cells, consistent with the inability of these cells to synthesize TK message. This analysis also showed that tk-related sequences are present in the genomes of several mouse strains, as well as in LTK-cells. These segmoents may represent pseudogenes.Thymidine kinase (TK; EC 2.7.2.21) catalyzes the phosphorylation of thymidine to thymidine 5'-monophosphate, which is subsequently used for DNA synthesis. Two major forms of TK have been identified in animal cells, one localized in mitrochondria and the other in the cytosol (16). The activities of these two forms of enzyme are regulated differentially during progression through the cell cycle and in association with the replicative state of the cell. Cytosolic TK activity is high in rapidly proliferating cells, and it peaks during the S phase of the cell cycle. Little activity is detectable in cells resting at confluence. The activity of cytosolic TK also increases after infection by simian virus 40, adenovirus 12, or Epstein-Barr virus (16). In addition, terminal differentiation of cells is associated with reduced expression (1,25). In contrast, the activity of the mitochondrial TK is relatively constant. Interest in TK has been further stimulated by the finding that the levels of the mitrochondrial and cytosolic enzyme activities in neoplastic tissue from patients with a variety of lymphoproliferative disorders are useful indicators for predicting either tumor behavior or the clinical course of patients (9).Our goal has been to resolve the molecular mechanism that underlies the regulatory pattern of TK expression associated with proliferation and also with cellular differentiation. cDNA sequences encoding human TK and chicken TK have been isolated (5, 24; Lin, Zhao, and Ruddle, unpublished data). In addition, tk genomic sequences have recently been cloned from chi...

We previously isolated and characterized the structure of murine thymidine kinase (tk) genomic and cDNA sequences to begin a study designed to identify regions of the tk gene important for regulated expression during the transition of cells from G0 to a proliferating state. In this report, we describe the stable transfection of the cloned gene into L-M(TK-) cells and show that both thymidine kinase (TK) enzyme activity and DNA synthesis increase in parallel when transfectants in G0 arrest are stimulated by serum. To define promoter and regulatory regions more precisely, we have constructed a series of tk minigenes and have examined their expression in stable transfectants after serum stimulation. We have identified a 291-base-pair DNA fragment at the 5' end of the tk gene that has promoter function, and we have determined its sequence. In addition, we have found that DNA sequences which mediate serum-induced expression of TK are transcribed, since expression of the murine tk cDNA, fused to a promoter from either the murine tk gene, the simian virus 40 early region, or the herpes simplex virus tk gene, is stimulated by serum. Our constructs also reveal that the murine tk polyadenylation signal is not required for regulation, nor is most of the 3' untranslated region. RNA dot blot analysis indicates that murine cytoplasmic tk mRNA levels always parallel TK enzyme activity. Nuclear runon transcription assays show less than a 2-fold increase in transcription from the cloned tk gene in serum-stimulated transfectants, but an 11-fold increase in mouse L929 cells, which are inherently TK+. These results taken together suggest that the murine tk gene is controlled in serum-stimulated cells by a transcriptional mechanism influenced by DNA sequences that flank tk and also by a posttranscriptional system linked to gene sequences that are transcribed.

Two functional cytosolic thymidine kinase (tk) cDNA clones were isolated from a mouse L-cell library. An RNA blot analysis indicated that one of these clones contains a nearly full-length tk sequence and that LTK- cells contain little or no TK message. The nucleotide sequences of both clones were determined, and the functional mouse tk cDNA contains 1,156 base pairs. An analysis of the sequence implied that there is an untranslated 32-nucleotide region at the 5' end of the mRNA, followed by an open reading frame of 699 nucleotides. The 3' untranslated region is 422 nucleotides long. Thus, the gene codes for a protein containing 233 amino acids, with a molecular weight of 25,873. A comparison of the coding sequences of the mouse tk cDNA with the human and chicken tk genes revealed about 86 and 70% homology, respectively. We also isolated the tk gene from a mouse C57BL/10J cosmid library. The structural organization was determined by restriction mapping, Southern blotting, and heteroduplex analysis of the cloned sequences, in combination with a mouse tk cDNA. The tk gene spans approximately 11 kilobases and contains at least five introns. Southern blot analysis revealed that this gene is deleted in mouse LTK- cells, consistent with the inability of these cells to synthesize TK message. This analysis also showed that tk-related sequences are present in the genomes of several mouse strains, as well as in LTK- cells. These segments may represent pseudogenes.