Induction of cellular thymidine kinase occurs at the mRNA level.

Abstract

The thymidine kinase (TK) gene has been isolated from human genomic DNA. The gene was passaged twice by transfection of LTK- Thymidine kinase (TK) is an enzyme in the pyrimidine salvage pathway that catalyzes the phosphorylation of thymidine to dTMP. Many mammalian cells, including human HeLa cells, contain both cytoplasmic and mitochondrial forms of the enzyme (3), but the cytoplasmic form alone concerns us here. The regulation of the synthesis of TK is interesting because it is typical of that seen for many enzymes involved in DNA metabolism. TK activity is closely linked to the growth state of the cell, being present in rapidly growing but not in resting cells (13). In synchronized populations of cells, the activity is low in resting or Gl phase cells, but increases dramatically 10 to 20 h after the cells are released from arrest by serum stimulation, in parallel with the onset of DNA synthesis and entry into S phase. This induction is not absolutely dependent upon DNA synthesis (13), but does require both RNA and protein syntheses, suggesting that induction may be at the level of transcription. TK can also be induced by infection of resting cells with papovaviruses such as simian virus 40 (SV40) and polyoma (16,17), and the viral genes required for this induction are the large T antigens (30). Whether viral induction occurs by the same or a different mechanism(s) as serum induction is a question that remains to be answered.The TK gene provides a useful model system for carrying out a molecular analysis of genes that are cell cycle regulated and induced by viral infection. First, TK shows a great increase in activity (10-to 20-fold) after both serum and viral induction. Moreover, TK enzyme assays are both sensitive and easily performed. We can genetically select both for (hypoxanthine-aminopterin-thymidine media) and against (bromodeoxyuridine media) the TK+ phenotype, and many TK-cell lines exist. It has been shown that LTK-cells transfected to a TK+ phenotype with heterologous (human, rat, or hamster) chromosomal DNA containing a functional TK gene exhibit normal cell cycle regulation of the gene (31). Recent experiments with a cloned human gene (5) that the sequences required for cell cycle regulation are closely linked to the gene and function after transfection into TK-cells. Thus, this system should offer the chance to dissect the sequences involved in cell cycle-specific gene regulation.The mechanisms by which the expression of cell cycle-dependent genes is controlled, and by which the papovaviruses override these controls, remain obscure, although many of the initial observations were made more than 15 years ago. To a large extent this is because molecular probes for these genes and their transcripts have not been available. In this paper we report the isolation of both the human chromosomal TK locus and a functional human TK cDNA clone. Isolation of the chromosomal locus has previously been reported by several investigators (5,19,22), and our mapping is essentially in agreement with their data. In a...

SV40 stimulates expression of the transacting factor Sp1 at the mRNA level.

“…The RNA level increased beginning at 12 hr postinfection and reached a plateau at -2 0 hr postinfection, remaining constant for at least another 16 hr. In our hands, the change in Sp 1 was at least 2-fold greater than the stimulation of thymidine kinase mRNA, although thymidine kinase showed a 10-to 20-fold increase in other reports (Stuart et al 1985;Stewart et al 1987). The induction of Spl by viral infection was not simply due to stimulation of cell growth, because equal change was observed in logarithmically growing cells.…”

“…In addition to using equal amounts of total RNA from the infected and uninfected cells, we have used an internal negative control, [3-2 microglobulin, whose expression is unaffected by SV40 infection (Stuart et al 1985;Stewart et al 1987}. The level of [3-2 microglobulin mRNA remained constant over the course of the experiment (Fig.…”

“…As a positive control, we have probed the RNA from the infected cells for thymidine kinase mRNA, which is stimulated 10-to 20-fold by SV40 infection of confluent cells (Stuart et al 1985;Stewart et al 1987). We confirmed the SV40 induction of this message but observed only a fourfold increase in our experiments (Fig.…”

“…For example, T antigen stimulates cellular DNA synthesis (Chou and Martin 1975) and causes G~-arrested cells to enter S phase (Soprano et al 1979). The viral oncogene product also affects the steady-state levels of cellular RNA species (Shutzbank et al 1982) including thymidine kinase (Stuart et al 1985;Stewart et al 1987), and enhances rRNA synthesis by RNA polymerase I (Soprano et al 1979). To define specific events in the transformation process, it is important to understand the mechanisms by which T antigen induces these cellular regulatory changes.…”

Genetic determinants of growth phase-dependent and adenovirus 5-responsive expression of the Chinese hamster thymidine kinase gene are contained within thymidine kinase mRNA sequences.

“…The recent clonings of the chicken (35), hamster (22), and human (2,19,24) TK genes have advanced the understanding of growth phase-dependent TK gene expression by providing molecular probes for Northern blot analyses which have established that growth phase-dependent TK gene expression, assayed as TK enzyme activity, is mediated by growth phase-dependent changes in the level of cytoplasmic poly(A)+ TK mRNA (22,39).…”