Recent evidence on the transcriptional regulation of the human thymidine kinase (TK) gene raises the possibility that cell-cycle regulatory sequences may be localized within its promoter. A hybrid gene that combines the TK 5' flanking sequence and the coding region of the bacterial neomycin-resistance gene (neo) has been constructed. Upon transfection into a hamster fibroblast cell line K12, the hybrid gene exhibits cell-cycle-dependent expression. Deletion analysis reveals that the region important for cell-cycle regulation is within -441 to -63 nucleotides from the transcriptional initiation site. This region ( -441 to -63) also confers cell-cycle regulation to the herpes simplex virus thymidine kinase (HSVtk) promoter, which is not expressed in a cell-cycle manner. We conclude that the -441 to -63 sequence within the human TK promoter is important for cell-cycle-dependent expression.One approach to understand the control of cell growth on a molecular level is to identify genes whose expression is modulated during the cell cycle and to study the underlying mechanisms of this regulation. The eukaryotic cell cycle has four distinct phases, G1, S, G2, and M (1). There is evidence revealing that several well-studied S-phase-specific genes, such as those encoding the replication-dependent histones, dihydrofolate reductase, and thymidylate synthase, are regulated at multiple control levels (2-4). Recently, it has been shown by DNA-mediated gene transfer that sequences flanking the replication-dependent histone genes can confer transcriptional (5-7) or posttranscriptional control (8) on the heterologous fusion genes, resulting in cell-cycle regulation of their mRNA levels in vivo.Another well-studied cell-cycle-regulated system is the thymidine kinase (TK) gene, which encodes a cytosol enzyme of the pyrimidine salvage pathway catalyzing the phosphorylation of thymidine to form thymidine 5' monophosphate. It has been documented that the activity of the cytosol TK is cell-cycle regulated and the increase in enzyme activity correlates with increases in DNA synthesis (9 (16,17). The implication is that at least part of the determinants of this regulation are contained within the TK mRNA sequence. With the direct demonstration that the half-life of TK mRNA decreases as S phase cells enter quiescence (18), posttranscriptional regulation of the TK transcripts clearly plays an important role in the cell-cycleregulated expression of the TK gene. The less well-understood level of control for the TK gene is at the step of transcriptional regulation. While it has been demonstrated that TK activity is sensitive to actinomycin D (9), the extreme low levels of the TK transcripts and its transient increase of transcriptional activity occurring only at a narrow period at the G1 and S border makes it difficult to directly measure its transcriptional rate. Thus, earlier attempts failed to detect an increase in TK transcriptional activity (19). Highly sensitive techniques have been used to demonstrate that TK gene expression is ...
