CCAAT-BOX CONTRIBUTIONS TO HUMAN THYMIDINE KINASE MESSENGER-RNA EXPRESSION

In order to examine the role of two inverted CCAAT boxes near the star t of transcription of the human thymidine kinase (TK) gene, a series o f constructs were prepared in which one or both CCAAT boxes were delet ed or mutated. These altered promoters (1.2 kb of 5'-flanking sequence ) were used to express a TK minigene containing the first two exons an d introns followed by the remainder of the cDNA. RNA blots were prepar ed from stable cell lines of ts13 cells containing these constructs un der three conditions: 1) serum deprived cells, 2) serum stimulated cel ls, and 3) cells that had been stimulated with serum, but were arreste d in the G(1) phase of the cell cycle by the temperature sensitive mut ation carried by these cells. TK mRNA expression from each construct w as suppressed by the temperature sensitive block to tell cycle progres sion. Measurement of protein expression from the various altered TK pr omoters indicated that both CCAAT boxes contribute to promoter strengt h. These experiments also suggested that the two CCAAT boxes were not equivalent and that the distal CCAAT could substitute for the proximal CCAAT, but the converse was not true, (C) 1995 Wiley-Liss, Inc.