Growth-responsive expression from the murine thymidine kinase promoter: genetic analysis of DNA sequences.
As a first step toward elucidating the biochemical basis of gene regulation at the G1-S boundary of the cell cycle, we have identified regions of the murine thymidine kinase (TK) promoter sufficient to confer appropriately growth-responsive expression to a heterologous gene. Using a series of TK promoter-chloramphenicol acetyltransferase (CAT) gene fusion constructs, we have identified sequences located between -174 base pairs upstream and +159 base pairs downstream of the TK translation initiation site that are sufficient to drive efficient S phase-specific expression of the CAT reporter gene in transfected murine fibroblasts. Both deletion analysis and site-specific mutagenesis experiments indicated that an Sp1 consensus binding site is critical to the activity of this promoter. Synchronized populations of BALB/c 3T3 cells stably transfected with either TK promoter-CAT fusion constructs or TK promoter-beta-globin fusion constructs expressed their respective reporter genes in an S phase-specific manner following serum stimulation. In each case, reporter gene expression was reduced during quiescence and G1 and rose upon entry of cells into S phase. The TK sequences included in these constructs therefore contained information sufficient to confer S phase-specific regulation to these two reporter genes. These results set the stage for a more detailed analysis of the sequences and trans-acting factors responsible for regulating murine TK gene expression and may lead to insights into the control of proliferation in normal and transformed cells.
Fridovich-Keil
J L
JL
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts.
Gudas
J M
JM
Dou
Q P
QP
Bouvard
I
I
Pardee
A B
AB
D000818:Animals; D001483:Base Sequence; D002455:Cell Division; D002478:Cells, Cultured; D002872:Chromosome Deletion; D004247:DNA / Q000235:genetics; D005347:Fibroblasts / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016196:S Phase; D013937:Thymidine Kinase / Q000235:genetics*; D014162:Transfection
D015345:Oligonucleotide Probes; D011993:Recombinant Fusion Proteins; D004247:DNA; D013937:Thymidine Kinase