Functional structure of the promoter region of the human thymidylate synthase gene and nuclear factors that regulate the expression of the gene.
To identify the essential motifs of the promoter of the human thymidylate synthase (hTS) gene, we constructed a set of deletion mutants that covers the region from -441 to +28 of the hTS gene. (The nucleotide positions are numbered from the first position of the initiation codon of the hTS gene.) From the results of chloramphenicol acetyltransferase (CAT) assay of these mutants, two positive elements for the promoter activity were identified: one contains CACCC box (CCACACCC) that is found in the SV40 enhancer motif and the other contains the sequence that is homologous to the Sp1 binding site in the mouse TS gene. Furthermore, two negative regulatory sequences were identified between the two positive elements and upstream from the CACCC box. Cassette mutations were introduced into these motifs and the function of the motifs was confirmed. From the results of gel mobility shift analysis, we found that three nucleoprotein complexes were formed in the promoter region of the hTS gene. The formation of one of the complexes was competed by the DNA fragment bearing the GC box. The gel mobility shift analyses using the DNA fragments with cassette mutations revealed that the complex was formed on the Sp1 binding site of the hTS gene and the formation of the complex correlated with the promoter activity of the fragments with cassette mutations measured by the CAT assay.
Horie
N
N
Laboratory of Genetic Engineering, School of Food and Nutritional Sciences, University of Shizuoka, Japan.
Takeishi
K
K
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D016254:Mutagenesis, Insertional; D009698:Nucleoproteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000378:metabolism; D013940:Thymidylate Synthase / Q000235:genetics*
D009698:Nucleoproteins; D016329:Sp1 Transcription Factor; D004247:DNA; D013940:Thymidylate Synthase