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In a previous study, we narrowed the region of the human ornithine decarboxylase (ODC) promoter responsive to 12-O-tetradecanoylphorbol-13-acetate (TPA) to nt -42 to +54 around the transcription initiation site (Kim YJ, Pan H, Verma AK, Mol Carcinog 10:169-179, 1994). Here we report defining the role of the TATA box in TPA-induced transcription from the -42/+54 ODC promoter fragment. A transversion mutation at the third position of the TATA box (TATAAGT-->TAAAAGT) reduced TPA responsiveness of the reporter construct -42/+54 ODC-Luc by 49%. Electrophoretic mobility shift assays (EMSAs) using HeLa cell nuclear protein extracts revealed no differences in the binding pattern between the natural -42/+54 ODC promoter element and the -42/+54 ODC promoter element containing the T-->A mutation. However, antibodies to the general transcription factor TFIIB disrupted the DNA-protein complexes normally formed with the -42/+54 ODC promoter element in EMSAs. A consensus TATA box oligonucleotide formed two bands, with the faster mobility band displaying enhanced binding with nuclear protein extracts from TPA treated cells. Furthermore, incubation of HeLa cell nuclear extracts with an oligonucleotide containing the ODC TATA box also caused formation of two specific bands in EMSA. Both bands exhibited augmented binding to nuclear proteins from TPA-treated cells. Introduction of the T-->A transversion mutation in the ODC TATA oligonucleotide eliminated binding of the faster migrating band formed with the natural ODC TATA oligonucleotide. These results indicate that TPA modulation of the general transcription machinery may play a role in the TPA-activated transcription of the human ODC promoter.
In a previous study, we narrowed the region of the human ornithine decarboxylase (ODC) promoter responsive to 12-O-tetradecanoylphorbol-13-acetate (TPA) to nt -42 to +54 around the transcription initiation site (Kim YJ, Pan H, Verma AK, Mol Carcinog 10:169-179, 1994). Here we report defining the role of the TATA box in TPA-induced transcription from the -42/+54 ODC promoter fragment. A transversion mutation at the third position of the TATA box (TATAAGT-->TAAAAGT) reduced TPA responsiveness of the reporter construct -42/+54 ODC-Luc by 49%. Electrophoretic mobility shift assays (EMSAs) using HeLa cell nuclear protein extracts revealed no differences in the binding pattern between the natural -42/+54 ODC promoter element and the -42/+54 ODC promoter element containing the T-->A mutation. However, antibodies to the general transcription factor TFIIB disrupted the DNA-protein complexes normally formed with the -42/+54 ODC promoter element in EMSAs. A consensus TATA box oligonucleotide formed two bands, with the faster mobility band displaying enhanced binding with nuclear protein extracts from TPA treated cells. Furthermore, incubation of HeLa cell nuclear extracts with an oligonucleotide containing the ODC TATA box also caused formation of two specific bands in EMSA. Both bands exhibited augmented binding to nuclear proteins from TPA-treated cells. Introduction of the T-->A transversion mutation in the ODC TATA oligonucleotide eliminated binding of the faster migrating band formed with the natural ODC TATA oligonucleotide. These results indicate that TPA modulation of the general transcription machinery may play a role in the TPA-activated transcription of the human ODC promoter.
The hallmark of cellular aging is the failure of senescent diploid cells to enter or to complete the S phase of the cell cycle. The cause for such failure may hold the key for our understanding of the molecular basis of cellular aging. We have previously shown that aging of IMR-90 human diploid fibroblasts in culture is accompanied by a five to sevenfold decrease in both thymidine kinase activity and thymidine kinase mRNA level (Chang and Chen, 1988, J. Biol. Chem., 263:11431-11435). To examine whether attenuation of gene expression at G1/S boundary is unique for thymidine kinase or it may involve most, if not all, of other G1/S genes, we compared the expressions of two classes of G1/S genes in young and in old IMR-90 cells following serum stimulation. We found that the expression of all these genes, including thymidylate synthase (TS), dihydrofolate reductase (DHFR), ribonucleotide reductase (PNR), proliferating cell nuclear antigen (PCNA), histone H1, histone H2A + 2B, histone H3, and histone H4, was induced to high levels in young IMR-90 cells but not in old IMR-90 cells. The mRNA levels of all G1/S genes in young cells were more than tenfold higher than that in old cells 12 hr after serum stimulation. The enzymes encoded by TS and DHFR genes and dUTPase also exhibited similar age-dependent attenuation in activities. In contrast, expression of growth-related genes such as eIF-5A, c-Ha-ras, and beta-actin did not show significant differences between young and old cells after serum stimulation. Computer analysis of the promoter region of these G1/S genes revealed an Sp-1 binding site as the most common cis-element. Taken together, our results suggest that the suppression of G1/S gene expressions during senescence may be a global phenomenon and that G1/S genes may be coordinately controlled.
Ornithine decarboxylase (ODC) is a homodimeric enzyme dependent on pyridoxal 5'-phosphate. We identified a complementary DNA clone corresponding to ODC from the brain of adult flounder (Paralichthys olivaceus). The flounder ODC cDNA consisted of 2939 bp encoding 272 amino acid residues. The flounder ODC showed 80.3% sequence identity to zebrafish and 70.8% to rat at the amino acid level. Comparison of the structure and nucleotide sequence of the ODC genes revealed that the gene is highly conserved in the flounder, zebrafish, and rat. The presence of ODC mRNA species in brain, kidney, liver, and embryo was confirmed using the reverse transcriptase polymerase chain reaction. The recombinant protein of flounder ODC containing a short histidine tag at the carboxyl terminus was overexpressed in Escherichia coli BL21 (DE3) codon plus using an inducible T7 expression system, and was purified by Ni-NTA affinity chromatography.
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