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Nucleic Acids Research, 2000, Vol. 28, No. 7 1525-1534
© 2000 Oxford University Press

Cloning and characterization of the 5'-upstream sequence governing the cell cycle-dependent transcription of mouse DNA polymerase {alpha} 68 kDa subunit gene

Naoko S. Nishikawa1,2, Masako Izumi2,3, Hiroshi Uchida1, Masayuki Yokoi1,4, Hiroshi Miyazawa1,5 and Fumio Hanaoka1,2,4,*

1Cellular Physiology Laboratory, 2Biodesign Research Group, 3Division of Radioisotope Technology, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama 351-0198, Japan, 4The Institute for Molecular and Cellular Biology, Osaka University, Suita, Osaka 565-0871, Japan and 5Department of Pharmaceutical Sciences, National Institute of Public Health, Minato, Tokyo 108-8638, Japan

We have isolated and determined the structure of the gene encoding the 68 kDa subunit (p68) of the mouse DNA polymerase {alpha}–primase complex. The p68 gene consists of four exons and the p68 promoter region lacks TATA and CAAT boxes, but contains a GC-rich sequence, two palindrome sequences and two putative E2F-binding sites. A series of transient expression assays using a luciferase reporter gene indicated that a region from nucleotide position –89 to –30 (–89/–30) with respect to the transcription initiation site is crucial for basal transcription of the p68 gene in proliferating NIH 3T3 cells. In particular, part of the GC-rich sequence (–57/–46) and the palindrome (–81/–62) elements were necessary for promoter activity, both of which share homology with the E-box sequence. Gel mobility shift assays using NIH 3T3 nuclear extracts revealed that the upstream stimulatory factor, known as an E-box-binding protein, binds to these sites. Moreover, we observed binding of E2F to two sites near the transcription initiation site (–11/–3 and +9/+16). A transient luciferase expression assay using synchronized NIH 3T3 cells in G0 phase revealed that these E2F sites are essential for transcription induction of the p68 gene after serum stimulation, but are dispensable for basal transcription. These results indicate that growth-dependent regulation of transcription of the mouse p68 and p180 genes is mediated by a common factor, E2F; however, basal transcription of the genes, interestingly, is regulated by different transcription factors.

* To whom correspondence should be addressed at: The Institute for Molecular and Cellular Biology, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan. Tel: +81 6 6879 7975; Fax: +81 6 6877 9382; Email: fhanaoka@imcb.osaka-u.ac.jp Present address: Hiroshi Uchida, Mitsui Chemicals Inc., Mobara, Chiba 297-0017, Japan


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