Sep 2, 1986 - Three major transcription initiation sites (cap sites) ... the PstI site as described previously (12) (Fig. ... 2012 on July 17, 2018 by Google Indexer.
MOLECULAR AND CELLULAR BIOLOGY, May 1987, p. 2012-2018 0270-7306/87/052012-07$02.00/0 Copyright © 1987, American Society for Microbiology
Vol. 7, No. 5
Murine DNA Polymerase 1 Gene: Mapping of Transcription Initiation Sites and the Nucleotide Sequence of the Putative Promoter Region MASAMITSU YAMAGUCHI,1 FUMIKO HIROSE,2 YUKO HAYASHI,1 YOSHIO NISHIMOTO,' AND AKIO MATSUKAGEl* Laboratory of Cell Biology, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya 464,1 and Laboratory of Cancer Cell Biology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Showa-ku, Nagoya 466,2 Japan Received 2 September 1986/Accepted 4 February 1987
The nucleotide sequence of the region (total, 2,512 base pairs [bp]) from intron 2 to the 5'-flanking region was determined for the mouse DNA polymerase 13 genomic clone, and the 300-bp region from intron 1 to the 5'-flanking region was also sequenced for the rat clone. At 51 bp upstream from the ATG codon which was previously suggested to be the translation initiation codon for the rat cDNA sequence, we found another ATG in the same reading frame in both mouse and rat genes. Three major transcription initiation sites (cap sites) each for rat and mouse DNA polymerase ,B mRNAs were localized precisely by primer extension analysis at 51, 41, and 0 bp upstream from the first ATG codon, suggesting that this codon is used for translation initiation. The 400-bp region around exon 1 was extremely G+C rich (about 70%). Although neither a TATA box nor a CAAT box was found within the 500-bp region upstream of the 5'-most cap site, triple repeats of 5'-CCGCCC were found within the 100-bp region flanking the cap site. DNA polymerase ,B purified from various animal cells consists of a single polypeptide of about 40,000 daltons (32, 33, 39), and its structure is highly conserved through the course of evolution (4, 33, 34), suggesting some vital function in the maintenance of living cells. The reaction properties of DNA polymerase 13 are well suited for an efficient gap-filling reaction (17, 37), and repair synthesis of damaged DNA has been considered to be at least one of the roles of this enzyme. DNA polymerase 1 is expressed in most growing and resting cells throughout various developmental stages (10, 16, 19), and its level does not fluctuate very much during the cell cycle (5, 31). These properties are in sharp contrast to those of some other housekeeping enzymes such as DNA polymerase a (5, 10, 16, 24, 31) or dihydrofolate reductase (DHFR) (8) whose levels vary with different physiological conditions and cell cycle stages. The following two questions remain to be resolved. (i) What cellular factor(s) regulates the expression of the DNA polymerase 13 gene? (ii) What structural features of the transcription regulatory region of the gene contribute to this highly constitutive expression of DNA polymerase 1? We have isolated a cDNA clone for rat DNA polymerase 1 (41) and used it to assign the human DNA polymerase 1 gene to chromosome 8 (18). In this study, we isolated rat and mouse genomic clones containing the DNA polymerase 13 gene sequence and focused our attention on the 5' upstream region of the gene which is supposed to contain the promoter. Mapping of transcription initiation sites defined the putative transcription regulatory region, and nucleotide sequence analysis revealed structural features of this region of the DNA polymerase 13 gene. DNAs extracted from mouse and rat cells were analyzed by Southern hybridization with nick-translated pUC9-1OF, which is specific for the 5' side of DNA polymerase , *
pUC19-1OSL, which is specific for the 3' side of the cDNA, or a mixture of both probes (Fig. 1). A 7.6-kilobase (kb) band of rat DNA was detected with the 5'-side-specific probe (Fig. 1B, lane 2), while 6.7- and 5.1-kb bands were detected with the 3'-side-specific probe (Fig. 1B, lane 4). With mouse DNA, 7.1- and 5.9-kb bands were detected with the 5'-sidespecific probe (Fig. 1B, lane 1), while doublet bands of 3.0 kb were detected with the 3'-side probe (Fig. 1B, lane 3). Previously, we observed that a single BamHI fragment hybridized with a pUC9-1OF probe in both mouse and rat DNA, suggesting the occurrence of a single copy of the DNA polymerase 13 gene per genome (18). Thus, both mouse and rat DNA polymerase 1 genes seem to distribute through about 19 kb of each genomic DNA. From the gel region containing DNA fragments that gave positive signals in the Southern analysis (30) (Fig. 1B, lanes 1 and 2), DNA was electrophoretically eluted and used for the construction of a genomic library in AgtWES AB vector (gift from Y. Nishida, Aichi Cancer Center Research Institute) as described previously (15). Positive plaques were selected by plaque hybridization (2) with nick-translated (26) pUC9-1OF DNA as a probe. The positive clone containing the 7.1-kb mouse DNA was designated AMG,B01, and that containing the 5.9-kb DNA was designated as XMG102. The positive clone containing the 7.6-kb rat DNA was designated XRG,O1. To determine the nucleotide sequence of the mouse genomic clone, we constructed a series of unidirectional deletion derivatives proceeding from the Hindlll site toward the PstI site as described previously (12) (Fig. 1C, arrows), and the nucleotide sequence of the individual clone was determined by the dideoxy sequencing method (28, 36). The total region of 2,512 base pairs (bp) including a 642-bp region upstream from the 5'-most cap site, exon 1 (111 bp), intron 1 (194 bp), exon 2 (58 bp), and a part of intron 2 (1,506 bp) was sequenced (Fig. 2A). For simplicity in describing the sequences, we used a numbering scheme in which +1 denotes
Corresponding author. 2012
VOL. 7, 1987
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FIG. 1. Southern blot hybridization analysis of DNAs from mouse and rat. (A) Structure of rat DNA polymerase E cDNA and subcloning of 5'-side and 3'-side fragments into pUC9 or pUC19 vector. 5'-side 442-bp and 3'-side 755-bp fragments were subcloned into pUC9 to construct the plasmids pUC9-1OF and pUC9-10S, respectively. In addition, the 3'-side 575-bp fragment was cloned to construct pUC19-1OSL. pUC9-1OF and pUC19-1OSL were used as probes in the Southern hybridization. (B) Autoradiogram showing results of Southern blot hybridization analysis. Genomic DNAs from mouse spleen (lanes 1, 3, and 5) and rat spleen (lanes 2, 4, and 6) were digested with EcoRI, fractionated by 0.6% agarose gel electrophoresis, transferred to a nitrocellulose membrane, and hybridized with 32P-labeled pUC9-10F (lanes 1 and 2), pUC19-1OSL (lanes 3 and 4), or mixture of the two probes (lanes 5 and 6). The size markers (in kilobases) were HindlIl-digested and 3'-end-labeled X DNA. Exposures were 16 h for lanes 1 and 2 and 36 h for lanes 3 to 7. (C) Restriction maps and sequencing strategy for genomic clones containing the 5' region of mouse and rat DNA polymerase P genes. Restriction mapping was performed by digesting subclones pUC19-MG001 and pUC19-RGp01 with restriction endonucleases and submitting the fragments to gel electrophoresis. Nucleotide sequences were determined by the Sanger method (31, 40) with sequential deletion derivatives of pUC19-MG301A containing the PstI-HindIII 4.5-kb fragment (thick bar) for the mouse gene with reverse 17mer universal primer (4-) or synthetic 15mer primer I (z-). pUC19-RGO01A containing the EcoRI-PstI 4.03-kb fragment (thick bar) for the rat gene was also sequenced with normal 17mer universal primer (-*) or synthetic 15mer primer I (
