INTRODUCTION

Reactive oxygen species produced during normal cellular metabolism damage DNA and its precursors (1). An oxidized form of guanine base, 8-oxo-7,8-dihydroguanine (8-oxoguanine),¹ is regarded as most critical in terms of mutagenesis as well as carcinogenesis (2-4). During DNA replication, the 8-oxoguanine nucleotide can pair with cytosine and adenine nucleotides, with an almost equal efficiency, and transversion mutation ensues (5-7).

Organisms are equipped with elaborate mechanisms to counteract such mutagenic effects of 8-oxoguanine, and enzymes responsible have been identified in the bacterium Escherichia coli. Two glycosylases, encoded by the mutM and the mutY genes, function to prevent mutation caused by 8-oxoguanine in DNA (8-12). The MutM protein removes 8-oxoguanine paired with cytosine, and the MutY protein removes adenine paired with 8-oxoguanine. Oxidation of guanine proceeds also in the form of free nucleotides, and an oxidized form of dGTP, 8-oxo-dGTP, is a potent mutagenic substrate for DNA synthesis (13). The MutT protein of E. coli hydrolyzes 8-oxo-dGTP to the monophosphate, and lack of the mutT gene increases the occurrence of A:T to C:G transversion several thousand-fold over the wild type level (13-15).

Mammalian cells contain enzymes similar to those of the MutM, MutY, and MutT proteins (16-20). Among them, the mammalian counterpart of MutT protein has been studied most extensively. The human enzyme specifically hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, with a relatively low K_m value, as compared with other deoxyribonucleoside triphosphates (20, 21). As the enzyme inhibits the misincorporation of 8-oxoguanine opposite the adenine residue of template DNA in an in vitro reconstituted DNA synthesis system, the mammalian 8-oxo-dGTPase probably has the same antimutagenic capacity as the E. coli MutT protein. The finding that expression of cDNA for mammalian 8-oxo-dGTPase in E. coli mutT mutated cells can revert the elevated level of spontaneous mutation frequency to normal (22, 23) would support this view.

To elucidate the roles of 8-oxo-dGTPase in carcinogenesis, it is necessary to construct an animal model with altered levels of the enzyme activity. It is of interest to determine whether the frequency of occurrence of tumors would increase in mice defective in the 8-oxo-dGTPase gene. We isolated the genomic sequence for mouse 8-oxo-dGTPase protein, identified the exon/intron region of the gene, and characterized the promoter in relation to the regulation of expression of the gene.

EXPERIMENTAL PROCEDURES

The mouse embryonic stem cell line CCE was obtained from M. Katsuki, and mouse fibroblast cell lines NIH 3T3 and Balb/c 3T3 were a gift from Y. Nakabeppu. CCE cells were cultured on a feeder layer in Dulbecco's modified Eagle's medium supplemented with 20% fetal bovine serum and 10³ units/ml of leukemia inhibitory factor (24), at 37 °C in a humidified atmosphere of 5% CO₂. Balb/c 3T3 and NIH 3T3 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and with 10% calf serum, respectively, at 37 °C in a humidified atmosphere of 5% CO₂.

[-³²P]dCTP and [-³²P]ATP were purchased from Amersham Corp., and [¹⁴C]chloramphenicol was purchased from DuPont NEN. DNA labeling kits were purchased from Nippon Gene (Toyama, Japan). Restriction enzymes, T4 DNA ligase, T4 polynucleotide kinase, T4 DNA polymerase, and Klenow fragment were obtained from Toyobo Co. (Osaka, Japan). Calf intestine alkaline phosphatase and acetyl coenzyme A were purchased from Boehringer Mannheim and Pharmacia Biotech Inc., respectively. The cloning vectors, pBluescript KS and pBluescript II KS⁺, were purchased from Stratagene. S1 nuclease and Moloney murine leukemia virus reverse transcriptase were obtained from Life Technologies, Inc., and recombinant RNasin was from Promega. Sources of other materials are given throughout.

Two genomic DNA libraries prepared from the 129Sv mouse and its embryonic stem cell line, CCE, were screened by plaque hybridization using as a probe the mouse MTH1 cDNA sequence (22). Positive clones were plaque-purified and subjected to further analyses. The region of the phage clones corresponding to the cDNA sequence were identified by Southern blot analysis. To obtain the 3-region of the MTH1 gene, the library was rescreened using as a probe an ~500-bp KpnI-EcoRI genomic fragment of LmMMTH2. Various DNA fragments derived from the phage clones were subcloned into pBluescript II KS⁺ for further analyses. Nucleotide sequence was determined by the dye terminator method with a model 373A automated DNA sequencer (Applied Biosystems, Inc.). Gene Works® Release 2.5 (nucleic acids and protein sequence analysis software) (IntelliGenetics) was used to handle the sequences.

Genomic DNA was isolated from CCE cells, as described (25). The DNA (8 µg) was digested with EcoRI and BamHI and applied to electrophoresis on 0.8% agarose gels and transferred onto a Hybond N⁺ nylon membrane (Amersham) by the alkali transfer method (25). The filter was hybridized with the 5- and/or the 3-region of the mouse MTH1 cDNA. These probes were labeled with [-³²P]dCTP, using a DNA labeling kit. The filter was washed in 0.2 × SSC and 0.1% SDS at 65 °C for 30 min. Data were processed using a Fujix BAS 2000 Bio-image analyzer.

Total RNAs was isolated from mouse cell lines, Balb/c 3T3 and CCE, using the guanidium thiocyanate/CsCl method (26). Poly(A)⁺ RNA was prepared with the use of an mRNA purification kit (Pharmacia). For Northern blot analysis, total RNAs were isolated from CCE cells and from various tissues of 10-week-old C57BL/6J mice (CLEA, Inc., Tokyo, Japan), using ISOGEN (Nippon Gene).

20 µg of total RNAs were applied to electrophoresis on a 1.2% agarose-formaldehyde gel, and the RNAs separated were transferred onto a nitrocellulose membrane (BA-85, Schleicher & Schuell) in 20 × SSC by capillary blotting (25). The filter was hybridized using a 503-bp NcoI-BamHI fragment of mouse cDNA as a probe (22). The labeled 18 S ribosomal RNA gene probe (27), obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan), was used to quantify amounts of RNA species on the blots. The filter was washed in 0.1 × SSC and 0.2% SDS at 37 °C for 30 min. Data were processed using a Fujix BAS 2000 Bio-image analyzer.

To generate probes for S1 mapping, a 330-bp BamHI-SmaI fragment containing part of exon 1 was excised from the genomic subclone ES5, and a 703-bp SmaI-EcoRI fragment was excised from the cDNA clone M-2 (22). The two fragments were ligated and inserted into the BamHI/EcoRI site of pBluescript KS to produce construct pHI201, which contains the 5 upstream region for the gene, placed adjacent to the cDNA portion. A 1-kb BamHI-EcoRI fragment, excised from pHI201, was inserted into the BamHI/EcoRI site of M13mp19. Single-stranded DNA was isolated from the phage and annealed with the 5-end-labeled primer HI-2, a 25-mer synthetic antisense oligomer primer (5-GTATAAAGCCTGGAGGTGCTCATGC-3), complementary to a region corresponding to positions 2 to +23 from the ATG codon. The annealed primer was used to elongate the sequence, and the product was digested with SacI. The digest was electrophoresed on a 6% denaturing polyacrylamide gel, and a labeled 267-base single-stranded DNA fragment, which can be used as a probe, was recovered from the gel.

The S1 mapping was carried out as described (28). Poly(A)⁺ RNA derived from CCE cells was annealed with a labeled probe (5 × 10⁴ cpm) by heating at 80 °C for 10 min, followed by additional incubation overnight at 55 °C. The resulting DNA/RNA hybrids were treated with S1 nuclease (100, 300, 500 units/reaction) at 16 °C for 30 min, and the products were applied to a 6% denaturing polyacrylamide gel. Sequences produced on pHI201 with the same primer (Sanger's dideoxytermination method) were applied as standards (29). Bands were monitored using a Fujix BAS 2000 Bio-image analyzer.

Poly(A)⁺ RNAs derived from CCE cells and Balb/c 3T3 cells were annealed with the primer HI-2, the 5-end labeled by [-³²P]ATP. Hybridization was performed in 10 mM Tris·HCl, pH 8.3, 0.25 M KCl, 1 mM EDTA at 65 °C for 1 h. The DNA strand was extended using 500 units of Moloney murine leukemia virus reverse transcriptase in an appropriate buffer in the presence of 2.1 µg of actinomycin D and 60 units of recombinant RNasin for each reaction at 42 °C for 90 min. The reaction products were ethanol-precipitated and analyzed on a 6% denaturing polyacrylamide gel. The data were processed using a Fujix BAS 2000 Bio-image analyzer.

pBLCAT30 and pBLCAT20 are derivatives of pBLCAT3 and pBLCAT2 (30), respectively, in which an additional polyadenylation signal derived from SV40 VP1 gene was placed upstream of the CAT gene to minimize the read-through transcription from criptic transcription initiation sites on the vector sequence. pBLCAT32, a derivative of pBLCAT30, has minimal CAT activity and thus served as a negative control in the CAT assay, while pBLCAT20 containing the herpes simplex virus thymidine kinase promoter served as a positive control. A 6.0-kb XhoI-SmaI fragment and a 6.5-kb XhoI-SpeI fragment, derived from genomic clone LmMMTH1, were subcloned in the sense orientation at the polylinker sites of the pBLCAT32 plasmid, and the resulting constructs were designated as pHI101 and pHI103, respectively. The constructs pHI102, pHI105, and pHI107 are the deletion mutants derived from pHI101, while pHI104, pHI106, and pHI108 are those from pHI103. pHI109 and pHI110 are deletion mutants derived from pHI104 and pHI103, respectively. A 477-bp SmaI-SpeI fragment was placed in the same orientation upstream of the 146 SacI site of pHI107, and the resulting plasmid was designated pHI111. When the same fragment was placed in reverse orientation, the resulting plasmid was named pHI113. pHI112 is a deletion mutant derived from pHI111.

NIH 3T3 cells (5 × 10⁵ cells in a 10-cm dish) were plated 24 h before transfection and transfected by the method of Chen and Okayama (31) with minor modifications. A mixture of DNA (30 µg) containing a CAT construct (2.8 pmol/dish), plasmid pYN3214:lacZ (2 µg) (32), and pBluescript KS was applied, together with 0.5 ml of 0.25 M CaCl₂ and 0.5 ml of 2 × N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid-buffered saline (pH 7.06), onto cells. After incubation at 35 °C for 24 h under 3% CO₂, the cells were washed with phosphate-buffered saline and incubated with a fresh culture medium at 37 °C for 24 h under 5% CO₂. Cells were harvested, and lysates were prepared for assays of -galactosidase and CAT activities. CAT assay was performed using 1.5 µl of [¹⁴C]chloramphenicol (50 mCi/mmol, 100 mCi/ml) and 20 µl of 4 mM acetyl coenzyme A. Nonacetylated and acetylated chloramphenicol spots on the TLC plates were quantified and processed using a Fujix BAS 2000 Bio-image analyzer. The -galactosidase assay was carried out as described (33).

RESULTS

To observe the gross structure of the mouse MTH1 gene, Southern blot analysis was performed using as probes the whole and the 5- and 3-regions of cDNA (Fig. 1). Genomic DNA was prepared from cultured cells of the mouse CCE line and digested with restriction enzyme EcoRI or BamHI. When examined with whole cDNA as a probe, the BamHI digestion yielded a single 5.4-kb band, whereas the EcoRI digest gave three fragments corresponding to sizes of 7.7, 2.0, and 1.3 kb. Judging from the intensity of the 5.4-kb BamHI band, we concluded that the band represented two independent fragments. Indeed, hybridization with either the 3-end or the 5-end probe gave the 5.4-kb band. In the case of EcoRI digestion, only the 7.7-kb band appeared upon hybridization with the 5-end probe, while the 2.0- and 1.3-kb bands appeared with the 3-end probe. Taken together, it appears that there is a single gene for MTH1 with an approximate size of 11 kb.

Two mouse genomic DNA libraries were screened by plaque hybridization, using as a probe ³²P-labeled mouse cDNA, which covers almost the entire transcribed region. Positive clones were plaque-purified and grown for preparation of DNAs. Among them, clone LmMMTH2 carried a region covering the 7.7-, 2.0-, and 1.3-kb EcoRI fragments. Since LmMMTH2 lacks the 3-most region of the gene, the library was rescreened, and two additional clones, LmMMTH3 and LmMMTH4, were isolated. The sequences were aligned according to patterns of restriction enzyme digestion, and the exon regions were identified by Southern blot hybridization (data not shown). As shown in Fig. 2, the gene spans about 9 kb and consists of five exons.

The nucleotide sequences of the exons and their flanking regions were then determined (Fig. 3). The coding sequence resides on exons 3, 4, and 5. Consensus sequences for splicing, i.e. 5-GT ... AG-3, are present at each exon/intron junction. Comparison of the genomic sequence and the cDNA sequence (22) revealed a complete match for both sequences.

Southern blot analysis of BamHI and EcoRI digests of the mouse genomic DNA gave faint bands with sizes of 11.5 and 6.0 kb, respectively (see Fig. 1). These fragments were not found in the region of the mouse MTH1 gene (Fig. 2).

In the course of isolation of the mouse genomic clones, we obtained one clone, named LmMMTH5, the restriction patterns of which were completely different from those of the MTH1 gene region (Fig. 4A). The sequence found in clone LmMMTH5 shows an 66.7% homology with the mouse cDNA sequence but carries many base changes, deletions and insertions (Fig. 4B). Several translation initiation codons and stop codons are found when this region is translated in any of three frames; thus, this sequence is regarded as a pseudogene for the mouse MTH1 gene. There are direct repeats of six bases, ACCACT, in both upstream and downstream flanking regions; these may be generated at integration sites when the processed gene for 8-oxo-dGTPase is inserted into the genome during the course of evolution.

Northern blot analysis was made to determine levels of expression of the MTH1 gene in mouse tissues. Total RNAs extracted from various tissues of C57BL/6J mice and also from mouse CCE embryonic stem cells were subjected to hybridization with MTH1 cDNA as well as an 18 S ribosomal RNA gene, as probes. As shown in Fig. 5, a band corresponding to 1.2-kb MTH1 mRNA was detected in all of the samples examined, although intensities of the bands differed considerably.

Intensities of the bands were quantified using an image analyzer, and the amounts of MTH1 mRNA, as standardized for 18 S ribosomal RNA, are shown in Table I. Relatively large amounts of mRNA were present in thymus, testis, heart, kidney, and lung. Lesser but significant amounts of MTH1 mRNA were present in other tissues, with the brain having the lowest value.

Table I. MTH1 mRNA in mouse tissues and cells Intensities of bands of MTH1 mRNA, measured by image analyzer and intensities of the bands of 18 S ribosomal RNA are shown. Ratios of MTH1 mRNA to 18 S ribosomal RNA intensity are listed in the last column. Tissues/cell MTH1 mRNA 18 S ribosomal RNA Ratio arbitrary units × 103 Brain 1.50 9.70 0.16 Thymus 7.06 12.30 0.57 Heart 3.30 9.88 0.34 Lung 2.97 10.80 0.28 Liver 4.13 18.96 0.22 Kidney 5.38 17.34 0.31 Spleen 3.79 19.21 0.20 Stomach 3.07 14.00 0.22 Small intestine 3.35 18.11 0.19 Large intestine 4.05 18.48 0.22 Testis 4.65 13.53 0.34 CCE (embryonic stem cell) 11.33 9.47 1.20

The content of MTH1 mRNA in CCE cells was exceedingly high, as compared with adult mouse tissues, a finding in accord with observations that CCE cells, with an intense proliferating capacity, have a high level of MTH1 protein (22). High oxygen consumption may correlate with high levels of oxidative damage (34), and the level of expression of the MTH1 gene might be regulated in this context.

To determine the transcription initiation site for the MTH1 gene, S1 mapping and primer extension were done (Fig. 6). Poly(A)⁺ RNA was prepared from CCE cells, which exhibit a sufficiently high level of expression, and used for analyses. In both S1 mapping and primer extension, multiple transcription initiation sites were detected over the 50-bp region, among which the most 5 site with a strong signal corresponds to the 5-end of the previously cloned cDNA (22). We obtained a similar result with poly(A)⁺ RNA prepared from Balb/c 3T3 cells. Based on these analyses, the major transcription initiation site was deduced and defined as +1.

To characterize the promoter, sequences of the EcoRI-SpeI and SpeI-ApaLI fragments, which were derived from clone LmMMTH2 and contain exons 1, 2, and 3 as well as introns 1 and 2, were determined (Fig. 7). Consensus sequences for some cis-elements are depicted in the figure, together with other relevant sequence data. The 350-bp region preceding the major initiation sites is rich in G and C. Although a CCAAT-like sequence was detected within this region, the distance between this sequence and the major transcription site was about 310 bp, far greater than the ordinary distance, 80-120 bp. Thus, a small exon(s) may be present in this region.

Nucleotide sequence of the 5-flanking region of the mouse MTH1 gene.

Upstream and downstream of the transcription initiation site there are putative binding sites for transcription factors AP-1, AP-2, Ets-1, HNF5, and Myb. Within intron 2, a putative binding site for transcription enhancer factor-1 was also found, which plays an important role in the activation of gene expression at the initiation of development of the mouse (35). As was frequently observed in many housekeeping genes (32, 36-38), a typical TATA box sequence is missing.

Various lengths of the upstream region for the MTH1 gene were placed upstream of the reporter CAT gene, and the constructs were introduced into NIH 3T3 cells together with reference plasmid pYN3214:lacZ, which expresses -galactosidase activity. In each assay, CAT and -galactosidase activities were determined, the former value being divided by the latter to express levels of CAT gene expression.

The results are summarized in Fig. 8. Inspection of data with a group of plasmids carrying various regions of promoters beginning from the +9 SmaI site, located 8 bases downstream of the major transcription initiation site, revealed that a 330-bp sequence from 321 to +9 carries the basic promoter activity (pHI105), as shown in Fig. 8A. Extension of the region up to 6 kb (for pHI101) caused only a 2-fold increase in transcription-promoting activity. On the other hand, shortening of the basic region led to a complete loss of the promoter activity (pHI107 carrying the 146 to +9 region).

Promoter activities of the 5-flanking and upstream regions of the mouse MTH1 gene as determined in NIH 3T3 cells.

When analyses were extended further downstream to the +525 site, a new picture emerged (Fig. 8B). A construct carrying a region from 146 to +525 (for plasmid pHI108) had a significantly high level of CAT expression. It should be noted that each of the two component sequences alone has no transcription-promoting activity, as evidenced by results with plasmid pHI107 (146 to +9) and pHI109 (+49 to +525). It appears that the potential transcription promoting-activity carried by pHI107 is activated by an enhancer present in pHI109.

A distinct enhancer activity was detected when placing the SmaI-SpeI fragment, carrying the +49 to +525 region, or a shorter one upstream of the 146 to +9 region of pHI107 (Fig. 8C). The same level of enhancer activity was retained even if the sequence was placed in the opposite direction. Since a shorter sequence, corresponding to the PstI-SpeI fragment, is effective for transcription promotion, a certain sequence within this region may be responsible for this activity.

Since there are putative AP-1 binding sites (TGACCTCA and TGACACA) in this region, the sequences were changed to GGGCCC (ApaI restriction sequence), and their transcription-promoting activities were examined; the mutant constructs retained full activity (data not shown). Some other sequence yet to be defined may be responsible for this transcription activation.

DISCUSSION

8-Oxo-dGTP can be generated not only by direct oxidation of dGTP but also by phosphorylation of 8-oxo-dGDP (39). Mammalian cells contain powerful nucleoside diphosphate kinase activity that converts ribo- and deoxyribonucleoside diphosphates, including 8-oxo-dGDP, to the corresponding nucleoside triphosphates. Once 8-oxo-dGTP is formed, it can be incorporated into cellular DNA to yield transversion mutations. The enzyme 8-oxo-dGTPase is present in bacteria and mammalian cells (13, 20-23, 40-42) and appears to function in order to prevent this misincorporation. The enzyme specifically hydrolyzes 8-oxo-dGTP to the monophosphate, and the 8-oxo-dGMP thus formed cannot be rephosphorylated. Guanylate kinase that acts on both GMP and dGMP for phosphorylation is totally inactive for 8-oxo-dGMP (39). By the action of nucleotidase, 8-oxo-dGMP is further degraded to 8-oxodeoxyguanosine, a form readily excretable through the cellular membrane.

The biological significance of the 8-oxo-dGTPase has been demonstrated by studies of E. coli mutant strains. Lack of the mutT gene encoding the enzyme causes an increased level of A:T to C:G transversion several thousand-fold over the wild type cells (13-15), and a significant increase in the 8-oxoguanine content in DNA of mutT cells occurs (12). Furthermore, there is evidence that the elevated level of spontaneous mutation frequency of mutT cells reverts to normal when cDNA for mouse 8-oxo-dGTPase is expressed in such cells (22). It seems likely that mouse 8-oxo-dGTPase has the same antimutagenic capacity as E. coli MutT protein.

To better understand the roles of mammalian 8-oxo-dGTPase in the control of spontaneous mutagenesis as well as carcinogenesis, mice defective in their own gene for 8-oxo-dGTPase (the MTH1 gene) would be useful. Isolation and characterization of the genomic sequence is the first step toward this goal, and this was achieved in the present work. We screened two genomic DNA libraries, derived from mouse 129Sv and its embryonic stem cell line, CCE, by using cDNA as a probe, and we isolated the gene encoding the 8-oxo-dGTPase protein. This gene has 5 exons, among which the coding sequence resides on the third through fifth exons, and spans approximately 9 kb.

Part of the human and rat gene for 8-oxo-dGTPase has been isolated (23, 42). The human gene is composed of four exons, while the rat gene has three exons. A comparison of the gross structures of the three types of genes is shown in Fig. 9. The overall structure of the mouse gene is similar to those of the human and rat counterparts. A comparison of DNA sequences revealed that sizes of the exons for the three species of the genes are practically identical, although sizes of the introns do differ.

To determine transcription characteristics of the MTH1 gene, we first determined expression levels of the gene in various mouse tissues. All organs examined contained substantial amounts of mRNA with a high value seen in the thymus, testis, heart, kidney, and lung, when comparisons were made on the basis of 18 S ribosomal RNA content. Even a higher level of expression was observed in the embryonic stem cell line CCE, with an intense proliferating capacity. On the other hand, the brain showed a low level of gene expression. These results of the mRNA level are almost in accord with the values of 8-oxo-dGTPase, established by Western blot analysis (22). The gene expression may be regulated to cope with oxidative stress.

We isolated the putative promoter region that resides upstream of the first exon and sequenced most of the region. In S1 mapping and primer extension analyses, multiple transcription initiation sites were detected. The promoter region is GC-rich and contains numerous AP-1 and AP-2 binding sites, while it lacks a typical TATA box, as is the case for many housekeeping genes.

To define the promoter region, CAT assays were performed in conjunction with deletion analyses. The basic promoter activity was found in the 330-bp sequence, located from 321 to +9, and the upstream region carried activity to enhance the level 2-fold over the basic level. When we extended the analyses into the transcribable region, pronounced modulator activities within the gene were evident. A 174-bp fragment, carrying parts of intron 1, exhibited strong, positive effects, and attachment of the fragment to a small region of the promoter led to a significant stimulation of the transcription. Modulation of the gene expression in vivo by manipulating these sequences is the subject of ongoing studies.