Characterization of mutations induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in the colon of gpt delta transgenic mouse: novel G:C deletions beside runs of identical bases

Ken-ichi Masumura1, Keiko Matsui1, Masami Yamada1, Mieko Horiguchi1,2, Kaori Ishida3, Masahiko Watanabe3, Keiji Wakabayashi3 and Takehiko Nohmi1,4

1 Division of Genetics and Mutagenesis, National Institute of Health Sciences, Setagaya-ku, Tokyo 158-8501,
2 Division of Bioregulation Studies, Tokyo University of Agriculture, Setagaya-ku, Tokyo 156-8502 and
3 Cancer Prevention Division, National Cancer Center Research Institute, Chuo-ku, Tokyo 104-0045, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mutations induced by one of the typical dietary mutagens/carcinogens, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), were characterized using gpt delta transgenic mice. This transgenic mouse model has two selection methods to efficiently detect different types of mutations, i.e. 6-thioguanine selection for point mutations and Spi selection for deletions. The mice were fed with a diet containing 400 p.p.m. PhIP for 13 weeks and gpt and Spi mutations were analyzed from the colon, where the highest mutant frequencies were detected. Concerning the types of gpt mutations from PhIP-treated mice, 81% were single base pair substitutions and G:C->T:A transversions predominated; single base pair deletions at G:C base pairs were also observed. In untreated mice G:C->A:T transitions predominated and >80% of these events involved 5'-CpG-3' sites. Concerning Spi mutants from PhIP-treated mice, 76% were G:C base pair deletions and more than half of these events occurred in monotonic G or C run sequences. Interestingly, a novel type of frameshift motif, i.e. G:C base pair deletions beside run sequences, was observed. The most frequently observed mutation in this class was the 5'-TTTTTTG-3'->5'-TTTTTT-3' event. These results suggest that PhIP induces point mutations, such as base substitutions and single base pair deletions, rather than larger deletions in vivo and that run sequences may play an important role in PhIP-induced G:C base pair deletions.

Abbreviations: dG-C8-PhIP, N-(deoxyguanosin-8-yl)-PhIP; MF, mutant frequency; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; 6-TG, 6-thioguanine.


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Humans are exposed to a variety of environmental chemicals, some of which are mutagens and carcinogens. In recent years a number of heterocyclic amines have been identified as mutagens from cooked food and most of these mutagens have been demonstrated to be carcinogenic in experimental animals (14). Among them, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) appears to be important with respect to human cancer development because PhIP has been estimated to be the most abundant heterocyclic amine in the daily diet (58). In fact, PhIP induces colon and prostate cancers in male Fischer 344 rats and mammary carcinoma in female rats (9,10) and these are the types of neoplasia associated with diet most commonly observed in western countries.

To better understand the molecular mechanisms of mutagenicity and carcinogenicity of PhIP, we have conducted mutation assays using the gpt delta transgenic mouse (11,12). A distinct feature of this mouse model is the incorporation of two selection methods, i.e. 6-thioguanine (6-TG) selection and Spi selection, to efficiently detect different types of mutations. 6-TG selection using the gpt gene of Escherichia coli mainly detects point mutations such as base substitutions and frameshifts (13). Spi selection using the red/gam genes of phage {lambda} detects deletions from 1 bp to ~10 kb (14). In previous work we treated mice with PhIP (400 p.p.m. in the diet) for 13 weeks and compared the mutant frequency (MF) in colon, spleen, liver, testis, brain and bone marrow. The highest MFs were observed in the colon, followed by the spleen and liver in both male and female mice (12): the MFs in the colon were 19 and 12 times higher than those in the colon of untreated mice in the gpt and Spi assays, respectively. In studies using other transgenic rodents treated with PhIP the colon also exhibited the highest MFs (1517). Thus, the colon appears to be the most sensitive organ with respect to the mutagenicity of PhIP in vivo.

To characterize PhIP-induced mutations at the molecular level we have analyzed PhIP-induced gpt and Spi mutations in the colon of male gpt delta mice by a DNA sequencing methodology. The results suggest that PhIP induces point mutations rather than larger deletions in vivo. We also report a novel type of mutation by PhIP that involves single G:C deletions beside runs of identical bases.


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 Materials and methods
 Results
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PhIP treatment and rescue of transgenes from mice
Homozygous gpt delta C57BL/6J transgenic mice were treated with PhIP as described previously (12). Briefly, three male mice at 7 weeks old were fed a diet containing 400 p.p.m. PhIP (CLEA, Tokyo, Japan) for 13 weeks; after PhIP treatment the mice were fed the CE-2 basal diet for 2 weeks and then killed after a total of 15 weeks. Three control male mice of the same age were fed with CE-2 basal diet for 15 weeks. Genomic DNAs were extracted from colon mucosa by the phenol/chloroform extraction method and {lambda}EG10 phages were rescued as described (11).

Mutation analyses
gpt mutation assay.
The gpt mutagenesis assay was performed as previously described (12). The gpt mutants recovered from colon DNA of untreated and PhIP-treated male mice were isolated and re-streaked on selection plates containing chloramphenicol and 6-TG to confirm the 6-TG-resistant phenotype. Confirmed 6-TG-resistant colonies were cultured and the cells collected by centrifugation. The bacterial pellets were stored at –80°C for sequencing analysis of the gpt gene. A 739 bp DNA fragment containing the gpt gene was amplified by PCR using two primers: primer 1 (forward), 5'-TACCACTTTATCCCGCGTCAGG-3'; primer 2 (reverse), 5'-ACAGGGTTTCGCTCAGGTTTGC-3'. PCR amplification was carried out using DNA polymerase ExTaq (Takara, Shiga, Japan). A small number of 6-TG-resistant cells were the source of DNA template. Amplification was performed with a DNA Engine PTC-200 (MJ Research, Cambridge, MA). The reaction was started by incubation at 94°C for 4.5 min, followed by 30 cycles of 30 s at 94°C, 30 s at 58°C and 120 s at 72°C. DNA sequencing of the gpt gene was performed with the Sequitherm Long-Read Cycle Sequencing Kit (Epicentre Technologies, Madison, WI) on an ALFred DNA sequencer (Amersham Pharmacia Biotech, Uppsala, Sweden) using a 5'-Cy5-labeled sequencing primer: either primer A (forward), 5'-GAGGCAGTGCGTAAAAAGAC-3', or primer C (reverse), 5'-CTATTGTAACCCGCCTGAAG-3'.

Spi mutation assay.
The Spi assay was carried out as described (11). Escherichia coli strain LE392 was infected with recovered Spi mutant phages to obtain the phage lysates (18). The lysates were used as templates for PCR analysis. The primers used were: primer 3, 5'-GTGAGGATGCGTCATCGCCATTGCTCCCC-3'; primer 4, 5'-GCGATGAAAAGATGTTTCGTGAAGCCGTCGACGC-3'; primer 5, 5'-AAACAGGCGCTGGGCATCAGCGTGGTCTGA-3'; primer 6, 5'-GGTTTGCCCAAAGCGCATTGCATAATCTTTCAGGG-3'. PCR was performed using the LA PCR kit Version 2 (Takara). The reaction was initiated by heating for 1 min at 94°C, followed by 29 cycles of 20 s at 98°C and 1 min elongation at 68°C per kb of the desired PCR fragment. The entire DNA sequence of {lambda}EG10 is available at http://dgm2alpha.nihs.go.jp. The DNA sequence was determined as described in the analysis of gpt mutants. Sequencing primers (5'-end-labeled with Cy5 dye) were: primer A, 5'-CCACTTTTATTGGCGATGAAAAGATGTTTC-3'; primer s201, 5'-CGCTTCGATAACTCTGTTGAATGGCTCT-3'; primer s202, 5'-TGAGTACGGTCATCATCTGACACTACAG-3'.


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 Materials and methods
 Results
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To characterize gpt mutations induced by PhIP in vivo, 99 and 71 mutants from three PhIP-treated and three untreated mice, respectively, were analyzed (Tables I and IIGoGo). The predominant PhIP-induced mutations were single base substitutions (80/99 total mutants = 81%); most of these events occurred at G:C base pairs (79/80 = 99%). The major type of PhIP-induced base substitution was G:C->T:A transversion (52/99 = 53%). G:C->A:T transitions and G:C->C:G transversions were 14 (14/99) and 13% (13/99) of the recovered mutations, respectively. Several mutational hot-spots were observed, including positions 108, 115, 140 and 208, which were hot-spots for G:C->T:A transversions. Nucleotide 402 was a hot-spot for all types of base substitutions at G:C base pairs. In this study hot-spots for base substitutions are defined as nucleotide positions where five or more mutations were observed from two or more mice. Not only base substitutions but also frameshift mutations were observed in the gpt gene (16/99 = 16%). The predominant frameshift mutations were single base pair deletions at G:C base pairs (14/16 = 88%), 64% (9/14) of which occurred at G:C sites in 5'-GGG-3' or 5'-GG-3' sequences. Single G:C deletions in the 5'-GGGA-3' sequence were also observed (three mutants). Hot-spots for deletions were not observed.


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Table I. Classification of the gpt mutations recovered from the colon of control and PhIP-treated mice
 

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Table II. Summary of the gpt mutations recovered from the colon of PhIP-treated and untreated mice
 
In control mice 90% (65/72) of the total mutations analyzed were base substitution mutations: G:C->A:T transitions predominated (31/65 = 48%) and >80% of these occurred at 5'-CpG-3' sites (25/31 = 81%). G:C->T:A transversions were also frequently observed in the controls (19/65 = 29%). Mutational hot-spots in the control mice, all being G:C->A:T transitions at 5'-CpG-3' sites, were observed at nt 64, 110 and 115 of the gpt gene.

To characterize deletion mutations in vivo, 96 and 13 Spi mutants from three PhIP-treated and three untreated mice, respectively, were analyzed (Table IIIGo). In the PhIP-treated group 78% of these were single base pair deletions in the gam gene (75/96). Most of these frameshifts were G:C deletions (73/75 = 97%). The observed single base pair deletions were classified into four groups. The first group includes deletions in repetitive sequences of identical bases (in run sequences in Table IIIGo). This group includes 44 mutants at 15 sites in the gam gene. Most of these events occurred in G:C run sequences (42/44 = 95%). The mutational hot-spots in this group were at nt 286–289, 308–310 and 348–349 in the gam gene; the sequence changes were GGGG->GGG, CCC->CC and GG->G, respectively. The second group includes deletions of G:C beside run sequences (beside run sequences in Table IIIGo). This group includes 19 mutants at 12 sites in the gam gene. The observed run sequences beside the deleted guanines were TTTTTT, CCCCC, TTT, CCC, AAA and AA. In 16 of the total of 19 mutants the deleted guanines were positioned on the 3'-side of the run sequences. The most frequently observed mutation in this group was 5'-TTTTTTG-3'->5'-TTTTTT-3' at nt 294 (six mutants). The third group includes nine mutants that have both single base pair deletions and single base substitutions within two or three neighboring base pairs in the gam gene (with base substitutions in Table IIIGo). At least one mutation in each mutant occurred at G:C base pairs. The fourth group includes three mutants that have single G:C base pair deletions without run sequences (other single base deletions in Table IIIGo).


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Table III. Summary of the Spi mutations recovered from the colon of PhIP-treated mice
 
In addition to single deletions, deletions of >2 bp (five mutants) and complex type mutations (five mutants) were observed (>2 base deletions in Table IIIGo). The former mutants included single deletions of –2 and –19 bp and a –5643 bp deletion that was observed three times. The latter complex mutants were not completely sequenced; these events might be identical or clonally expanded mutants because they were recovered from the same mouse (mouse no. 1) and the sequence characteristics so far determined are very similar.

Besides deletion mutants, 11 mutants that had only base substitutions in the gam gene were also observed (base substitutions in Table IIIGo). Interestingly, the majority of these mutants could make plaques on recA mutants of E.coli, whereas all the other Spi mutants (groups 1–4 described above) could not.

In untreated mice, 77% of the Spi mutants analyzed were single base pair deletions in the gam gene (10/13). All of these events were –1 frameshifts in runs of identical bases (Table IVGo). The predominant event was AAAAAA->AAAAA at nt 295–300. Three independent deletions with sizes of 6–7 kb were also found in the untreated mice. Two of these deletions have short homologous sequences (3 or 4 bp) in their junction regions; the other deletion exhibited a flush junction. In the three large deletion mutants no characteristically common sequences were found in the junctions or in the neighboring regions.


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Table IV. Summary of the Spi mutations recovered from the colon of untreated mice
 

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Mutational spectra of the gpt mutations
The predominant PhIP-induced mutations in the gpt gene were single base substitutions (Table IGo). Most of these occurred at G:C base pairs and the major types of base substitutions were G:C->T:A transversions, followed by G:C->A:T transitions and G:C->C:G transversions. In addition, single G:C deletions were also observed. Similar PhIP-induced mutation spectra have been reported in the hprt gene in Chinese hamster cells (19) and human lymphoblastoid cells (20) and in the lacI gene in the colon of Big Blue transgenic mice and rats (17,21). Thus, it seems clear that PhIP predominantly induces single base substitutions and deletions at G:C base pairs. N-(deoxyguanosin-8-yl)-PhIP (dG-C8-PhIP) has been identified as a major DNA adduct in tissues of rats treated with PhIP (2224). The characteristic PhIP-induced mutations at G:C base pairs could be due to the PhIP–DNA adduct.

Several mutational hot-spots for base substitutions were observed in the gpt gene (Table IIGo). Nucleotides 108 (5'-GCC-3'), 115 (5'-CGG-3'), 140 (5'-GCG-3') and 208 (5'-CGA-3') were the hot-spots for G:C->T:A transversions and nt 402 (5'-GGA-3') was the hot-spot for all types of base substitutions (the underlined base is mutated). Although mutations at the hot-spots were identified in more than two mice, we have to be careful that some of them might have arisen as the product of clonal expansion within the colon. However, hot-spots having similar sequence contexts [5'-C(G)G-3'] have been reported in the lacZ, hprt and lacI loci (19,21,23,25). It has been reported that high frequencies of mutations were observed when G = C > T > A is located at the 5'-flanking position to dG-C8-PhIP (26). The numbers of nucleotides positioned on the 5'-side of the 78 base substituted guanines of the gpt mutants were 39 for C, 23 for G, 11 for T and five for A. These results suggest that there are some preferred sequence contexts for PhIP-induced base substitutions in the colon of mice.

Other than base substitutions, single G:C deletions were also observed (14/99 = 14%), 64% (9/14) of which occurred in G:C run sequences. In the lacZ and lacI loci in the colon of PhIP-treated transgenic mice and rats, single G:C deletions are also frequently (>20% of total) detected (15,17,21). This high incidence of single base pair deletions has not been reported after 2-amino-3,4-dimethylimidazo[4,5-f]quinoline or 2-amino-9H-pyrido[2,3-b]indole treatment in the colon of Big Blue mice (21). A high frequency of single G:C deletions is one of the characteristic mutations associated with PhIP treatment. Kakiuchi et al. reported that four of the eight PhIP-induced colon tumors in F344 rats had a 5'-GGGA-3'->5'-GGA-3' frameshift mutation in the Apc gene (27). In the gpt mutants, 3% (3/99) of the total mutations were 5'-GGGA-3'->5'-GGA-3'. Single G:C deletions in this motif are also observed in 5–10% of lacI and lacZ mutants in other transgenic rodents and hprt mutants in Chinese hamster cells (17,19,21,25). Thus, the mutation 5'-GGGA-3'->5'-GGA-3' may be commonly induced by PhIP in the colon as a minor frameshift event.

In control mice 90% (65/72) of the total gpt mutants were base substitutions (Table IGo). G:C->A:T transitions predominated (31/65) and >80% of these occurred at 5'-CpG-3' sites (25/31 = 81%), namely nt 64, 110 and 115 of the gpt gene (Table IIGo). Similar hot-spots were also observed in the skin of untreated gpt delta mice (13). This suggests that deamination of methylated cytosines at CpG sites contributes to spontaneous mutations in the gpt gene in vivo (28). Besides transitions, G:C->T:A transversions were also frequently observed in the control gpt mutants. These mutations may reflect oxidative damage in DNA, such as 8-oxoguanine lesions (29) or abasic sites (30). The remaining 10% of mutants recovered from the untreated mice were frameshifts and short deletions. These mutational characteristics are largely consistent with the spontaneous mutation spectra in the lacI gene of Big Blue mice (31,32).

Mutational spectra of the Spi mutations
PhIP-induced deletion mutations were investigated by Spi selection, which preferentially detects deletion mutations (11,14). Spi selection positively detects defective {lambda} phage whose gam and redBA gene functions are simultaneously inactivated. Thus, the most typical Spi mutants are deletions in the region of the gam and redBA genes (14). However, the mutants having –1 base frameshifts between nt 121 and the termination codon of the gam gene also exhibit an Spi phenotype. This is because these mutations create a stop codon from the mutated gam gene in the downstream redB gene. Since translation of the gam and redBA genes is probably linked and the gam gene is transcribed first, extension of the coding frame of the mutated gam gene into downstream may interfere with the start of translation of the red genes, thereby functionally inactivating not only gam but also redBA. Mutants having –1 frameshifts before nt 120 do not exhibit an Spi phenotype because the mutations create stop codons within the coding sequence of the gam gene. Hence, Spi selection can detect –1 frameshifts in the gam gene as well as large deletions in the red and gam genes. In the PhIP-treated group, most frequently occurring mutations were single G:C deletions in the gam gene (Table IIIGo and Figure 1Go). Since large deletions were not frequently observed in the Spi mutants recovered from PhIP-treated mice despite the ability of Spi selection to detect large deletions, we suggest that PhIP mainly induces single base pair deletions rather than large deletions in vivo. This is consistent with the report that PhIP exhibits only weak or negative genotoxicity in chromosomal aberration experiments and micronucleous assays in bone marrow and peripheral blood cells using C57BL/6 mice (33).



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Fig. 1. The single base pair deletions in the gam gene found in the Spi mutants recovered from control and PhIP-treated mice. The coding sequence of the gam gene is boxed. The mutants recovered from control and PhIP-treated mice are indicated above and below the coding sequence, respectively. Triangles represent single base pair deletions. The start codon ATG of the gam and red genes and the stop codon TAA of the gam gene are underlined. The region after nucleotide position 121 from the gam start codon detects frameshift mutations by Spi selection and is in bold. The stop codon TGA for gam mutants created by out-of-frame short deletions is underlined and in bold.

 
The single base pair deletions in the Spi mutants were classified according to their sequence context (Table IIIGo). The most prominent group includes 42 mutants in G:C run sequences. The numbers of mutants occurring at GGGG, GGG and GG runs were 13, 11 and 18, respectively. PhIP-induced single base pair deletions in G:C run sequences are also reported in the lacI and lacZ transgenic rodents and the hprt gene in Chinese hamster cells (15,17,19,21). The numbers of repetitive guanines in the gam gene (after position 121) are two sites of GGGG, three sites of GGG and 21 sites of GG, suggesting that single G deletions occur more frequently in longer G:C runs. It has been reported that the dG-C8-PhIP adduct at the 3'-end of a run sequence is more mutagenic than are adducts at the 5'-end of run sequences in mammalian cells (26). There is no 5'-GGGA-3' sequence in the gam gene and therefore our study cannot address the importance of single G:C deletions at this motif which was found in PhIP-induced colon tumors in the rat (27). The next most frequently observed group includes G deletions beside T, C and A run sequences. In most cases that we observed, the deleted guanine is on the 3'-side of runs, such as 5'-TTTTTTG-3'. These results indicate that run sequences are important for PhIP-induced G:C deletions and that neighboring sequences strongly influence these events. We suggest that a slipped mutagenic intermediate is formed after the incorporation of cytosine opposite the dG-C8-PhIP adduct for the induction of –1 frameshifts in run sequences (Figure 2aGo). Similar models have been proposed for frameshift mutations induced by N-acetyl-2-aminofluorene (34) and by 1-nitroso-8-nitropyrene (3537). In the case of deletions beside T:A run sequences, we speculate that adenine is first incorporated opposite the dG-C8-PhIP adduct and then the adenine correctly pairs with the neighboring thymine in the template strand by looping out the adducted guanine (Figure 2bGo). Replication from the correctly paired A:T primer terminus, with the adducted dG-C8-PhIP looped out, could generate single G:C deletions. It seems reasonable to assume that adenine is incorporated opposite the dG-C8-PhIP adduct because G:C->T:A transversions are the most predominant type of gpt mutation observed in these PhIP-treated mice. In addition, it has been reported that the bases A >> T > G, in that order of preference, are misincorporated opposite the dG-C8-PhIP adduct in DNA in mammalian cells (26). A similar frameshift model was discussed with respect to lacI mutations induced by 1-nitroso-8-nitropyrene in an E.coli strain harboring pKM101 (35). The run sequences may stabilize the slipped mutagenic intermediates when they are on the 5'-side of the adducted guanine. It is worth noting that these single G:C deletions besides run sequences are novel and have not been observed in systems using the gpt, lacI or lacZ loci. This is probably because these genetic systems mainly detect point mutations, which occur more frequently than deletions, and thus largely ignore rare mutagenic events such as deletions. An advantage of Spi selection is the ability to selectively detect such rare but potentially important mutations occurring in the mouse genome.



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Fig. 2. A proposed mechanism for single base pair deletions in runs or beside run sequences. (a) In G run sequences, incorporation of cytosine opposite a PhIP adduct leads to a misaligned intermediate by looping out the adducted guanine. The intermediate is stabilized by formation of the correct G:C base pair at the template/primer terminus. (b) In the case of a –1 frameshift beside the run sequences, adenine is first misincorporated opposite an adduct and then forms correct base pairs with the neighboring thymine in the template strand. Slipped mutagenic intermediates are stabilized by the following monotonous run sequences.

 
We also revealed that various types of deletions and base pair substitutions occurred in the motif 5'-GGGCGCG-3' at nt 164–170 in the gam gene (Table IIIGo). The sequence changes observed were –1G deletions in runs (GGGCGCG->GGCGCG), a –1G deletion + a G->C transition (GGGCGCG-> GGCCCG), –1G deletions in non-run sequences (GGGCGCG->GGGCCG and GGGCGCG->GGGCGC) and a –CG deletion (GGGCGCG->GGGCG) (the underlined base is mutated). In particular, the –1G + G->C mutation (GGGCGCG-> GGCCCG) occurred four times in three different mice, suggesting that they are mostly independent. This suggests that either the DNA damage by PhIP is highly localized in this motif or that DNA replication in this motif is highly mutagenic when it is modified by PhIP. It is worth noting that the motif GGGCGCG resembles the NarI site sequence, i.e. GGCGCC, in which –CG mutation (GGCGCC->GGCC) is efficiently induced by N-acetoxy-N-acetyl-2-aminofluorene (38). However, since not only simple –1G or –CG deletions but also –1 G plus base substitutions are efficiently induced, we speculate that complex events such as loop-back DNA synthesis may occur when a PhIP adduct is present in the guanine bases of GGGCGCG (36).

Besides these single base deletion mutants, 11 Spi mutants recovered from PhIP-treated mice have base substitutions at G:C base pairs in the gam gene. These mutants are remarkable because no deletions were detected in the gam gene. We can rule out the possibility that deletions in the gam gene were overlooked due to a technical error because most of this class of mutants exhibited a distinct phenotype: they could make plaques on a recA E.coli strain, whereas other types of Spi mutants could not. The phenotype of phage {lambda} that cannot produce a plaque on a recA strain is termed Fec (39). {lambda} red and gam double deletion mutants are Fec. We suspect that there are other mutations in the {lambda} DNA among the Spi mutants having a single base change without deletions in the gam gene. However, the molecular mechanisms of the mutations which exhibit Spi but not Fec are currently unknown.

In untreated mice 77% of the mutants analyzed were single base deletions in the gam gene (10/13). All of them were –1 frameshifts in runs of identical bases (Table IVGo). The predominant event was AAAAAA->AAAAA at nt 295–300. This mutation was repeatedly identified in Spi mutants recovered not only from the colon but also from the spleen of other untreated gpt delta transgenic mice (14). Therefore, this site appears to be a hot-spot for spontaneous Spi mutants. Three independent deletions, 6–7 kb in size, were also observed in the untreated mice. Two of these deletions have short homologous sequences (3 or 4 bp) in their junction regions and the other deletion has flush junctions. These are common characteristics of Spi mutants recovered from the spleen of {gamma}-irradiated transgenic mice (14).

In conclusion, gpt and Spi mutation spectra were generated in the colon of PhIP-treated gpt delta transgenic mice. The predominant mutations were G:C->T:A transversions and single G:C deletions in the gpt mutants. We observed that PhIP-induced single G:C deletions occur not only in runs but also beside run sequences using Spi selection. In the published p53 database, which includes 7433 p53 mutants recovered from various tumors (40), 1108 G:C->T:A transversions, 126 single G:C deletions in runs and 30 single G:C deletions beside run sequences are listed. However, the relevance of the G:C deletions that occur beside runs in colon carcinogenesis remains to be established. The gpt delta transgenic mouse model may be useful for the detailed molecular analysis of various types of mutations and for the investigation of the mechanisms of mutagenesis and carcinogenesis in multiple organs of rodents.


    Notes
 
4 To whom correspondence should be addressed Back


    Acknowledgments
 
We thank Dr Alasdair Gordon (RIKEN, Saitama, Japan) for critically reading the manuscript and helpful comments. This work was supported by Grants-in-Aid for Cancer Research from the Ministry of Health and Welfare, Japan, and from the Japan Human Science Foundation.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received April 11, 2000; revised June 19, 2000; accepted July 24, 2000.