Molecular characterization of X-ray-induced mutations at the HPRT locus in plateau-phase Chinese hamster ovary cells

CHO-K1 cells were irradiated in plateau phase to determine the effect of dose, dose fractionation, and delayed replating on the type, location and frequency of mutations induced by 250 kVp X-rays at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus. Independent HPRT-deficient cell lines were isolated from each group for Southern blot analysis using a hamster HPRT cDNA probe. When compared with irradiation with 4 Gy and immediate replating, dose fractionation (2 Gy + 24 h + 2 Gy) the entire gene. Since an increase in survival was noted under these conditions, these data suggest that repair of sublethal and potentially lethal damage acts equally on all premutagenic lesions, regardless of type or location. Differences in the mutation spectrum were noted when cells were irradiated at 2 Gy and replated immediately. The location of the deletion breakpoints was determined in 15 mutants showing partial loss of the HPRT locus. In 12 of these cell lines one or both of the breakpoints appeared to be located near the center of the gene, indicating a nonrandom distribution of mutations. These results indicate that damage induced by ionizing radiation results in a nonrandom distribution of genetic damage, suggesting that certain regions of the genome may be acutely sensitive to the mutagenic effects of ionizing radiation.     

Molecular characterization of mutations at the hprt locus in V79 Chinese hamster cells induced by bleomycin in the presence of inhibitors of DNA repair.

The molecular basis of bleomycin (BLM)-induced mutations in the absence and presence of inhibitors of DNA repair was investigated in V79 cells with Southern hybridization techniques. 43% of the BLM-induced thioguanine-resistant mutants suffer from large alterations of hprt DNA sequences. To understand the role of DNA repair in the process of mutagenesis, the effect of inhibitors of DNA repair on the frequency and types of BLM-induced mutations was tested. The inhibitors used were arabinofuranosyl cytosine (araC), didesoxythymidine (ddThd) and 3-aminobenzamide (3AB), which inhibit different steps of excision repair. Only 3AB caused a comutagenic effect. The increased mutation frequency was mainly due to additionally induced gene deletions. In the presence of 3AB, 70% of the HPRT-deficient mutants revealed partial or total deletions of the hprt coding sequences. Thus, it could be shown that BLM induces a broad range of types of mutation and that inhibited repair of BLM-induced DNA damage leads to specific types of mutations.     

Molecular analysis of mutation at the hprt locus of Chinese hamster V79 cells induced by ethyl methanesulphonate and mitomycin C

Mutation at the hprt locus of Chinese hamster V79 cells were induced by treatment with ethyl methanesulphonate (EMS), considered primarily a point mutagen and mitomycin C (MMC), a potent clastogen. EMS gave a dose-dependent induction of mutants while MMC induced a poor mutagenic response. Mutations were analysed using Southern and Northern blotting.Analysis of 9 EMS-induced and 4 spontaneous mutants yielded no detectable alterations in the hprt locus after digestion of DNA with 6 restriction enzymes. Mutants without detectable changes carried presumptive point mutations. In contrast, 4 out of 12 MMC-induced mutants had detectable alterations. 2 of these appeared to have lost the entire hprt gene while the other 2had prodable partial deletions. For these 4 deletion mutants no hprt mRNA was detected. 3 MMC-induced and 1 EMS-induced mutants had reduced levels of hprt mRNA. All the other mutants showed normal levels of hprt mRNA and the message detected was always of the correct size.It is suggested that the poor mutagenic response induced by MMC may be due to the lethal nature of large deletions involving both the hemizygous hprt locus and adjacent essential genes. This may lead to an underestimate of the mutagenicity of clastogenic agents such as MMC in the V79 HPRT mutation assay.     

Adenovirus-induced mutations at the hypoxanthine phosphoribosyltransferase locus of Chinese hamster cells.

Hpt-13 is a Chinese hamster cell line deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) and sensitive to a medium containing 10(-4) M hypoxanthine, 5.5 X 10(-6) M aminopterin, and 10(-4) M thymidine. In this cell line there is a high incidence of cells resistant to this selective medium after an incubation with either ethyl methane sulfonate or adenovirus type 2 complete virions or their incomplete particles. The rate of reversion in the presence of these agents was 34-fold higher with ethyl methane sulfonate and 2.5- to 5.6-fold higher with adenovirus particles than the spontaneous rate of reversion. The revertant phenotypes were stable for many generations without selective pressure. All of the revertants tested recovered the hypoxanthine phosphoribosyltransferase activity. Most of them, however, carried an enzyme of lower activity and faster electrophoretic mobility than that of the wild type. The preferential reversion to this type of enzyme was observed among spontaneous revertants as well as among those induced by mutagenesis with ethyl methane sulfonate or exposure to viral particles. Our results suggest that adenovirus particles and ethyl methane sulfonate induce mutations at the hpt locus of Hpt-13 cells through similar mechanisms.     

Molecular analysis of spontaneous mutations at the gpt locus in Chinese hamster ovary (AS52) cells

AS52 cells are Chinese hamster ovary (CHO) cells that carry a single functional copy of the bacterial gpt gene and allow the isolation of 6-thioguanine-resistant (6TGr)mutants arising from mutation at the chromosally integrated gpt locus. The gpt locus in AS52 cells is extremely stable, giving rise to 6TGr mutants at frequencies comparable to the endogenous CHO hprt locus. In this study, we describe the spectrum of spontaneous mutations observed in AS52 cells by Southern blot and DNA sequence analyses. Using the polymerase chain reaction (PCR) and the Thermus aquaticus (Taq) polymerase, we have enzymatically amplified 6TGr mutant gpt sequences in vitro. The PCR product was then sequenced without further cloning manipulations to directly identify gpt structural gene mutations. Deletions predominant among the 62 spontaneous 6TGr-AS52 mutant clones analyzed in this study. Of these, 79% (49/62) of the mutations were identified as deletions either by Southern blotting, PCR amplification or DNA sequence analysis. Among these deletions is a predominant 3-base deletion that was observed in 31% (19/62) of the mutants. These data provide a basis for future comparisons of induced point mutational spectra derived in the AS52 cell line, and demonstrate the utility of PCR in the generation of DNA sequence spectra derived from chromosomally integrated mammalian loci.     

Molecular analysis of spontaneous and ethyl methanesulphonate-induced mutations of the hprt gene in hamster cells

Independent spontaneous or ethyl methanesulphonate (EMS)-induced mutants lacking HPRT enzyme activity were analysed for changes in hprt gene structure. Of 21 spontaneous mutants, 6 had total gene deletions, 2 had partial gene deletions, and 13 were indistinguishable from wild-type by Southern analysis. In contrast a sample of 23 EMS-induced mutants, each of which showed potentially interesting characteristics (e.g. high reversion frequency, X-chromosome rearrangement), showed no detectable hprt gene changes. RNA isolated from 59 mutants with presumptive point mutations (13 spontaneous, 46 EMS-induced) was analysed on dot blots for changes in the amount of hprt mRNA. A wide range of mRNA levels was found, from mutants with undetectable amounts to those with more than wild-type amounts. However, Northern blots of all these mutant RNAs revealed only one (EMS-induced) mutation with a change in hprt mRNA size. Taken with our previously-published data on these mutants, it is argued that they represent a broad range of mutational types, and that the hprt gene mutation system provides a sensitive means of distinguishing mutational spectra of different DNA-damaging agents.     

Spontaneous splicing mutations at the dihydrofolate reductase locus in Chinese hamster ovary cells.

We isolated and characterized three spontaneous mutants of Chinese hamster ovary cells that were deficient in dihydrofolate reductase activity. All three mutants contained no detectable enzyme activity and produced dihydrofolate reductase mRNA species that were shorter than those of the wild type by about 120 bases. Six exons are normally represented in this mRNA; exon 5 was missing in all three mutant mRNAs. Nuclease S1 analysis of the three mutants indicated that during the processing of the mutant RNA, exon 4 was spliced to exon 6. The three mutant genes were cloned, and the regions around exons 4 and 5 were sequenced. In one mutant, the GT dinucleotide at the 5' end of intron 5 had changed to CT. In a second mutant, the first base in exon 5 had changed from G to T. In a revertant of this mutant, this base was further mutated to A, a return to a purine. Approximately 25% of the mRNA molecules in the revertant were spliced correctly to produce an enzyme with one presumed amino acid change. In the third mutant, the AG at the 3' end of intron 4 had changed to AA. A mutation that partially reversed the mutant phenotype had changed the dinucleotide at the 5' end of intron 4 from GT to AT. The splicing pattern in this revertant was consistent with the use of cryptic donor and acceptor splice sites close to the original sites to produce an mRNA with three base changes and a protein with two amino acid changes. These mutations argue against a scanning model for the selection of splice site pairs and suggest that only a single splice site need be inactivated to bring about efficient exon skipping (a regulatory mechanism for some genes). The fact that all three mutants analyzed exhibited exon 5 splicing mutations indicates that these splice sites are hot spots for spontaneous mutation.     

Quantification and analysis of reverse mutations at the hgprt locus in Chinese hamster ovary cells

We describe an assay for the quantification of reverse mutations at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus in Chinese hamster ovary cells utilizing the selective agent L-azaserine (AS). Conditions are defined in terms of optimal AS concentration, cell density, and phenotypic expression time. After treatment, replicate cultures of 10⁶ cells are allowed a 48-h phenotypic expression time in 100-mm plates. AS (10 μM) is then added directly to the growing culture and AS-resistant (AS^r) cells form visible colonies. This assay is used to quantify ICR-191-, ICR-170-, and N-ethyl-N-nitrosourea-induced reversion of independently isolated HGPRT^? clones. The AS^r phenotype is characterized both physiologically and biochemically. All AS^r clones isolated are stably resistant to AS and aminopterin but sensitive to 6-thioguanine. They also have re-expressed HGPRT enzyme. In addition, several revertants are shown to contain altered HGPRT. The data provide further evidence that ICR-191 and ICR-170 cause structural gene mutations in mammalian cells and also suggest that ICR-191, ICR-170, and N-ethyl-N-nitrosourea induce similar types of mutations in Chinese hamster ovary cells.     

The nature of ultraviolet light-induced mutations at the heterozygous aprt locus in Chinese hamster ovary cells

A system for studying mutational specificity at a heterozygous locus in Chinese hamster ovary (CHO) cells is described. The strategy employed is based on restriction fragment analysis and DNA sequencing of enzymatically amplified mutant adenine phosphoribosyltransferase (aprt) alleles. We have demonstrated the usefulness of this approach through the characterization of a collection of aprt- mutants with respect to the role played by loss of heterozygosity events in ultraviolet light (UV) induced mutagenesis. A similar strategy has also been applied to speculate on the identity of the premutational lesion responsible for a UV-induced mutational hotspot at the aprt locus.     

Molecular analysis of X-ray-induced mutants at the HPRT locus in V79 Chinese hamster cells

Spontaneous and X-ray-induced mutants at the hypoxanthine phosphoribosyl transferase (HPRT) locus have been isolated from V79 Chinese hamster cells and characterized at the biochemical and cytogenetic levels. Fourteen spontaneous and 24 X-ray-induced clones were azaguanine and thioguanine resistant, did not grow in HAT medium (AZRTGRHATS) and failed to incorporate significant levels of [14C]hypoxyanthine. Cytogenetic analysis of two spontaneous and eight X-ray-induced mutants revealed no major X chromosome rearrangements. In two induced mutants, one of which was hypotetraploid (mode 35-39) with 2 X chromosomes, the short arm of the chromosome (Xp) was slightly shorter than normal. A third mutant was hyperdiploid (mode 22-23) compared with the parental clone (mode 21). When compared with wild-type clones, no other cytogenetic changes were evident in the remaining mutants. Analysis at the DNA level using a Chinese hamster HPRT cDNA probe showed major deletion of HPRT sequences in two and partial deletion in another two induced mutants. In two of the mutants with deletions of HPRT sequences there was a visible shortening of the Xp arm. In the other six mutants two spontaneous and four induced) no karyotypic changes or alterations in restriction fragment patterns were detected suggesting that they carry small deletions or point mutations at the HPRT locus.     

Isolation and characterization of spontaneously occurring mutations at the HPRT locus in V79 Chinese hamster cells

The aim of the present investigation was to screen for rare types of spontaneously occurring mutational events in order to provide information on the organization of the mammalian genome. For this purpose a hierarchical sequence of analyses is used with a first step utilizing a forward reverse mutation approach. The present paper deals with the characterization of 22 isolated mutants from 2 groups, 11 spontaneously appearing mutants and, in comparison, 11 ethyl methanesulfonate-induced mutants at the HPRT locus in V79 Chinese hamster cells, by means of reverse mutation analyses using selection with medium containing L-azaserine. Nine out of the 11 mutant clones of each group could be reverted either spontaneously or induced by treatments with ethyl nitrosourea (ENU), ICR191 or 5-azacytidine (5AC), which indicates that they were caused by point mutations. Two of the revertible mutant clones of spontaneous origin were found to be resistant to HAT but not HAsT medium. These 2 6TGrHATr mutants were the only mutants isolated which could be affected by 5AC with a significant increase in reversion frequency. Chromosome aberration analysis did not indicate any enhancement in aberration frequency in the X-chromosome by 5AC treatment. Studies on the mutagenicity at the OUA locus indicated that the 5AC- and ENU-induced mutation frequencies in these 2 mutants were comparable to the effects in the parent wild-type cell line. Their cellular incorporation of 3H-hypoxanthine was enhanced in the presence of aminopterin, but decreased with L-azaserine indicating that they were phosphoribosyl pyrophosphate (PRPP) mutants. On the basis of these results, it is hypothesized that reversion of these 2 6TGrHATr mutants may occur by a gene amplification mechanism and that this process may be facilitated by 5AC treatment.     

The dose-response relationship for ultraviolet-light-induced mutations at the hypoxanthine-guanine phosphoribosyltransferase locus in Chinese hamster ovary cells.

Exposure of Chinese hamster ovary (CHO) cells clone K1BH4 to ultraviolet (UV) light at doses up to 86 ergs/mm2 did not significantly reduce cell survival, but UV doses of 86-648 ergs/mm2 produced an exponential cell killing. Observed mutation frequency ro 6-thioguannine resistance induced by UV increases approximately in proportion to increasing doses up to 260 ergs/mm2 in a range of 5-648 ergs/mm2 examined. The pooled data of mutation frequency f(X) as a function of dose X from 0-260 ergs/mm2 is adequately described by f(X)=10(-6) (13.6 + 2.04 X). That the UV-induced mutations to 6-thioguanine resistance affects the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus is supported by the observation that all randomly isolated drug-resistant colonies contained highly reduced or undetectable HGPRT activity.     

Molecular basis of spontaneous mutation at the aprt locus of hamster cells

Mutations occurring spontaneously at the hamster aprt locus were examined at the base-pair level by amplifying target sequences using the polymerase chain reaction and then directly sequencing the double-stranded products. In a collection of 89 sequenced genes, all types of mutations were found, with transitions (mostly G.C to A.T) constituting the largest class (35%), transversions accounting for 27%, and small deletions/duplications for 25%. Simple base substitutions were distributed throughout the aprt structural gene with few sites having recurring mutations and G.C base-pairs being the predominant substitution target. Small deletions, on the other hand, were not distributed so evenly, being concentrated in a region of aprt rich in short direct and inverted repeat sequences. The base substitutions were predominantly missense, while about 10% produced nonsense codons. Splice junctions, and start and stop codons were also significant targets for mutation. No alterations were detected in three aprt-deficient strains after sequencing all exons and substantial upstream and downstream regions.     

DNA sequence analysis of spontaneous mutations at the aprt locus of hamster cells.

To determine the nature of spontaneous mutational events in cellular genes in hamster cells, mutant adenine phosphoribosyltransferase (aprt) genes were cloned and the regions to which we mapped alterations were sequenced. A variety of nucleotide changes were found to occur in the 12 mutant genes analyzed. Most mutations were simple base-pair substitutions-transitions (both G X C----A X T and A X T----G X C) and transversions. The only multiple mutation was a simple transition next to a single-base-pair insertion. Of the 12 mutations, 4 were more complex, involving small deletions or duplications. Two of these were similar to previously described deletions in that they occurred between short direct sequence repeats. No hot spots were detected. Three independent mutations were characterized at one restriction endonuclease site, although no other mutations were detected in the nucleotides surrounding this site in other mutant strains. At a functional level, sequence changes were either in exons (resulting in missense and, in one instance, nonsense mutations) or at splicing sites.     

The dose-response relationship for ethyl methanesulfonate-induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells.

The frequency of ethyl methanesulfonate (EMS)-induced mutations to 6-thioguanine resistance in a Chinese hamster ovary cells clone K1-BH4 was studied at many EMS doses including the minimally lethal range (0-100 microng/ml) as well as the exponential killing portion (100-800 microng/ml) of the survival curve. The mutation frequency increases approximately in proportion with increasing EMS concentration at a fixed treatment time. The pooled data for the observed mutation frequency, f(X), as a function of EMS dose X, is adequately described by a linear function f(X)=10(-6)(8.73+3.45 X), where 0 less than or equal to X less than or equal to 800 microng/ml. One interpretation of the linear dose-response is that, as a result of EMS treatment, ethylation of cellular constituents occurs, which is directly responsible for the mutation. Biochemical analyses demonstrate that most of the randomly isolated 6-thioguanine-resistant variants possess a highly reduced or undetectable level of HGPRT activity suggesting that the EMS-induced mutations to 6-thioguanine resistance affect primarily, if not exclusively, the HGPRT locus.     

Structure of mutant alleles at the aprt locus of Chinese hamster ovary cells

To determine the types of gene structural alterations causing deficiency of adenine phosphoribosyl transferase (aprt) activity in spontaneous and chemically induced mutations of cultured somatic cells, we analyzed the restriction enzyme cleavage patterns of aprt gene sequences in mutant strains selected from Chinese hamster ovary cells. Patterns of aprt-containing fragments in Southern blots were mostly unchanged in our collection of 280 ethyl methane sulfonate-induced and spontaneous aprt- mutants, suggesting that base-pair changes or other alterations below our limit of resolution on agarose gels (approximately 50 base-pairs) are responsible for the great majority of mutations at the aprt locus. Occasionally, these mutations could be localized when they resulted in the loss or gain of a restriction enzyme site and the generation of new fragments of predictable size. Deletions of aprt-containing sequences were detected in only eight of 119 spontaneous mutants and in only one ethyl methane sulfonate-induced mutant. An insertion of 300 base-pairs near the 5' end of the aprt structural gene was found in one spontaneous aprt- strain. This insertion mutant was stable with a reversion frequency of less than 2 X 10(-7). Several unstable aprt- mutants were detected in our collection, but these had no observable alterations of aprt coding or flanking sequences.