Aprt Locus Research Articles

The restriction enzyme Mse I applied for the detection of a possibly common mutation of the APRT locus.

The restriction enzyme Mse I applied for the detection of a possibly common mutation of the APRT locus.

Differences in the rate of DNA adduct removal and the efficiency of mutagenesis for two benzo[ a]pyrene diol epoxides in CHO cells

The initiation of carcinogenesis by carcinogens such as 7 r,8 t-dihydroxy-9,10 t-oxy-7,8,9,10-tetrahydrobenzo[ a]pyrene (BPDE-I) is thought to involve the formation of DNA adducts. However, the diastereomeric diol epoxide, 7 r,8 t-dihydroxy-9,10 c-oxy-7,8,9,10-tetrahydrobenzo[ a]pyrene (BPDE-II), also forms DNA adducts but is inactive in standard carcinogenesis models. We have measured the formation and loss of DNA adducts derived from BPDE-II in a DNA-repair-proficient line of Chinese hamster ovary (CHO) cells, AT3-2, and in two derived mutant cell lines, UVL-1 and UVL-10, which are unable to repair bulky DNA adducts. BPDE-II adducts were lost from cellular DNA in AT3-2 cells with a half-life of 13.8 h; this was about twice the rate found for BPDE-I adducts. BPDE-II adducts were also lost from DNA in UVL-1 and UVL-10 cells, but at a much slower rate. When purified DNA was modified in vitro with BPDE-II and then held at 37°C, DNA adducts were removed at a rate identical to that seen in UVL-1 and UVL-10 cells, suggesting that the loss in these cells was not due to enzymatic DNA-repair processes but to chemical lability of the adducts. Mutant frequencies at the APRT and HPRT loci were measured at BPDE-II doses that resulted in > 20% survival, and were found to increase linearly with dose. In the DNA-repair-deficient cells, the HPRT locus was moderately hypermutable compared with AT3-2 cells (about 5-fold); the APRT locus was extremely hypermutable, giving about 25-fold higher mutant fractions in UVL-1 and UVL-10 than in AT3-2 cells at equal initial levels of binding. When we compared the mutational efficiency of BPDE-II at both loci in AT3-2 cells (the mutant frequency in mutants/10 6 survivors at a dose that resulted in one adduct per 10 6 base pairs) with our previous studies of BPDE-I, we found that BPDE-II was 4–5 times less efficient as a mutagen than BPDE-I. This difference in mutational efficiency could be explained in part by the increased rate of loss of BPDE-II adducts from the cellular DNA, part of which was due to an increased rate of enzymatic removal of these lesions compared with the removal of BPDE-I adducts.

Low absolute mutagenic efficiency but high cytotoxicity of a non-bay region diol epoxide derived from benzo[ a]pyrene

Insights into the mechanisms of chemical carcinogenesis can sometimes be gained by comparing the effects of closely related chemicals which differ in carcinogenic potency. We have treated Chinese hamster ovary (CHO) cells with a non-carcinogenic metabolite of benzo[ a]pyrene, 9 r,10 t-dihydroxy-7 c,8 c-oxy-7,8,9,10-tetrahydrobenzo[ a]pyrene (BPDE-III), and measured the formation and persistence of DNA adducts. We have correlated this binding data with cytotoxicity and mutagenicity in a DNA-repair-proficient CHO cell line (AT3-2) and in two derived lines, UVL-1 and UVL-10, which are unable to repair bulky DNA adducts. These data are compared with similar studies of the effects of the carcinogenic metabolite, 7 r,8 t-dihydroxy-9 t,10 t-oxy-7,8,9,10-tetrahydrobenzo[ a]pyrene (BPDE-I). Synchronous fluorescence spectroscopy was used to measure the levels of BPDE-III-DNA adducts in treated cells. Adduct levels increased linearly with dose, but the absolute binding levels were about 30-fold lower than in comparable incubations with BPDE-I. Measurements of the removal of adducts derived from these two diol epoxides indicated no significant difference in the rate of repair measured 24 h post-treatment. When cells were treated with increasing doses of BPDE-III, survival curves were obtained which exhibited a shoulder region at low doses and an exponential decrease in plating efficiency at higher doses. By comparison of the D 0's, the DNA-repair-deficient cell lines were found to be 4–5 fold more sensitive to the killing effects of BPDE-III than were the repair-proficient AT3-2 cells. Mutants at the APRT and HPRT loci were selected 1 week after treatment with BPDE-III at doses which resulted in > 10% survival. Mutant frequencies at both loci were found to increase linearly with dose. In the DNA-repair-deficient cells, both the HPRT and APRT loci were hypermutable, giving 9–15-fold higher mutant fractions in UVL-1 and UVL-10 than in AT3-2 cells at equal initial doses. When we compared the mutational efficiency of BPDE-III at both loci in AT3-2 cells (the mutant frequency in mutants/10 6 survivors at a dose which results in 1 adduct per 10 6 base pairs) with out previous studies of BPDE-I, we found that BPDE-III was 4–5 times less efficient as a mutagen than BPDE-I. However, a similar comparison of cytotoxic efficiency on a per adduct formed basis, revealed that BPDE-III was 2–3-fold more effective than BPDE-I. Since we have found no difference in the rate of removal of BPDE-I and BPDE-III adducts, these data suggest that BPDE-III adducts are inherently less mutagenic but more cytotoxic than are BPDE-I adducts.

Ionizing radiation and genetic risks III. Nature of spontaneous and radiation-induced mutations in mammalian in vitro systems and mechanisms of induction of mutations by radiation

This paper (1) presents an analysis of published data on the molecular nature of spontaneously arising and radiation-induced mutations in mammalian somatic cell systems and (2) examines whether the molecular nature and mechanisms of origin of radiation-induced mutations, in mammalian in vivo and in vitro systems, as currently understood, are consistent with expectations based on the biophysical and microdosimetric properties of ionizing radiation. Depending on the test system (CHO cells, human T lymphocytes and human lymphoid cell line TK6), 80–97% of spontaneous HPRT mutations show normal Southern patterns; the remainder is due to gross changes, predominantly partial (intragenic) deletions. Total gene deletions at the HPRT locus are rare except in the TK6 cell line. At the APRT locus in CHO cells, 80–97% of spontaneous mutations are due to base-pair changes, the remainder being, mostly, partial delections. The latter can extend upstream in the 5′ direction but not beyond the APRT gene in the 3′ direction. At the human HLA-A locus (T lymphocytes), the percentage of mutations with normal Southern patterns is lower than that for HPRT, and in the range of 50–60%. At the HLA-A locus, mitotic recombination contributes substantially to the mutation spectrum (∼ 30% of mutations recovered) and this is likely to be true of the TK locus in the TK6 cell line as well. With a few exceptions, most of the radiation-induced mutations show altered Southern patterns and are consistent with their being deletions and/or other gross changes (HPRT, 70–90% (CHO); 50–85% (TK6); 50–75% (T lymphocytes); TK, 60–80% (TK6); HLA-A, 80% (T lymphocytes); DHFR, 100% (CHO)). The exceptions are APRT mutations in CHO cells (16–20% of mutants with deletions or other changes) and HPRT mutations in T lymphocytes from A-bomb survivors (15–25%); the latter finding is consistent with the occurrence of in vivo selection against HPRT mutant cells. In cases of HPRT intragenic deletions analyzed (CHO cells and V79 Chinese hamster cells), there is evidence for a non-random distribution of breakpoints. The spontaneous mutation frequencies vary widely, from about 0.04/10 6 cells (sickle cell mutations at the human HBB locus) to 30.8/10 6 cells HLA-A mutations in T lymphocytes) and are dependent on the locus, the system employed and a number of other factors. Those for the other loci fall between these limits. The rates of induction by X-rays again are quite low for the HBB locus (0.07–1.5/10 6 cells/Gy; only two types of specific point mutations were scored), low for DHFR (1.6/10 6 cells/Gy) and APRT (4.3/10 6 cells/Gy), in the range of about 10–30/10 6 cells/Gy for most others, and are highest for the HLA-A, and glycophorin A locus (the NØ variant) (about 60/10 6/cells/Gy). Gene size alone cannot explain the differences between either spontaneous frequencies or induction rates. On the basis of chromatin and DNA organization in cells and the biophysical and microdosimetric properties of ionizing radiation, one can qualitatively explain the predominance of deletions among radiation-induced mutations. For spontaneously arising and radiation-induced point mutations, there may be common elements in mechanisms, but for spontaneously arising and induced deletions, the extent of overlap in mechanisms is difficult to discern at present.

Introduction of cytochrome P450IA2 metabolic capability into cell lines genetically matched for DNA repair proficiency/deficiency.

We introduced into the CHO cell line the cDNA of the mouse cytochrome P3450 (P450IA2) gene, which oxidizes aromatic amines. A cDNA clone of P3450 was transfected into mutant UV5 cells, which is defective in nucleotide excision repair. Expression of the P3450 cDNA was measured using 9000 x g supernatant (S9) fractions from CHO cells to evaluate Salmonella TA1538 mutagenicity with the mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). The P3450-expressing clone UV5P3 was reverted to repair proficiency using ethyl methanesulfonate to obtain the UV-resistant clone 5P3R2, which maintained the same level of P3450 protein activity as UV5P3. These genetically similar cell lines were compared for toxicity and mutation induction at the aprt locus. With 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (the most prevalent mutagen found in fried beef) the differential sensitivity due to repair deficiency/proficiency was approximately 40-fold, and with IQ there were smaller, but significant, differences in sensitivity. These genotoxic effects occurred at doses that were approximately 10 times lower than those that previously gave similar effects in S9-mediated assays. Thus, these cell lines should be valuable for genotoxicity analysis as well as important for assessing DNA repair when evaluating compounds that undergo metabolic activation.

A very large spontaneous deletion ataprt locus in CHO cells: Sequence similarities with smallaprt deletions

Spontaneous deletion mutants can be isolated from CHO cell lines heterozygous at the adenine phosphoribosyltransferase locus at frequencies up to 10(-4), i.e., about 100-fold higher than spontaneous point mutations. Indirect evidence has suggested that most deletions were in the megabase range. We have fully characterized one such mutant, Del 155, and shown that it resulted from an illegitimate recombination that took place between overlapping tetranucleotides. Comparisons with sequences of other deletions at various loci revealed a number of similarities, most striking of which was a CHI-like motif found within 6 bp of the upstream breakpoint of Del 155 and breakpoints of 8/21 previously described short deletions at the CHO aprt locus. Homology also existed between the downstream breakpoint of Del 155 and breakpoints of retinoblastoma gene deletions (3/6 cases) and also a 20-bp stretch of an Alu sequence in which breakpoints at the low-density lipoprotein receptor locus have been shown to cluster. The magnitude of the deletion event in Del 155 was assessed by pulsed field (PF) gel analysis and found to be at least 2100 kb long. PF analysis also indicated that the downstream breakpoint was near a region of structural differences between the two chromosomes carrying apart. These structural differences were probably not implicated in the mechanism of the high frequency event, since no indication of breakpoint clustering among a large collection of mutants was found either by conventional or PF electrophoretic analyses.

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 in described. The strategy employed is based on restriction fragment analysis and DNA sequencing of enzymatically amplified mutant adenine phosphoribosyltransferase ( arpt) 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 resposible for a UV-induced mutational hotspot at the aprt locus.

Targeted gene replacement at the endogenousAPRT locus in CHO cells

We demonstrate the feasibility of targeted gene replacement at an endogenous, chromosomal gene locus in cultured mammalian cells, employing a two-step strategy similar to an approach routinely used for genetic manipulation in yeast. Utilizing an APRT+ recombinant generated by targeted integration of plasmid sequences (including a functional copy of the gpt gene) at the CHO APRT locus, we have been able to select gpt- "pop-out" recombinants that have arisen by intrachromosomal recombination between APRT direct repeats at the targeted integration site. Reciprocal exchanges leading to "pop-out" of integrated plasmid/gpt gene sequences occur at a rate of approximately 6.3 x 10(-6) per cell generation. Depending on the site of crossover, such "pop-out" events result in either replacement or restoration of the original APRT target gene sequence.

Loss of heterozygosity in mammalian cell mntagenesis: molecular analysis of spontaneous mutations at the aprt locus in CHO cells

Loss of heterozygosity at previously heterozygous loci may occur by one of several possible mechanisms and account for a large fraction of all mutations occurring at such loci. In order to investigate loss of heterozygosity events, we have chosen the aprt locus of Chinese hamster ovary (CHO) cells as our model since it is readily available in either heterozygous or hemizygous form. Cloning and sequencing of the two heterozygous aprt alleles from the CHO derivative D423 identified a single polymorphic site, which does not create a restriction fragment length polymorphism. In order to evaluate the loss of heterozygosity events at this locus, we devised a method that creates an artificial restriction fragment length polymorphism in one of these two alleles as a direct consequence of enzymatic amplification. Restriction enzyme digestion of the amplified sequences can then conveniently identify the genotype of the DNA sample. This same methodology also provides for the selective cloning of only one allele of a heterozygous pair into a plasmid vector for subsequent DNA sequence analysis, and can be easily adapted to other situations requiring the analysis of single base changes at a particular position within known sequences. Using this technique, we have determined that 16/37 (43%) spontaneous APRT- mutants had undergone a loss of heterozygosity event.

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 region.

DNA sequence determination of γ-radiation-induced mutations of the hamster aprt locus

From a collection of 85 independent γ-radiation hamster aprt − mutants, 27 having no major structural alterations were analysed at the nucleotide level by using the polymerase chain reaction to amplify mutant exons and then directly sequencing the double-stranded products. The majority of these mutations were simple base substitutions of all types, particularly transversions (11/27). Frameshifts and small deletions were also induced. The ‘spectrum’ of mutations produced by γ-radiation was not significantly different from that occurring spontaneously at this locus. Differences with respect to the target and structure of frameshifts and small deletions occurring in the two collections were apparent.

Perspectives on UV light mutagenesis: investigation of the CHO aprt gene carried on a retroviral shuttle vector.

The extent to which the cellular processing of shuttle vector-carried genes reflects that of endogenous chromosomal loci has been a subject of considerable controversy. In order to address this issue, we have developed a retroviral-based shuttle vector carrying the Chinese hamster ovary (CHO) adenine phosphoribosyltransferase (aprt) gene stably integrated into the genome to be used for studying mutational specificity in mammalian cells. Initially, we have characterized a collection of UV-induced mutants in a CHO cell background. We have therefore been able to directly compare this shuttle vector data to that previously obtained for UV-induced mutation at the endogenous CHO (aprt) locus. Although some potential differences between the two spectra have been noted, there appears to be a remarkable similarity in the distribution and site specificity of UV-induced mutations. These similarities extend to extrachromosomal shuttle vectors as well and consolidate the role of shuttle vectors as powerful analytical tools for studying mechanisms of point mutagenesis in mammalian cells.

Mutational specificity studies of endogenous mammalian cell loci: methodological aspects

We report here the details of a modified cloning method first described by Seed et al. (1983) for use in an extensive study of mutational specificity at the aprt locus of Chinese hamster ovary cells. The technology depends on homologous recombination between a suppressor probe plasmid and the desired insert in a genomic library constructed in a double amber mutant bacteriophage λ vector. Recombinant phage form blue plaques on a non-suppressor, lacZ amber host. We have determined the sensitivity of the method. Homologous recombination frequency is in the order of 10 −3, 6–7 orders of magnitude above non-homologous events. Recombination efficiency is unaffected when the target phage is diluted 100,000-fold with parental vector. A background of plaques is observed along with the desired blue plaques. Although the color discrimination permits us to easily avoid these background plaques, we have characterized the frequency and the nature of these events.

Analysis of APRT deficient mutants of friend erythroleukaemia cells

Cytogenetic analysis of mutants of the Friend Erythroleukaemia cell line deficient in adenine phosphoribosyl transferase (Aprt) was undertaken to ascertain whether nondisjunctional events were involved in the production of this mutation. All mutant clones examined were found to carry two copies of chromosome 8, the chromosome to which Aprt maps. Since the two homologues are distinguishable by silver staining, it was also clear that no mutants contained two copies of one homologue having lost the other. Treatment of mutants with 5-azacytidine failed to reactivate the Aprt locus.

A Novel Pathway for Transversion Mutation Induced by dCTP Misincorporation in a Mutator Strain of CHO Cells

Intracellular imbalances of dCTP produce both T----C transitions and an unusual class of transversions (A----C) at the aprt locus of CHO cells. Our data suggest that this transversion pathway is the consequence of dCTP:T mispairs which are not efficiently proofread during DNA replication.

A novel pathway for transversion mutation induced by dCTP misincorporation in a mutator strain of CHO cells.

Intracellular imbalances of dCTP produce both T----C transitions and an unusual class of transversions (A----C) at the aprt locus of CHO cells. Our data suggest that this transversion pathway is the consequence of dCTP:T mispairs which are not efficiently proofread during DNA replication.

CHO mutant UV61 removes (6-4) photoproducts but not cyclobutane dimers.

The CHO mutant UV61 was previously assigned to complementation group 6 of UV-sensitive rodent cell mutants. UV61 is less sensitive to killing by UV radiation than mutants such as UV5, which is highly defective in the incision process that acts on UV-induced lesions. The D37 for cell survival is approximately 4 J/m2 for UV61, compared with 10 J/m2 for the parental AA8 line and approximately 2 J/m2 for UV5. Similarly, mutation induction at the hprt and aprt loci shows an intermediate response to UV61. In a post-replication recovery assay, the kinetics of maturation of pulse-labelled nascent DNA were normal after UV irradiation in UV61. Data from alkaline elution and alkaline unwinding assays showed that the rates of break accumulation and resealing, measured 0-120 min after irradiation, were also normal in the mutant. This repair incision correlated with the rapid, normal removal of pyrimidine(6-4)pyrimidone photoproducts in UV61 measured using a radioimmunoassay that is specific for this class of damage. In contrast, after exposure to 10 or 15 J/m2, no detectable removal of cyclobutane dimers from DNA was found in UV61 while AA8 cells removed 32% by 24 h. We suggest that the mutation in UV61 specifically lowers the affinity of a repair protein for cyclobutane dimers, which are also inefficiently removed from the bulk DNA of normal CHO cells. The resistance of UV61 to killing by the direct acting chemical 7-bromomethylbenz[a]anthracene was only slightly greater than that of UV5, indicating defective repair of bulky chemical adducts in addition to cyclobutane dimers.(ABSTRACT TRUNCATED AT 250 WORDS)

High-frequency deletion event at aprt locus of CHO cells: detection and characterization of endpoints.

Two mechanisms are implicated in generating recessive drug resistance mutants at the adenine phosphoribosyltransferase (aprt) locus of Chinese hamster ovary (CHO) cells, one of which is a spontaneous high-frequency deletion of the entire gene. We have isolated and mapped a 19-kb fragment carrying aprt and its flanking sequences. A Southern blot study of 198 independent deletion mutants revealed that two different mutants have one of their breakpoints within the 19-kb region analyzed. One of these has an upstream breakpoint which could be narrowed down to a 4-kb fragment containing repetitive sequences. The other mutant has a breakpoint within a 410-bp sequence located 8.5 kb downstream of the aprt gene and which carries several elements similar to those signaling V-(D)-J joining in immunoglobulin and T-cell receptor gene rearrangements. In each case the other breakpoint lay outside of the analyzed region. These results support the previous indications that the deletions created by this spontaneous event are large.

A genetic analysis of the adenine phosphoribosyl transferase locus in Chinese hamster V79-AP4 cells: relevance to mutagenesis studies

The chromosomal location of the autosomal locus aprt has been investigated in the permanent Chinese hamster cell line V79-AP4 by standard somatic cell genetics methodologies. Aprt is functionally dizygous in V79-AP4 and the a alleles map on 2 chromosome 3 homologs, in agreement with the chromosome assignment of the gene in Chinese hamster primary cells. Chromosome G-banding and a Southern blot analysis of V79-AP4 DNA, using as a probe the cloned Chinese hamster aprt gene, have not revealed any structural alteration at either of the 2 aprt alleles. One of the chromosomes 3 has, however, a terminal deletion in its long arm and is therefore morphologically marked. These findings could make V79-AP4 an interesting cell system for the study of mutational mechanisms at the aprt locus in Chinese hamster.

Gene dosage mutants at adenine phosphoribosyltransferase locus induced by colcemid in Chinese hamster V79-AP4 cells.

Pseudodiploid Chinese hamster V79-AP4 cells, functionally diploid at the adenine phosphoribosyltransferase (aprt) locus, were treated with colcemid, a well-known aneuploidizing agent, under various experimental conditions. Aneuploid and tetraploid cells and variants resistant to 10 micrograms/ml of 2,6-diaminopurine (DAP), which selects for presumptive aprt+/- heterozygotes in the untreated cells, were induced. Many of the induced variants were hypotetraploid with three (rather than four) chromosomes carrying the aprt gene. Dot-blot and Southern analysis of the DNA of these clones confirmed that they had three copies of the aprt gene. Their APRT specific enzymatic activity was 60-80% of that of wild-type V79-AP4. The results of these and other experiments suggest that in these variants resistance to DAP is due to an altered aprt gene dosage and point to a possible genetic effect of colcemid and other aneuploidizing agents in somatic mammalian cells.