Assay to measure CD59 mutations in CHO A(L) cells using flow cytometry.

BACKGROUND: A sensitive mammalian cell mutation assay was developed previously using a Chinese hamster ovary cell line (CHO A(L)) that stably incorporates human chromosome 11. The assay measures mutations in the CD59 gene on chromosome 11 but it requires the use of rabbit complement and colony growth for mutant selection. We have developed a more rapid flow cytometry-based mutation assay with CHO A(L) cells that uses monoclonal antibodies against CD59 to detect mutants and does not require colony formation. METHODS: CHO A(L) cells were treated with gamma-radiation or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and then allowed to grow for various times for mutant expression. Cells were labeled with monoclonal antibodies against CD59 and analyzed by flow cytometry. RESULTS: Negative and positive populations were separated by over 100-fold. Mixing various proportions of CD59-positive and -negative cells demonstrated that the assay is highly linear (r2 = 0.9999) and sensitive (<0.05% background mutants). The yield of CD59-inducible mutants was linearly related to dose for a clastogen (gamma-radiation) and point mutagen (MNNG). The mutant yield was time and treatment specific. CONCLUSIONS: Mutations induced by genotoxic agents can be rapidly and sensitively measured in CHO A(L) cells using flow cytometry.     

Multiparameter analysis of CHO AL mutant populations sorted on CD59 expression after gamma irradiation

The flow cytometry mutation assay is based on detecting mutations in the CD59 gene on human chromosome 11 in CHO A(L) cells with flow cytometry, but the kinetics of mutant expression and the histogram region for mutant selection have not been studied in detail. CHO A(L) cells were analyzed by flow cytometry for CD59 expression at various times after irradiation. The mutant fraction increased to a maximum at day 6 but decreased to near background levels by day 20. Cells were sorted from six different regions on the CD59 histograms after irradiation. The growth rate was similar for cells from all regions, and the surviving fraction was 50% of that for control cells. By 14 days the CD59 expression of cells from regions 2-5 was reduced to that of region 1. Cells were also analyzed for simultaneous expression of CD59, CD44 and CD90 (all on chromosome 11) to roughly characterize the size of the mutations. Triple mutants from the sorted populations were reduced from 41% on day 6 to 8% on day 24. We conclude that the mutant region should be increased to include cells with intermediate CD59 expression; also, the loss of CD59 mutant expression over time could be explained in part by the loss of triple mutants from the population.     

Quantification of CD59- mutants in human-hamster hybrid (AL) cells by flow cytometry

Mutation assay is an important approach in evaluating the genotoxic risk of potentially harmful environmental chemicals. The human-hamster hybrid (A(L)) cell mutagenesis system, based on the complement/antibody-mediated cytotoxicity principle, has been used successfully to evaluate the mutagenic potential of a variety of environmental toxicants. The A(L) cells contain a standard set of CHO chromosomes and a single human chromosome 11, which expresses several cell surface proteins including CD59 encoded by the CD59 gene at 11p13.5. A modified mutation assay by flow cytometry was developed to determine the yield of CD59- mutants after either radiation or chemical treatment. After incubation with phycoerythrin-conjugated mouse monoclonal anti-CD59 antibody, the CD59- mutant yields were determined by quantifying the fluorescence of the cells using flow cytometry. This method is faster and eliminates the commonly encountered toxicity problems of the complements with the traditional complement/antibody assay. By comparing the mutant fractions of radiation or chemically treated A(L) cultures using the two methods, we show here that the flow cytometry assay is an excellent substitute in providing an efficient and highly sensitive method in mutant detection for the traditional complement/antibody assay.     

Measuring the spectrum of mutation induced by nitrogen ions and protons in the human-hamster hybrid cell line A(L)C

Astronauts can be exposed to charged particles, including protons, alpha particles and heavier ions, during space flights. Therefore, studying the biological effectiveness of these sparsely and densely ionizing radiations is important to understanding the potential health effects for astronauts. We evaluated the mutagenic effectiveness of sparsely ionizing 55 MeV protons and densely ionizing 32 MeV/nucleon nitrogen ions using cells of two human-hamster cell lines, A(L) and A(L)C. We have previously characterized a spectrum of mutations, including megabase deletions, in human chromosome 11, the sole human chromosome in the human-hamster hybrid cell lines A(L)C and A(L). CD59(-) mutants have lost expression of a human cell surface antigen encoded by the CD59 gene located at 11p13. Deletion of genes located on the tip of the short arm of 11 (11p15.5) is lethal to the A(L) hybrid, so that CD59 mutants that lose the entire chromosome 11 die and escape detection. In contrast, deletion of the 11p15.5 region is not lethal in the hybrid A(L)C, allowing for the detection of chromosome loss or other chromosomal mutations involving 11p15.5. The 55 MeV protons and 32 MeV/nucleon nitrogen ions were each about 10 times more mutagenic per unit dose at the CD59 locus in A(L)C cells than in A(L) cells. In the case of nitrogen ions, the mutations observed in A(L)C cells were predominantly due to chromosome loss events or 11p deletions, often containing a breakpoint in the pericentromeric region. The increase in the CD59(-) mutant fraction for A(L)C cells exposed to protons was associated with either translocation of portions of 11q onto a hamster chromosome, or discontinuous or "skipping" mutations. We demonstrate here that A(L)C cells are a powerful tool that will aid in the understanding of the mutagenic effects of different types of ionizing radiation.     

Frequency of CD59 mutations induced in human-hamster hybrid A(L) cells by low-dose X-irradiation

Determination of the genotoxic effects of ionizing radiation, especially at low-doses, is of great importance for risk assessment, e.g. in radiological diagnostics. The human-hamster hybrid A(L) cell line has been shown previously to be a well-suited in vitro model for the study of mutations induced by various mutagens. The A(L) cells contain a standard set of hamster chromosomes and a single human chromosome 11, which confers the expression of the human cell surface protein CD59. Using CD59 specific antibodies, cells mutated in the CD59 gene can be detected and quantified by the loss of the cell surface marker. In contrast to previous studies, prior to irradiation we removed spontaneous mutants by magnetic cell separation (MACS) which allows analysis of radiation-induced mutation events only. We exposed A(L) cells to 100kV X-rays at 0.1 to 5Gy. The proportions of X-irradiation-induced CD59(-) mutants were quantified by flow cytometry after immunofluorescence labeling. Between 0.2 and 5Gy the yield of CD59 mutants was a linear function of dose. The molecular analysis of individual CD59-negative clones induced after exposure of 1, 3 and 5Gy of X-ray revealed a dose-dependent linear increase of large deletions (>6Mbp), whereas, point mutations could be seen only in spontaneous CD59 mutants or after low-dose exposure (< or =1Gy). We conclude that the modified A(L) assay presented here is appropriate for detection and quantification of non-lethal DNA lesions induced by low-dose ionizing radiation.     

Comparison of the mutagenic potential of 17 physical and chemical agents analyzed by the flow cytometry mutation assay

Several methods to assess genotoxicity of physical and chemical agents have been developed, most of which depend on growing colonies in selective medium. We recently published a new method for detecting mutations in the CD59 gene in a Chinese hamster ovary cell line that contains a single copy of human chromosome 11 (CHO A_L). The assay is based on detecting the surface expression of CD59 with monoclonal antibodies using flow cytometry. The capabilities of this flow cytometry mutation assay (FCMA) to detect mutations from a wide variety of genotoxic agents are described here. There was a 400-fold separation between CD59⁻ and CD59⁺ populations based on fluorescence intensity. Small numbers of negative cells mixed in with positive cells were detected in a highly linear fashion. Mutation dose response curves over a dose range yielding 80% to 20% survival are shown for ethyl methane sulfonate (EMS), mitomycin C (MMC) and lead acetate. EMS and lead acetate exhibited a threshold in response while MMC had a linear dose response over the full dose range. The mutant fraction was measured over time periods ranging up to 35 days following treatment. The mutant fraction peaked at different times ranging from 6 to 12 days after treatment. An additional 14 chemical and physical agents including point mutagens, heavy metals, ionizing and UV radiation, and DNA intercalators and cross linkers, were analyzed for mutagenic potential after doses giving 80% to 20% survival. The results presented here demonstrate the sensitivity and broad-ranging capability of the FCMA to detect mutations induced by a variety of genotoxic agents.     

Gamma-ray mutagenesis studies in a new human-hamster hybrid, A(L)CD59(+/-), which has two human chromosomes 11 but is hemizygous for the CD59 gene.

Kraemer, S. M., Vannais, D. B., Kronenberg, A., Ueno, A. and Waldren, C. A. Gamma-Ray Mutagenesis Studies in a New Human-Hamster Hybrid, A(L)CD59(+/-), which has Two Human Chromosomes 11 but is Hemizygous for the CD59 Gene. Radiat. Res. 156, 10-19 (2001).We have developed a human-CHO hybrid cell line, named A(L)CD59(+/-), which has two copies of human chromosome 11 but is hemizygous for the CD59 gene and the CD59 cell surface antigen that it encodes. Our previous studies used the A(L) and A(L)C hybrids that respectively contain one or two sets of CHO chromosomes plus a single copy of human chromosome 11. The CD59 gene at 11p13.5 and the CD59 antigen encoded by it are the principal markers used in our mutagenesis studies. The hybrid A(L)CD59(+/-) contains two copies of human chromosome 11, only one of which carries the CD59 gene. The incidence of CD59 (-) mutants (formerly called S1(-)) induced by (137)Cs gamma rays is about fivefold greater in A(L)CD59(+/-) cells than in A(L) cells. Evidence is presented that this increase in mutant yield is due to the increased induction of certain classes of large chromosomal mutations that are lethal to A(L) cells but are tolerated in the A(L)CD59(+/-) hybrid. In addition, significantly more of the CD59 (-) mutants induced by (137)Cs gamma rays in A(L)CD59(+/-) cells display chromosomal instability than in A(L) cells. On the other hand, the yield of gamma-ray-induced CD59 (-) mutants in A(L)CD59(+/-) cells is half that of the A(L)C hybrid, which also tolerates very large mutations but has only one copy of human chromosome 11. We interpret the difference in mutability as evidence that repair processes involving the homologous chromosomes 11 play a role in determining mutant yields. The A(L)CD59(+/-) hybrid provides a useful new tool for quantifying mutagenesis and shedding light on mechanisms of genetic instability and mutagenesis.     

A role for long-lived radicals (LLR) in radiation-induced mutation and persistent chromosomal instability: counteraction by ascorbate and RibCys but not DMSO.

Miazaki, Watanabe, Kumagai and their colleagues reported that induction of HPRT(-) mutants by X-rays in cultured human cells was prevented by ascorbate added 30min after irradiation. They attributed extinction of induced mutation to neutralization by ascorbate of radiation-induced long-lived mutagenic radicals (LLR), found using spectroscopy to have half-lives of minutes or hours. We find that post-irradiation treatment with ascorbate reduces, but does not eliminate, induction of CD59(-) mutants in human-hamster hybrid A(L) cells exposed to high-LET carbon-ions (LET of 100KeV/microm). A(L) cells contain a standard set of Chinese hamster ovary (CHO) chromosomes and a single copy of human chromosome 11 containing the CD59 gene which encodes the CD59 cell surface antigen, a convenient marker for mutation. RibCys [2(R, S)-D-ribo-(1',2',3',4'-tetrahydroxybutyl)thiazolidine-4(R)-carboxylic acid] a 'prodrug' of l-cysteine which also scavenges LLR, had a similar but lesser effect on induced mutation. DMSO, which scavenges classical radicals like H* and OH* but not LLR, also reduced mutation, but only when it was present during irradiation. The lethality of carbon-ions was not altered by ascorbate, RibCys no matter when added. Post-radiation addition of ascorbate and RibCys also affected the quality of CD59(-) mutations induced by carbon-ions. The major change in mutant spectra was a reduction in the prevalence of small, intragenic mutations (mutations not detected by PCR) and in the prevalence of unstable, complicated mutants, which display high levels of persistent chromosomal instability. Thus, ascorbate and RibCys may suppress some kinds of mutations induced by ionizing radiation including those displaying aspects of radiation-induced genomic instability. Countering the effects of both classical radicals and LLR may be important in preventing genetic diseases.     

Increased chromosomal radiosensitivity of a Chinese hamster ovary cell line that inducibly expresses the Eco RI restriction endonuclease

We have transfected a Chinese hamster ovary cell line (CHO 6) with a plasmid that inducibly expresses the Eco RI restriction endonuclease gene in the presence of cadmium sulfate (CdSO4). Expression of Eco RI results in DNA double-strand breaks, which can lead to chromosome aberrations. The new line, designated CHO 10, also has a low level of constitutive expression of Eco RI in the absence of CdSO4 without any cytogenetic effect. This suggested that these cells may be efficient at repairing low levels of DNA double-strand breaks. To test this, both cell lines were exposed to ionizing radiation, and aberration yields were analyzed with or without induction of Eco RI. CHO 10 cells showed increased radiosensitivity after G1 irradiation, but after G2 exposure, only doses greater than or equal to 0.4 Gy caused more damage in CHO 10 cells. We conclude that CHO 10 cells can tolerate constitutive expression of Eco RI, but that when the cells are subjected to additional stress, in this case ionizing radiation, they become very sensitive to DNA double-strand breaks.     

Molecular basis of X-ray-induced mutation at the HPRT locus in human lymphocytes

Human lymphocytes lacking functional HPRT enzyme after a dose of 300 rad X-radiation were cloned and the monoclonal populations expanded so that sufficient genomic DNA was obtained for Southern analysis. A total of 33 mutant clones were analysed. Wild-type clones showed no evidence of changes to the HPRT gene resolvable by Southern banding patterns whereas 17 of 33 mutant clones showed changes. The alterations observed included total gene deletions (3 clones) and partial gene deletions with or without the appearance of novel bands (12 clones). Two clones showed the appearance of novel bands only. There were no changes observed in 16 of the 33 mutant clones. Three clones showed changes inconsistent with deletion of portions of the gene. In these clones inversion seems to have been the most likely cause of the mutation. The spectrum of gene alterations following ionizing radiation appears different to that previously observed for spontaneous mutations. Consequently, ionizing radiation or radiomimetic agents would appear to be aetiologic, at the most, for only a minor proportion of in vivo somatic mutations.     

Molecular characterization of thymidine kinase mutants of human cells induced by densely ionizing radiation

In order to characterize the nature of mutants induced by densely ionizing radiations at an autosomal locus, we have isolated a series of 99 thymidine kinase (tk) mutants of human TK6 lymphoblastoid cells irradiated with either fast neutrons or accelerated argon ions. Individual mutant clones were examined for alterations in their restriction fragment pattern after hybridization with a human cDNA probe for tk. A restriction fragment length polymorphism (RFLP) allowed identification of the active tk allele. Among the neutron-induced mutants, 34/52 exhibited loss of the previously active allele while 6/52 exhibited intragenic rearrangements. Among the argon-induced mutants 27/46 exhibited allele loss and 10/46 showed rearrangements within the tk locus. The remaining mutants had restriction patterns indistinguishable from the TK6 parent. Each of the mutant clones was further examined for structural alterations within the c-erbA1 locus which has been localized to chromosome 17q11-q22, at some unknown distance from the human tk locus at chromosome 17q21-q22. A substantial proportion (54%) of tk mutants induced by densely ionizing radiation showed loss of the c-erb locus on the homologous chromosome, suggesting that the mutations involve large-scale genetic changes.     

Molecular characterization of hprt mutants induced by low- and hgh-LET radiations in human cells

Southern blotting techniques were employed to examine the spectrum of molecular alterations in DNA induced by internally emitting iodine isotopes and X-rays at and around the hprt locus in a human lymphoblastoid cell line. We analyzed 165 mutant clones using a cDNA probe for the human hprt locus, and 3 anonymous sequenceprobes for regions of the X-chromosome which are linked to hrpt. The results were compared with those for 35 spontaneously arising mutant clones. The majority of ionizing radiation-induced mutants showed changes in the normal restriction patterns at the hprt locus, whereas very few alterations were seen at linked markers along the X chromosome. Total hprt coding sequence deletions comprised 30–48% of the changes observed at this locus, while partial deletions and rearrangements comprised 14–54% of the observed changes. In the case of mutants induced by [^125I]dUrd, a densely ionizing radiation, the spectrum of alterations was dose-dependent; at low doses it was not significantly different from that seen after sparsely ionizing X-ray exposure, whereas a higher proportion of gene deletions and rearrangements occurred after high doses of this incorporated isotope. Changes were rarely observed in the 3 linked markers examined. Overall, these results indicate that the distribution of mutational events at the hprt locus in irradiated human cells may not only be LET-dependent but dose-dependent, and that deletions involving large regions of the X chromosome surrounding the hprt locus are rae events.     

Gene for human CD59 (likely Ly-6 homologue) is located on the short arm of chromosome 11

The CD59 (MEM-43) antigen, which probably is a human homologue of mouse Ly-6 antigens, is a broadly expressedM _r 18000–25000 human leucocyte surface glycoprotein recognized by monoclonal antibody MEM-43. Ten mouse-human T-lymphocyte hybrids, carrying all mouse chromosomes and a limited number of human chromosomes, were analyzed for expression of CD59 by indirect immunofluorescence and immunoblotting with MEM-43 antibody. Karyotypic analysis of the tested clones showed that the presence of human chromosome 11 correlated with the expression of CD59 in all clones tested. Three other human chromosome 11-encoded antigens, 4F2 (Trop-4), Leu 7 (HNK-1, CD57), and lymphocyte homing receptor, were expressed concordantly with CD59. A more exact localization of the gene for CD59 was obtained by the study of Chinese hamster-human cell hybrids containing short or long arm deletions of human chromosome 11. CD59 segregated with hybrids containing part of the short arm of human chromosome 11, but not with the hybrids containing the long arm. Based on these studies we assign the gene for CD59 to regionP14–p13 of the short arm of chromosome 11.     

Refinement of the dichlorofluorescein assay for flow cytometric measurement of reactive oxygen species in irradiated and bystander cell populations

Reactive oxygen species (ROS) have been implicated in many ionizing radiation-related phenomena, including bystander effects. The oxidation of 2'7'-dichlorofluorescin (DCFH) to fluorescent 2'7'-dichlorofluorescein (DCF) is commonly used for the detection of radiation-induced ROS. The DCF assay was adapted for efficient, systematic flow cytometry quantification of low-linear energy transfer (LET) gamma-radiation-induced ROS in vitro in Chinese hamster ovary (CHO) cells. This method is optimized for increased sensitivity to radiation-induced ROS and to discriminate against measurement of extracellular ROS. This method can detect a significant increase in ROS in cells exposed to gamma radiation at doses as low as 10 cGy. The antioxidants N-acetyl-cysteine and ascorbic acid (vitamin C) significantly reduced the amount of ROS measured in cells exposed to 5 Gy ionizing radiation. This method was used to measure the intracellular ROS in unirradiated CHO bystander cells co-cultured with low-LET-irradiated cells. No increase in ROS was measured in bystander cell populations co-cultured with the irradiated cells beginning 9 s after radiation exposure.     

Alteration of N-terminal phosphoesterase signature motifs inactivates Saccharomyces cerevisiae Mre11.

Saccharomyces cerevisiae Mre11, Rad50, and Xrs2 function in a protein complex that is important for nonhomologous recombination. Null mutants of MRE11, RAD50, and XRS2 are characterized by ionizing radiation sensitivity and mitotic interhomologue hyperrecombination. We mutagenized the four highly conserved phosphoesterase signature motifs of Mre11 to create mre11-11, mre11-2, mre11-3, and mre11-4 and assessed the functional consequences of these mutant alleles with respect to mitotic interhomologue recombination, chromosome loss, ionizing radiation sensitivity, double-strand break repair, and protein interaction. We found that mre11 mutants that behaved as the null were sensitive to ionizing radiation and deficient in double-strand break repair. We also observed that these null mutants exhibited a hyperrecombination phenotype in mitotic cells, consistent with previous reports, but did not exhibit an increased frequency of chromosome loss. Differential ionizing radiation sensitivities among the hypomorphic mre11 alleles correlated with the trends observed in the other phenotypes examined. Two-hybrid interaction testing showed that all but one of the mre11 mutations disrupted the Mre11-Rad50 interaction. Mutagenesis of the phosphoesterase signatures in Mre11 thus demonstrated the importance of these conserved motifs for recombinational DNA repair.     

Assignment of a human DNA-repair gene associated with sister-chromatid exchange to chromosome 19

The Chinese hamster ovary (CHO) cell mutant, EM9, is defective in rejoining strand breaks, hypersensitive to chlorodeoxyuridine (CldUrd), and has a high frequency of sister-chromatid exchange (SCE). Somatic cell hybrids constructed from fusion of EM9 cells with normal human lymphocytes and fibroblasts, and selected in CldUrd, extensively segregate human chromosomes but preferentially retain markers of human chromosome 19. The SCE frequency in the hybrid clones is low as in normal CHO cells, but in CldUrd-sensitive subclones, which lose the human chromosome 19 markers, SCE frequencies return to mutant levels. We therefore assign a human gene designated repair complementing defective repair in Chinese-hamster (RCC) to chromosome 19. Since this is the second (of two) human genes complementing repair-deficiency mutations in CHO cells assigned to the 19, the assignment and organization of DNA-repair genes is discussed in the light of hemizygosity in CHO cells and the evolutionary conservation of mammalian linkage groups.     

Intrachromosomal changes and genomic instability in site-specific microbeam-irradiated and bystander human-hamster hybrid cells

Exposure to ionizing radiation may induce a heritable genomic instability phenotype that results in a persisting and enhanced genetic and functional change among the progeny of irradiated cells. Since radiation-induced bystander effects have been demonstrated with a variety of biological end points under both in vitro and in vivo conditions, this raises the question whether cytoplasmic irradiation or the radiation-induced bystander effect can also lead to delayed genomic instability. In the present study, we used the Radiological Research Accelerator Facility charged-particle microbeam for precise nuclear or cytoplasmic irradiation. The progeny of irradiated and the bystander human hamster hybrid (A(L)) cells were analyzed using multicolor banding (mBAND) to examine persistent chromosomal changes. Our results showed that the numbers of metaphase cells involving changes of human chromosome 11 (including rearrangement, deletion and duplication) were significantly higher than that of the control in the progeny of both nuclear and cytoplasmic targeted cells. These chromosomal changes could also be detected among the progeny of bystander cells. mBAND analyses of clonal isolates from nuclear and cytoplasm irradiations as well as the bystander cell group showed that chromosomal unstable clones were generated. Analyses of clonal stability after long-term culture indicated no significant change in the number of unstable clones for the duration of culture in each irradiated group. These results suggest that genomic instability that is manifested after ionizing radiation exposure is not dependent on direct damage to the cell nucleus.     

Brca2 (XRCC11) Deficiency Results in Radioresistant DNA Synthesis and a Higher Frequency of Spontaneous Deletions

We show here that the radiosensitive Chinese hamster cell mutant (V-C8) of group XRCC11 is defective in the breast cancer susceptibility gene Brca2. The very complex phenotype of V-C8 cells is complemented by a single human chromosome 13 providing the BRCA2 gene, as well as by the murine Brca2 gene. The Brca2 deficiency in V-C8 cells causes hypersensitivity to various DNA-damaging agents with an extreme sensitivity toward interstrand DNA cross-linking agents. Furthermore, V-C8 cells show radioresistant DNA synthesis after ionizing radiation, suggesting that Brca2 deficiency affects cell cycle checkpoint regulation. In addition, V-C8 cells display tremendous chromosomal instability and a high frequency of abnormal centrosomes. The mutation spectrum at the hprt locus showed that the majority of spontaneous mutations in V-C8 cells are deletions, in contrast to wild-type V79 cells. A mechanistic explanation for the genome instability phenotype of Brca2-deficient cells is provided by the observation that the nuclear localization of the central DNA repair protein in homologous recombination, Rad51, is reduced in V-C8 cells.     

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 deletions. 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 (approximately 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.(ABSTRACT TRUNCATED AT 400 WORDS)