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.     

Induction of 6-thioguanine resistance in human cells treated with N-acetoxy-2-acetylaminofluorene

Induction of 6-thioguanine resistance was studied in human cells treated with the direct-acting chemical carcinogen N-acetoxy-2-acetylaminofluorene (NA-AAF). At low concentrations (2.5–7.5 μM) induction of resistant clones was linear and followed one-hit kinetics, while at 10 μM the yield of resistant clones was higher and appeared to result from the combination of one-hit and two-hit kinetics. A study of about 50 resistant clones revealed that most had reduced levels of hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity (25–85% of controls) and were able to use exogenous hypoxanthine for growth (“Type II mutants,” deMars, 1974); a few had very low HGPRT activity (1–8% of controls) and were unable to use exogenous hypoxanthine (“Type I mutants”). Use of [9¹⁴-C]NA-AAF allowed us to examine the frequency of induction of thioguanine resistance as a function of binding to DNA (μmole AAF/mole DNA-P). Calculations from these data suggest that most “hits” on the HGPRT locus do not result in detectable mutations: At three different levels of binding and induced mutation frequency, the yield was 2.5–3 detectable mutants/10 000 molecules of acetylaminofluorene bound to the HGPRT locus. These data suggest that most bound acetylaminofluorene molecules either produce no change in the primary sequence of DNA (possibly as a result of repair or correct “read through” by the DNA polymerase) or result in changes which are phenotypically undetectable.     

The cytotoxic and mutagenic effects of 5-bromodeoxyuridine-plus-black-light treatment in cultured mammalian cells

The cytotoxic and mutagenic effects of the incorporation of 5-bromodeoxyuridine (BrdU)_followed by exposure to black light were investigated with Chinese hamster ovary (CHO) cells in cell culture. Mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (hgprt) locus was determined by selection for 6-thioguanine resistant (TG^r) mutants (CHO/HGPRT system). BrdU alone has been shown to be mutagenic only at concentrations of 50 μM or greater. This study was performed in an effort to determine whether BrdU is actually incorporated into the hgprt gene when lower, nonmutagenic concentrations are employed. Neither BrdU (1–20 μM) nor exposure to black light alone was mutagenic, but the combined treatment did result in the induction of TG^r mutants. The mutant frequency increased with increasing light exposure at constant BrdU and with inreasing BrdU at constant light exposure. These results show that BrdU is incorporated into the hgprt gene, but that this does not result in mutation induction in the absence of light exposure. Such a BrdU-plus-light procedure might be applied to studies of DNA repair at this locus, since mutation induction requires both BrdU incorporation and subsequent exposure to black light.     

Induction of 6-thioguanine resistance in human cells treated with N-acetoxy-2-acetylaminofluorene

Induction of 6-thioguanine resistance was studied in human cells treated with the direct-acting chemical carcinogen N-acetoxy-2-acetylaminofluorene (NA-AAF). At low concentrations (2.5–7.5 μM) induction of resistant clones was linear and followed one-hit kinetics, while at 10 μM the yield of resistant clones was higher and appeared to result from the combination of one-hit and two-hit kinetics. A study of about 50 resistant clones revealed that most had reduced levels of hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity (25–85% of controls) and were able to use exogenous hypoxanthine for growth (“Type II mutants,” deMars, 1974); a few had very low HGPRT activity (1–8% of controls) and were unable to use exogenous hypoxanthine (“Type I mutants”). Use of [9-¹⁴C]NA-AAF allowed us to examine the frequency of induction of thioguanine resistance as a function of binding to DNA (μmole AAF/mole DNA-P). Calculations from these data suggest that most “hits” on the HGPRT locus do not result in detectable mutations: At three different levels of binding and induced mutation frequency, the yield was 2.5-3 detectable mutants/10 000 molecules of acetylaminofluorene bound to the HGPRT locus. These data suggest that most bound acetylaminofluorene molecules either produce no change in the primary sequence of DNA (possibly as a result of repair or correct “read through” by the DNA polymerase) or result in changes which are phenotypically undetectable.     

Recovery of V79 cell clones with different phenotypes in aminopterin- or azaserine-containing media used for the selection of HGPRT revertants

Three 6-thioguanine (6TG)-resistant mutants were mutagen-treated and selected for clones capable of growing in 2 selective media: HAT medium, containing aminopterin (AP) and HAS medium, containing L-azaserine (AS). Both 6TG-sensitive, wild-type clones and 6TG-resistant mutants were found among colonies growing in HAT medium, while only 6TG-sensitive clones grew in HAS medium. Time for expression was required by 6TG-resistant but not by 6TG-sensitive clones, that were fully expressed immediately after treatment. All HAT-resistant, 6TG-resistant clones which were analyzed proved to be resistant to AP. These data were interpreted as follows: in HAT medium, both HGPRT⁺ revertants and double mutants (HGPRT^?, AP-resistant) were selected, while only HGPRT⁺ revertants were selected in HAS medium. Not all 6TG-resistant mutants were able to produce both classes of HAT-resistant clones.     

Quantitative mutagenesis at the adenosine kinase locus in Chinese hamster ovary cells. Development and characteristics of the selection system

Stable mutants exhibiting high degree of resistance (100-1000-fold) to various nucleoside analogs viz, toyocamycin, tubercidin, 6-methyl mercaptopurine riboside (6-MeMPR) and pyrazofurin, are obtained at similar frequency (congruent to 1 X 10(-4] in CHO cells. The mutants resistant to any of the above analogs exhibit similar degree of cross-resistance to the other three nucleoside analogs, and all of the mutants examined contained no measurable activity of the purine salvage pathway enzyme adenosine kinase (AK) which converts these analogs to their phosphorylated derivatives. These results indicate that very similar mutants are selected using any of these analogs. The recovery of AK- mutants in CHO cells is not affected by cell density (up to at least 5 X 10(5) cells per 100-mm diameter dish) and after treatment with mutagen(s) maximum mutagenic effect is observed after 7-8 days, which then remains unchanged for the next several days. Treatment of CHO cells with a number of mutagenic agents e.g. ethyl methanesulfonate, ICR170, ultraviolet light, and benzo[a]pyrene, led to a nearly linear concentration-dependent increase in the frequency of the AK- mutants in cultures. The mutagenic response of the AK locus to these agents compared favorably with that of the HGPRT locus (6-thioguanine resistance) within the same experiments. These results show that the selection system for AK- mutants provides an additional valuable genetic marker for quantitative mutagenesis studies in CHO cells.     

Genetic complementation in hybrid cells derived from mutagen-induced mouse clones deficient in HGPRT activity.

Cultured mouse clonal cells, H-5, were treated with two different mutagens, ethyl methanesulfonate (EMS) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Then two selective procedures using 8-azaguanine (8-AZ) or 6-thioguanine (6-TG) were compared in an effort to isolate hypoxanthine-guanine phosphoribosyl-transferase (HGPRT)-deficient cells containing different gene alterations. While many 8-AZ resistant cells were induced by EMS treatment, considerably more 6-TG resistant cells were induced by the same treatment. MNNG treatment induced many 8-AZ resistant mutants but induced hardly any 6-TG resistant mutants. After a fusion experiment of 91 sets involving 13 HGPRT-deficient mouse clones, 7 of which were resistant to 8 AZ and 6 of which were resistant to 6TG with subsequent selection on HAT medium, complementation occurred only in those hybrid mixtures formed between 8-AZ- and 6-TG-resistant clones, while it did not occur at all in hybrid mixtures formed between different 8-AZ-resistant clones and mixtures formed between different 6-TG-resistant clones. The clonally isolated HGPRT-positive cells, characterized by tetraploid karyology, had an apparent activity of HGPRT ranging from 25 to 30% of that of the wild-type parental cells. Heat-inactivation of HGPRT at 65 °C revealed that HGPRT from wild-type cells was heat stable and HGPRT from some 8-AZ-resistant clones were heat labile, while HGPRT from hybrid cells had intermediate stability. These results indicate that there would be alterations in the structural gene of HGPRT in the 8-AZ- or 6-TG-resistant mutants, and also that two selective procedures with 8-AZ or 6-TG alone can isolate different alterations in the structural gene of HGPRT. Moreover, this indicates that some of these gene alterations were mutually complementary. It is most likely that there would be at least 3 cistrons in the locus responsible for HGPRT activity in the mouse cells.     

Mutagenicity of an antitumor protein, neocarzinostatin, in Escherichia coli.

An assay is described for the measurement of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells utilizing resistance to 6-thioguanine (TG). Optimal selection conditions are defined for such parameters as phenotypic expression time prior to selection, and TG concentration and cell density which permits maximum mutant recovery. The nature of the TG-resistant mutants is characterized by several physiological and biochemical methods. The data demonstrate that more than 98% of the mutant clones isolated by this selection procedure contain altered HGPRTase activity. The CHO/HGPRT system thus shows the specificity necessary for a specific gene locus mutational assay.     

Biological and biochemical characterisation of purine analogue resistant clones of V79 Chinese hamster cells

Purine analogue resistant clones have been selected from the closely related Chinese hamster lines V79A and V79S. Clones were of either spontaneous origin or induced by EMS or ultraviolet light. The majority of clones selected in 8-azaguanine showed stable cross resistance to 6-thioguanine. Clones derived from V79A and selected for 6-thioguanine resistance were cross resistant to 8-azaguanine: however a group of 6-thioguanine resistant mutants selected from V79S cells were 8-azaguanine sensitive. All clones except two were unable to grow in HAT medium. The two exceptions were 8-azaguanine resistant, showed partial sensitivity to 6-thioguanine, and also differed in other biochemical characteristics. HGPRT activity was measurable in extracts of all clones under standard conditions. In many clones, HGPRT activity increased as the hypoxanthine concentration was reduced. Whole cell uptake of [14C] hypoxanthine was low in all cases examined and was not modified by incubation in the presence of amethopterin. The heat sensitivity and electrophoretic mobility of HGPRT in extracts of some clones was compared to that in wild-type extracts. All clones tested except one, which was consistently HAT positive, showed enhanced heat sensitivity and reduced electrophoretic mobility. None of the mutants reverted spontaneously at detectable frequency but some could be induced to revert by EMS. The presence of measurable enzyme with altered properties in all clones suggests that these revertable drug resistant clones represent missense mutants.     

Further studies on recombination in diploid clones from Bacillus subtilis protoplast fusion

Summary Diploid prototrophs were obtained from protoplast fusion of Bacillus subtilis strains. They are unstable but upon further cultivation they stabilize retaining diploidy but are genetically inactive. It has been suggested that recombination between the parental chomosomes is involved in the production of stable prototrophs and recombinants. In this work the occurrence of this recombination was searched for by determining genetic linkages in transformation experiments. In prototrophs two alleles: hisH2 and trpE8 carried originally on each parental chromosome, were shown to be 48% co-transformable in a stable clone whereas they were only cotransformed in 10% of the unstable colonies. For Trp^- recombinants (the most frequent type of a Leu^- Met^- Thr^- x Ade^- Ura^- Trp^- fusion pair) lysed protoplasts were used as donor DNA for the transformations. High values of co-transfer for Ura⁺ Met⁺ were obtained. These results confirm the occurrence of recombination in stable diploid clones, prototrophs or recombinants.     

Expression of SV40 Large T Antigen Stimulates Reversion of a Chromosomal Gene Duplication in Human Cells

Transformation of human cells is characterized by altered cell morphology, frequent karyotypic abnormalities, reduced dependence on growth factors and substrate, and rare “immortalization”—clonal acquisition of unlimited proliferative potential. We previously reported a marked increase in DNA rearrangements, arising between two duplicated segments in a transfected plasmid substrate, for five immortal human cell lines relative to three normal fibroblast strains [Finnet al.(1989)Mol. Cell. Biol.9, 4009–4017]. We have now assessed reversion of a 14-kilobase-pair duplication within the hypoxanthine phosphoribosyl transferase (HPRT) gene locus, in a fibroblast strain during its normal replicative lifespan and after stable transformation with SV40 large-T antigen. Revertants, selected under HPRT-dependent growth conditions immediately after purging preexistingHPRT⁺cells, were confirmed asHPRT⁺by hypoxanthine incorporation and 6-thioguanine sensitivity. Southern blot analyses indicate loss from most revertant clones of a restriction fragment representing the duplicatedHPRTregion, as predicted for homologous recombination between the 14-kilobase-pair repeats. Amplification of a subregion ofHPRTmRNA implicated deletion of duplicated exons in 93% of revertant colonies. Reversion toHPRT⁺was unaltered during the normalin vitrolifespan of these cells, but increased in 9 clones stably transformed with large-T antigen (mean = 3.8-fold; eachP< 10⁻⁵). Stimulation ofHPRT-reversion is abrogated in a variety of T-antigen mutants, and depends on continued induction of T antigen by glucocorticoid in two clones tested 10–30 doublings before replicative senescence. Since no immortal subclones arose from these clones, elevated reversion must precede immortalization. Increased DNA rearrangements, in cells expressing T-antigen, could facilitate the rare concurrence of multiple mutations necessary for immortalization.     

Excess thymidine is not mutagenic in Chinese hamster V79 fibroblasts

The ability of thymidine to enhance the frequency of HGPRT⁻, 6-thioguanine resistant V79 cell mutants has been investigated as a function of post treatment growth interval. We have also studied the relative sensitivities towards exogenous thymidine of wild type and mutant cell lines. Our data imply that increases in mutant frequency following exposure of V79 cells to thymidine can be explained on the basis of a greater sensitivity of wild type cells compared with HGPRT deficient cells.     

Factors affecting the growth of Chinese hamster cells in halt selection media

Factors affecting the efficiency of selection of “reverants” of salvage pathway mutants in media containing amethopterin have been examined. Our V79 Chines hamster cell line was found to require a significantly higher level of thymidine for optimal growth in such media than has been reported for other cell lines. Hypoxanthine (but not glycine) was also required for reversal of amethopterin toxicity, but levels did not differ significantly from those reported elsewhere. Growth in HAT was also dependent on plating density and serum batch. Our modification (VHAT) was compared with published HAT recipies in back selection reconstruction experiments. A sharp fall in EOR (efficiency of recovery) of wild type cells from mixtures with mutants at plating densities greater than 3500 cells/cm² (10⁵ cells/6 cm dish) was observed for VHAT. EOR with other HAT recipes was lower still, and was affected also by the particular mutant used in the mixture.EMS induced “revertants” were isolated from three 8AZ^r mutants by plating in VHAT. All. revertants were however amethopterin resistant, they were also 8AZ resistant and the mobility of residual HGPRT (as measured by polyacrylamide gel electrophoresis) was similar to that of their 8AZ^r parents i.e. dissimilar from that in wild type. The modal chromosome number of V79 wild type cells was 21. No significant deviation from this mode was detected in any of the mutant lines examined. The data indicate that the recovery of colonies in HAT from 8AZ^r mutants does not necessarily indicate that a back mutation in the structural gene for HGPRT has occurred. Thus, the frequency of HAT⁺ colonies cannot be taken as a direct indication of reversion frequencies.     

Plasmid mediated mutagenesis of a cellular gene in transfected eukaryotic cells.

NIH3T3 cells are widely used in transformation assays and readily take up transfected DNA. A system has been devised using NIH3T3 cells to measure the mutagenic effect of transfected DNA on recipient cell genes. NIH3T3 cells can be mutated to 6-thioguanine resistance at a frequency which suggests that at least a portion of the cells have only one functional copy of the HGPRT gene. They have a low spontaneous background mutation frequency (approximately 1 X 10(-7)). Transfection of three different plasmids into NIH3T3 cells induced 6-thioguanine resistant mutants at frequencies ranging from 3 to 11 fold above background. The mutant phenotype is stable and reversion frequencies of several mutants are less than or equal to 1 X 10(-7). Southern blot analysis of the HGPRT gene in several mutants showed that 4 of 26 mutants (15.4%) had detectable alterations in the structure of the HGPRT gene. Interestingly 3 of the 4 mutants showing rearrangements were obtained by transfection of the HSV-2 morphological transforming region.     

Mutagenicity testing in mammalian cells: I. Derivation of a chinese hamster ovary cell line heterozygous for the adenine phosphoribosyltransferase and thymidine kinase loci

As a first step in the development of a multiple-marker, mammalian cell mutagenesis assay system, we have isolated a Chinese hamster ovary (CHO) cell line that is heterozygous for both the adenine phosphoribosyltransferase (aprt) and thymidine kinase (tk) loci. Presumptive aprt^+/? heterozygotes with intermediate levels of APRT activity were selected from unmutagenized CHO cell populations on the basis of resistance to low concentrations of the adenine analog, 8-azaadenine. A functional aprt^+/? heterozygote with ～50% wild-type APRT activity was subsequently used to derive sublines that were also heterozygous for the tk locus. Biochemical and genetic characterization of one such subline, CHO-AT3-2, indicated that it was indeed heterozygous at both the aprt and tk loci. CHO-AT3-2 cells permitted single-step selection of mutants resistant to 8-azaadenine or 5-fluorodeoxyuridine, allowing quantitation and direct comparison of mutation induction at the autosomal aprt or tk loci, as well as in the gene involved in ouabain resistance or at the X-linked, hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus. Significant dose-dependent increases in mutation frequency were observed for all 4 genetic markers after treatment of CHO-AT3-2 cells with ethyl methanesulfonate.     

Forward mutation assay of V79 cells to 6-thioguanine resistance in a soft agar technique that eliminates effects of metabolic co-operation

A technique involving culture in soft agar was used for the assay of forward mutation of V79 cells to 6-thioguanine (6TG) resistance. The main reason for the use of soft agar was to prevent reduction in recovery of mutants depending on the cell density plated for mutation selection, which is the chief problem in the liquid method, and which results mainly from metabolic co-operation due to cell-to-cell contact.V79 cells grew well in fortified soft agar medium (DMEM + 20% FBS) showing cloning efficiencies (>80%) as high as in liquid culture. Therefore, V79/HGPRT mutagenesis could be assayed quantitatively in soft agar culture.The frequency of 6TG-resistant colonies in agar selective medium increased linearly with increase in concentration of EMS. Toxicity and mutagenic responses were greater in soft agar than in liquid culture.In cultures of untreated and EMS-treated cells, more than 95% of the 6TG-resistant colonies isolated were aminopterin-sensitive.Use of soft agar for selection prevented the reduction in the number of mutants with increase in the size of incula on plating up to 1?2 × 10⁶ cells per 9-cm dish: in liquid culture, even with a lower plating number (2 × 10⁵ cells per 9-cm dish), a notable reduction in numbers of mutants was observed. This character was re-examined in a reconstruction experiment. The results show that, when up to 2 × 10⁶ cells were plated per 9-cm dish, 6TG-resistant cells were almost completely recovered from the soft agar medium, whereas only 10% were recovered from liquid culture.     

Influence of edeine on intergenic and interallelic recombination in Neurospora crassa

Summary The effect of edeine and the mutation ed ^r-2 to edeine resistance on genetic recombination in Neurospora crassa was investigated. For this purpose crosses between pairs of edeine sensitive and edeine resistant strains respectively were set up without or in the presence of the drug (0–750 g/ml). The genetic markers ylo-1, ad-1, pan-2 (B ₃ and B ₅) and tryp-2, all on linkage group VI, were used for scoring recombinants. These were ad ⁺, tryp ⁺ (intergenic recombination) and pan ⁺ (interallelic recombination).Frequencies of about 6–7% for intergenic and of about 0.4% for interallelic recombination were found in crosses between ed^s strains and ed^r strains respectively, if edeine was absent. However, crosses in the presence of edeine gave higher frequencies of both intergenic and interallelic recombination (about 12% intergenic and 1% interallelic with 180 to 200 g ed/ml).The pan⁺ prototrophs (interallelic recombinants) obtained in the different crosses were tested for distribution of outside markers. The data thus obtained revealed, that under the effect of both the mutation to edeine resistance and edeine itself the relative number of non-crossover (gene conversion) recombinants decrease in favour of crossover recombinants, and the relative number of double crossover recombinants (events outside the pan locus) decreases in favour of single crossover recombinants.It is concluded that a) edeine and the mutation ed ^r-2 to edeine resistance affect recombination via related pathways, and b) noncrossover and crossover recombinants are caused by different molecular mechanisms, in agreement with the work of other authors.     

8-Azaguanine versus 6-thioguanine: influence on frequency and expression time of induced HGPRT- mutations in Chinese hamster V79 cells

Chinese hamster V79 cells were mutagenized with ethyl methanesulfonate at various concentrations. Clones resistant to 8-azaguanine (20 and 80 micrograms/ml) or 6-thioguanine (4 micrograms/ml) were selected at different times after the treatments. The total yield of induced mutations was only slightly affected by the kind and concentration of purine analog used in the selection. However, full phenotypic expression of the mutants selected with 8-azaguanine was achieved earlier than that of mutants resistant to 6-thioguanine. This result seems to be best explained by the reported lower affinity of 8-azaguanine for the wild-type HGPRT enzyme, thus providing evidence that, in this gene-mutation assay, the phenotypic expression time has a physiological component.     

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.