Proc. Natl. Acad. Sci. USA

Vol. 76, No. 12, pp. 6505-6509, December 1979

Genetics

Characterization of a mutator gene in Chinese hamster ovary cells (somatic cell genetics/dCTP/thymidine auxotroph)

MARK MEUTH, NICOLE L'HEUREUX-HUARD, AND MARIE TRUDEL Institut de Recherches Cliniques de Montreal, 110 ouest, avenue des Pins, Montreal (Quebec) H2W 1R7, Canada

Communicated by Howard Green, September 10, 1979

ABSTRACT We have recently reported the isolation of a class of mutants (called thy-) that is both resistant to arabinosyl cytosine and auxotrophic for thymidine. thy- mutants have a 5- to 10-fold elevated pool of dCTP and are deficient in the synthesis of dTTP as an apparent consequence of a single mutation in the gene for ribonucleoside-diphosphate reductase (2'-deoxyribonucleoside-diphosphate:oxidized-thioredoxin 2'oxidoreductase, EC 1.17.4.1). Here we show that three independent thy- lines have a 5- to 50-fold higher frequency and rate of spontaneous mutation for two genetic markers, 6-thioguanine resistance and ouabain resistance. The higher rate of mutation is site specific because two other genetic markers, reversion of proline auxotrophy to proline prototrophy and emetine resistance, are unaffected. Ouabain- and 6-thioguanine-resistant mutations occur at a much lower rate in revertants of thy- to the wild-t state, so the increased rate of mutation is the consequence of the thy- mutation. Both the increased mutational rate and the increased intracellular pools of dCTP are dominant or codominant in hybrid cells, andalterations of the ratio of the pools of dCTP to dTTP in thy-49 produce corresponding changes in the rate of mutation. Thus, thy is a mutator gene in Chinese hamster ovary cells, apparently as a consequence of the imbalance of deoxynucleoside triphosphate pools created by the expanded pool of dCTP. A continuous supply of deoxynucleoside triphosphates is essential for the replication of cellular DNA. These deoxyribonucleoside triphosphates are synthesized by reduction of the ribonucleoside diphosphate to the corresponding deoxyribonucleoside diphosphate and phosphorylation to the triphosphate (1, 2). dTTP is synthesized by reduction of UDP to dUDP, phosphorylation to dUTP, hydrolysis to dUMP, methylation to dTMP, and finally phosphorylation to the triphosphate. Pools of deoxynucleoside triphosphates accumulate in animal cells, but they are very small and are capable of sustaining the maximal rate of DNA synthesis for only 30 sec to 3 min (3, 4). Drugs or even other deoxynucleosides that interfere with the synthesis of the deoxynucleoside triphosphates also immediately arrest the synthesis of DNA (4-7). Thus, the synthesis of DNA during the S phase of the cell cycle is tightly coupled to the supply of deoxynucleoside triphosphates. Recent experiments from several laboratories have suggested that balanced pools of the triphosphates are essential for the accurate replication of cellular DNA. dCTP starvation of cells caused by excess thymidine (5) or bromodeoxyuridine (8) treatment significantly increases the frequency of cell mutations at two loci (9, 10). We (11) have recently reported the isolation of a class of arabinosyl cytosine (araC)-resistant Chinese hamster ovary (CHO) mutants that require thymidine for growth. This mutation (called thy-) expands the pool of dCTP 5- to 10-fold, but it also makes the cells dependent upon an exogenous source of thymidine for the maintenance of the pool of dTTP. The thymi-

dine requirement of thy can also be satisfied by deoxyuridine or deoxycytidine but not by the other ribo- or deoxyribonucleosides. In somatic cell hybrids, resistance to araC is dominant or codominant whereas the thymidine requirement is recessive. These observations led us to suggest that thy- was a mutation of ribonucleoside-diphosphate reductase (2'-deoxyribonucleoside diphosphate:oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1)-a mutation that both increased the reduction of CDP and decreased reduction of UDP (11). Experiments with revertants of thy to the wild-type state (thy +, araC sensitive) showed that the complex phenotype of these mutants was the result of a single mutation. However, the frequency of reversion of thymidine auxotrophy to thymidine prototrophy was high. To determine whether the mechanism causing the high reversion frequency could also affect other genetic markers, we examined the rate of mutation at four independent genetic loci in thy- mutants. Here we show that the rate of mutation is increased at two of the four loci and that this increased rate is the consequence of the thy mutation. MATERIALS AND METHODS Materials. Powdered medium and fetal calf serum for cell culture were purchased from GIBCO. Aminopterin, hypoxanthine, 6-thioguanine, ouabain, and emetine were purchased from Sigma. Thymidine and deoxynucleoside triphosphates were products of P-L Biochemicals. Plastic dishes for cell culture were obtained from NUNC and Costar. Cell Lines and Culture Techniques. The origin and characteristics of the CHO cell lines we used in these experiments are presented in Table 1. The techniques used to culture CHO cells have been described in detail elsewhere (15). Cells were routinely maintained in suspension culture at 37°C in minimal essential medium (MEM) alpha and 10% fetal calf serum. Thymidine (10 ,uM), but not the other ribo- or deoxyribonucleosides, was added to all cultures. During serial cultivation, cultures were diluted to 20,000 cells per ml and then allowed to grow to about 600,000 cells per ml before redilution. Genetic Markers. In the experiments to determine the frequency and rate of mutation in our cell lines, four genetic markers independent of the thy- phenotype were used: 6-tgr, ouaR, emtr, and reversion of the proline auxotrophy of CHO (pro-) to proline prototrophy (pro + ). 6-tgr mutants have been shown to be deficient or altered in hypoxanthine phosphoribosyltransferase (HPRT-) by previous investigators (16). For determination of 6-tgr mutants, cultures were plated in medium containing 10,uM 6-tg in MEM alpha supplemented with 10,MM thymidine and 10% dialyzed fetal calf serum. Plating densities were not greater than 500,000 cells per 100-mm dish. Reconstruction experiments indicated that this cell density did Abbreviations: CHO, Chinese hamster ovary cell line; 6-tg7, 6-thioguanine resistance or resistant; ouaR, ouabain resistance or resistant; emtr, emetine resistance or resistant; araC, arabinosyl cytosine; HPRT, hypoxanthine phosphoribosyltransferase marker; HAT, hypoxanthine/aminopterin/thymidine; MEM, minimal essential medium.

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Table 1. Origin and property of Chinese hamster cell lines Cell line Origin and characteristics proA proline-requiring Chinese hamster line from ovarian tissue (CHO) established in Puck's laboratory (12). A review of the genetic properties (13) and a detailed chromosomal analysis (14) of the line used have been published. The line was obtained from Louis Siminovitch. thy 49 pro-thy 49, a thymidine-requiring mutant of pro - obtained by selection in the presence of araC and thymidine (11). thy-l90 pro thy-190 and pro-thy 303, independent thy 303 araC-resistant thymi Ine auxotrophs of pro-. thy-49-2r5 pro thy 49-2r5 and pro thy 49-81rl, thy-49-81rl revertants of two independent subclones of thy 49 selected by their ability to grow in the absence of thymidine. These revertants have also regained sensitivity to araC. pro- X glyA A hybrid cell line of pro and the glycine auxotroph of the A complemention group (glyA). thy-49 X glyA A hybrid cell line between thy 49 and glyA. This hybrid line no longer requires thymidine for growth but remains resistant to araC.

not reduce the frequency of 6-tgr mutations in our cultures. Because HPRT- cells are unable to grow in hypoxanthine/ aminopterin/thymidine (HAT) medium (17), we also tested a number of our 6-tgr mutants in HAT medium consisting of 0.1 mM hypoxanthine, 1 uM aminopterin, and 10 ,M thymidine. Ouabain is an inhibitor of the Na+,K+ transport system of mammalian cells, and cells resistant to this drug have a Na+,K+-ATPase more resistant to ouabain (18). To determine the number of ouabain-resistant mutants in a culture, up to 106 cells were plated per 100-mm dish in 2 mM ouabain in MEM alpha/10% dialyzed fetal calf serum/10,uM thymidine. Emetine is a potent protein synthesis inhibitor in mammalian cells (19). CHO cells resistant to this drug have alterations of the 40S ribosomal subunit, rendering protein synthesis, measured in vitro, resistant to inhibition by emetine (19-21). About 2 X 106 cells per 100-mm dish were plated in 0.2 ,uM emetine in the experiments to determine the number of emetine-resistant cells. Our CHO cell line has an absolute growth requirement for proline (12). Consequently, the last genetic marker we used in these experiments was the reversion of this proline growth requirement (auxotrophy, pro-) to proline independence (prototrophy, pro + ). In these experiments it was important to keep cell density below 500,000 per 100-mm dish. Before plating, the cell population was washed twice with MEM alpha lacking proline but supplemented with 10% dialyzed fetal calf serum and 10 ,M thymidine. In all these experiments, cells were incubated for 7 days in the selective medium and then plates were fixed and stained with 0.5% methylene blue/50% methanol. Only colonies with more than 50 cells were counted. Occasionally, drug-resistant clones were taken and serially cultivated in nonselective medium for further screening. Pool Measurements. Deoxyribonucleoside triphosphates were extracted from cell monolayers by using ice-cold 60% methanol and were measured by using the defined copolymers

Proc. Nati. Acad. Sci. USA 76 (1979)

poly[d(I-C)] and poly[d(A-T)] (Boehringer Mannheim) as described (22, 23). A minimum of two determinations were done on each sample. The amount of DNA per culture was measured on the methanol precipitate (5). RESULTS Frequency of 6-tgr and oua R Clones in pro and thyCells. We first examined the frequency of 6-tgT and ouaR mutants in pro- and thy- cultures to be certain that thyg- cells were as sensitive to these drugs as the parental pro- line. We also wanted to determine the effects of thy- on these markers. Consequently, we plated serially passaged cultures of pro and thy- cells in increasing concentrations of 6-thioguanine or ouabain. Fig. 1 shows that both cell lines were equally sensitive to the drugs. The concentration of drug reducing cellular plating efficiency to 10% of the control for the two lines was about 0.3 ,M for 6-thioguanine and 0.4 mM for ouabain. However, thy- had a 10-fold higher frequency of 6-tgt mutants than wild-type cells. Furthermore, mutants resistant to 2 mM ouabain could be isolated from thy 49 and thy 190 at a frequency of about 10-5 whereas the frequency in procultures ranged from 6 X 10-7 to less than 1O-7. In contrast, the frequency of pro + revertants in thy cultures was roughly the same as that in wild-type pro cultures. Spontaneous Rate of Mutation of pro and thy-. The above experiments indicated that the frequency of 6-tgr and ouaR mutants was higher in the thy mutants. However, this is not firm evidence of a higher mutation rate in the thymutants, because the frequency of mutants can vary significantly in parallel cultures (15). Consequently, we next examined the spontaneous rate of mutation in pro and thy by the Luria-Delbruck fluctuation test (24). Four markers were tested, 6-tgr, ouaR, the reversion of proline auxotrophy to proline prototrophy, and emtr. For each fluctuation experiment, about 30 replicate cultures of each cell line were grown in nonselective medium from a single cell to the indicated density (Table 2). The entire cell population was then plated in the appropriate selective medium. Mutation rates were calculated from the fraction of cultures containing no mutants. The results of these

t

.t 0.1 0.2 0.4 1.0.I 2.0

0.1 0.2 0.4 1.0 2.0 4.0 6-Thioguanine, ,M

Ouabain, mM

FIG. 1. Effect of 6-thioguanine and ouabain on colony-forming ability of wild-type pro and thy- lines. 0, pro-; *, thy 49; A, thy-l 90. ? indicates that the relative plating efficiency of pro- at these ouabain concentrations was less than this value. In this particular experiment no ouaR colonies were detected in pro - cultures after plating more than 107 cells in 2 mM ouabain. In other experiments the frequency of ouaR clones has been about 6 X 10-7. We have found, as others (15), that mutation frequency varied considerably with culture history. These measurements were done on cultures that were carried for 2 weeks in suspension after thawing.

Genetics: Meuth et al. Marker

Proc. Natl. Acad. Sc. USA 76 (1979) Table 2. Fluctuation analyses for pro- and thy- cell lines prothy 303 thy-190 thy-49

Pro- - pro+ reversion Replicate cultures tested Initial cell number Final cell number Number of revertants per culture: Range Mean Variance PO Mutation rate

33

28

1

1

5 x 105

32

6507

thy-49-2r5

thy-49-81rl

N.D.

N.D.

5 X 105

31 1 6 X 105

6 X 105

0-9 0.88 3.5 0.67 5.5 X 10-7

0-5 0.57 1.4 0.74 4.1 X 10-7

0-7 1.1 4.1 0.65 5.0 X 10-7

0-4 0.47 0.96 0.75 3.3 X 10-7

31

32 1 5 X 105

34

37

1 2.5 X 106

1

5 x 105

1 2.5 X 105

28 1 1 X 106

25 1 2 X 106

0-6 0.32 1.2 0.84 4.7 X 10-8

0-7 0.34 1.6 0.88 1.8 X 10-7

0-25 0.94 18. 0.85 2.2 X 10-7

0-29 1.5 26. 0.84 4.7 X 10-7

0-15 1.4 13. 0.75 2 X 10-7

0-28 2.1 38. 0.80 7.6 X 10-8

33 1

29

30 1

32

26

24

1

1

1

1

5 X 106

1 x 105

5 X 105

1 X 106

5 x 106

5 X 106

0-7 0.75 2.8 0.70

0-21 1.8 17. 0.53

4.8 X 10-8

0-12 1.0 5.9 0.69 2.6 X 10-6

0-5 0.62 1.8 0.53 4.4 X 10-7

0-21 1.0 17. 0.81 2.9 X 10-8

0-14 1.0 8.2 0.75 4.0 X 10-8

22 1 1 X 107

22 1 1 X 107

29 1 1 X 107

N.D.

N.D.

1

6-tgr

Replicate cultures Initial cell number Final cell number Number of resistant mutants per culture: Range Mean Variance

Po Mutation rate oua R Replicate cultures Initial cell number Final cell number Number of resistant mutants per culture: Range Mean Variance

PO Mutation rate

8.7 X 10-7

emtr

Replicate cultures Initial cell number Final cell number Number of resistant mutants per culture: Range Mean Variance

N.D.

0-5 0-7 0.24 0.64 0.91 2.6 0.86 0.90 PO 1 X 10-8 7.6 X 10-9 Mutation rate Clones originating from single cells of the above cell lines were grown in 96-well cloning dishes in MEM alpha supplemented with fetal calf serum and 10 uM thymidine. These clones were picked and transferred to culture tubes and were grown to the indicated density in the same medium. The number of mutant cells for each culture was determined. N.D., not determined; P0, fraction of cultures with zero resistant colonies. Mutation rate was a P0 calculation. 0-3 0.27 0.6 0.86 1 X 10-8

experiments are presented in Table 2. The rate of mutation to 6-tgr was about 5- to 10-fold higher for the thy- lines compared to pro-. Similarly, ouaR mutants occurred at a 10- to 50-fold higher rate in the thy- mutants. In contrast, the mutation rate to emtT and the rate of reversion to proline prototrophy were not significantly different from either wild type or thy-. The mutation rates we have determined from the fluctuation experiments for pro- are very close to those reported for 6-tgr (16), ouaR (18), and emt7 (19) in CHO. Furthermore, the variances in the experiments exceeded the means by factors of 2- to 20-fold. Those determinations with a small increase of the variance relative to the mean also had a small number of replicate cultures with resistant colonies. This high variance is consistent with the random appearance of resistant mutant cells (24). Rate of Mutation of Revertants of thy-. To determine if the observed increase of the spontaneous mutation rate was the consequence of the thy - mutation, we examined the rate of

mutation to 6-tgT and ouaR in two revertants of thy-49. These revertants were selected by their ability to grow in the absence of exogenous thymidine (11). In addition to being prototrophic for thymidine, they were sensitive to araC and thymidine. The revertants also had decreased pools of dCTP relative to thy-, although the dCTP pools did not return to the level of the pa-

rental pro- line (Table 3). Both revertants had decreased spontaneous mutation rates relative to thy-49 for the two markers tested (Table 2). The rate of mutation of both revertants to ouaR was decreased to a level less than that of the parental pro-. The rate of mutation to 6-tgt was decreased 1:2 in one revertant, whereas in the second it returned to the level of the wild type, pro-. Spontaneous Rate of Mutation to ouaR of Hybrids of thy-49 and pro- Cells. To determine if the increased mutation rate of thy- was recessive or dominant, we examined the rate of mutation to ouaR in cell hybrids between pro- and glyA and between thy-49 and glyA. The isolation and properties

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Proc. Nati. Acad. Sci. USA 76 (1979)

Genetics: Meuth et al. Table 3. Deoxynucleoside triphosphate pools pmol per yg of DNA dATP dGTP dCTP dTTP dCTP/dTTP

pro

3.8

1.8

2.6

5.1

0.51

6.3 3.5 22.0 0.8 1.8 thy-303 4.2 3.8 16.0 1.1 3.4 thy-190 2.3 7.3 17.0 1.8 3.0 thy-49 0.5 10.0 5.0 2.2 2.8 thy 49-2r5 1.3 4.2 5.6 4.0 1.1 thy-49-81rl 1.1 3.6 4.0 0.9 6.9 pro- X glyA 2.4 8.5 3.6 1.0 thy 49 X glyA 4.0 Cultures growing exponentially in medium supplemented with dialyzed fetal calf serum and 0.01 mM thymidine were harvested and assayed for deoxyribonucleoside triphosphate content. The values above for parental lines (pro-, thy 49, etc.) represent the average from eight separate experiments. Those for the revertants and hybrids are the average of determinations from duplicate cultures.

of these hybrids have been described (11). Thy 49 X glyA hybrids remain resistant to araC and thymidine but no longer require thymidine for growth. Thus, the araC and thymidine resistance of thy 49 are dominant or codominant and the thymidine auxotrophy is recessive. Similarly, the pool of dCTP in the thy 49 x glyA hybrid is intermediate between pro and thy whereas the ratio of dCTP to dTTP is almost identical to that of thy 49 (Table 3). The rates of mutation to ouaR of the two hybrids are presented in Table 4. thy 49 X glyA cells had a 30-fold higher mutation rate for oua R than did pro X glyA cells. This rate in the thy hybrid was almost equal to that of thy 49. Thus, the increased mutation rate of thy 49 is dominant or codominant like the excess pool of dCTP. Effect of Alterations of the dCTP Pool on Mutation Rate in thy 49. To further examine the relationship between the mutation rate in the thy mutants and the intracellular pools of dCTP and dTTP, we altered the pools of these deoxynucleoside triphosphates by growing thy 49 cells in the presence of different external thymidine concentrations. When thy 49 cells were grown at 1-100 ,uM thymidine, the dTTP pool decreased 1:4 and dCTP increased 17-fold, and the ratio of dCTP to dTTP increased 60-fold (Table 5). Similarly, the rate of mutation to ouabain resistance in thy 49 increased 30-fold over this range of thymidine concentrations. The correspondence of mutation rate with the dCTP-to-dTTP ratio was particularly good at 10-100 ,M thymidine. Thus, intracellular manipulations of the dCTP and dTTP pools directly affect the rate of mutation to ouaR in thy 49 cells. Table 4. Fluctuation analysis of mutation to ouaR by hybrid cells thy 49 X pro- X glyA glyA Exp. 2 Exp. 1 39 15 8 Replicate cultures tested 1 1 1 Initial cell number 8 X 106 10 X 106 2 X 106 Final cell number No. ouaR colonies per culture: 0-20 0-2 0-8 Range 0.53 1.18 0.25 Mean 11.9 0.5 4.2 Variance 0.43 0.93 0.875 Po 1.1 0.48 29 Mutation rate (X10-8) Clones originating from single cells of the above cell lines were grown in 96-well cloning dishes in medium supplemented with fetal calf serum and 10 AM thymidine. These clones were picked and transferred to culture tubes and were grown to the indicated density in the same medium. The number of ouaR clones for each culture was determined. Po, fraction of cultures with zero resistant colonies. Mutation rate is a Po calculation.

Table 5. Effect of dCTP pool on rate of mutation to ouaR in thy 49 cells Deoxynucleoside triphosphate Mutation pools, pmol/gg of DNA Thymidine rate dCTP/ concentrato ouaR dTTP dTTP dCTP tion, uM 1.4 X 10-6 16. 3.8 1 60.8 6.0 X 10-7 2.3 8.2 18.6 10 4.4 X 10-8 0.24 14.5 3.5 100 For the measurement of deoxynucleoside triphosphates, exponentially growing cultures of thy-49 were plated in medium supplemented with 10% dialyzed fetal calf serum and the concentration of thymidine specified above. After 24 hr, the cells were harvested and assayed for deoxynucleoside triphosphate content. The mutation rates were determined by a Po calculation as in Table 2. In these experiments, 20-30 replica cultures of thy-49 cells were grown from a 100-cell innoculum to the desired density in growth medium containing 10% dialyzed fetal calf serum and the specified thymidine concentration.

Stability of Mutants Isolated During Fluctuation Analyses. We have isolated and passaged several clones from the above experiments to be sure they were authentic 6-tgr, ouaR, and pro + mutants. Of 20 6-tgT clones isolated from pro and thycells, all remained resistant to the drug after cloning and growth to mass culture in nonselective medium. A few of these have also been recloned and carried in culture for up to 30 days. These, too, remained resistant to thioguanine. Most of these clones were sensitive to HAT medium with a survival frequency of less than 10-5. However, some clones formed small colonies in HAT medium at a considerably reduced efficiency (about 10% of control). These two 6-tgr mutant phenotypes have been described (25) for CHO cells. Of 15 ouaR clones isolated, all remained resistant to ouabain after serial cultivation in the absence of the drug. And, similarly, five pro + revertants were stable. The 6-tgT and oua R mutants of thy 49 isolated in these experiments also remained auxotrophic for thymidine. DISCUSSION In this paper we present evidence that the thy is a mutator gene in CHO cells. Of four markers tested in three independent thy mutants, two markers had 5- to 50-fold higher rates of mutation. Two observations show that the increased mutation rate is a result of the thyp mutation: (i) three independent thymutations showed increased rates of mutation at the same two genetic loci and (ii) revertants of thy 49 to the wild-type state also had reduced rates of mutation for the two markers. Two lines of evidence, genetic and biochemical, show that the mutator activity of thy 49 is the consequence of the excess pool of dCTP over dTTP. The mutator activity of thy 49 is dominant or codominant in hybrid cells like the excess pool of dCTP. The correlation of mutation rate with the ratio of dCTP to dTTP in these experiments seems particularly good. Secondly, we have manipulated the pools of dCTP and dTTP in thy-49 cultures by altering the external thymidine concentration. When we increased the ratio of the pool of dCTP to that of dTTP, there was a corresponding increase in the rate of mutation. An interesting aspect of these experiments is the specificity of action of the thy mutator. The mutator increases the rate of mutation to ouabain resistance [missense mutations (18)] and thioguanine resistance [missense and perhaps nonsense mutations (16)]. But it does not increase the rate of emetine-resistant mutations [which also appear to be missense mutations (21)] or the rate of reversion of pro to pro +. It is not clear why this mutator gene would be "site specific" particularly with the

Genetics: Meuth et al.

markers we are using. This observation could prove useful in elucidating the mechanism by which deoxynucleoside triphosphate pool alterations produce mutations. Exactly how the thy- mutation increases the rate of mutation at these loci is not clear. Previous observations in bacterial systems suggest two possible mechanisms. (i) The excess of dCTP over dTTP in our cells could increase the frequency of mispairing. For example, dCTP could be mistakenly inserted in place of dTTP, thus creating the transition gene rating A-C mispair (26, 27). This pathway has been shown to occur in T4 phage infected Escherichia coli starved for thymidine (28, 29). (ii) It is also possible that the alterations of pools of deoxynucleoside triphosphates, particularly dCTP, induce an errorprone repair mechanism. Thymidine starvation in E. coli appears to increase the mutation frequency by induction of the SOS repair system (30, 31). A clear test of these and other possibilities requires specific well-characterized mutations that do not yet exist in animal cells. If our first suggestion is correct (as our present evidence would imply), the thyj- mutation could prove useful to somatic cell genetics by allowing the isolation of such mutants with known base changes. Manipulations of the pools of deoxynucleoside triphosphates combined with the marker specificity of thy should provide a good test of these possibilities. But what becomes clear from these experiments is the importance of the pools of deoxynucleoside triphosphates in maintaining the fidelity of replication in animal cells. Monica Meuth and Perin Sankar are thanked for their critical reviews. This work was funded by grants from the Medical Research Council of Canada and the Conseil de la Recherche en Sante du

Quebec.

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71,39-43. 7. -Reichard, P. (1978) Fed. Proc. Fed. Am. Soc. Exp. Biol. 37, 9-14. 8. Meuth, M. & Green, H. (1974) Cell 2, 109-112. 9. Bradley, M. D. & Sharkey, N. A. (1978) Nature (London) 274, 607-608. 10. Kaufman, E. R. & Davidson, R. L. (1978) Proc. Natl. Acad. Sci. USA 75, 4982-4986. 11. Meuth, M., Trudel, M. & Siminovitch, L. (1979) Somat. Cell Genet. 5, 303-318. 12. Kao, F. T. & Puck, T. T. (1967) Genetics 55,513-524. 13. Siminovitch, L. (1976) Cell 7, 1-11. 14. Worton, R. G., Ho, C. C. & Duff, C. (1977) Somat. Cell Genet.

3,27-45. 15. Thompson' L. H. & Baker, R. M. (1975) in Methods in Cell Biology, ed. Prescott, L). M. (Academic, New York), Vol. 6, pp. 209-281. 16. Caskey, C. T. & Kruh, G. D. (1979) Cell 16, 1-9. 17. Szybalski, W. & Szybalski, C. (1962) Univ. Mich. Med. Bull. 28, 277-293. 18. Baker, R. M., Brunette, D. M., Mankovitz, R., Thompson, L. H., Whitmore, G. F., Siminovitch, L. & Till, J. E. (1974) Cell 1, 9-21. 19. Gupta, R. S. & Siminovitch, L. (1976) Cell 9,213-219. 20. Gupta, R. S. & Siminovitch, L. (1977) Cell 10, 61-66. 21. Boersma, D., McGill, S., Mollenkamp, J. & Roufa, D. J. (1979) J. Biol. Chem. 254,559-567. 22. Skoog, L. (1970) Eur. J. Biochem. 17,202-208. 23. Lindberg, U. & Skoog, L. (1970) Anal. Biochem. 34, 152-160. 24. Luria, S. E. & Delbruck, M. (1943) Genetics 28,491-511. 25. Chasin, L. A. & Urlaub, G. (1976) Somat. Cell Genet. 2, 453467. 26. Watson, J. D. & Crick, F. H. C. (1953) Cold Spring Harbor Symp. Quant. Biol. 18, 123-131. 27. Watson, J. D. & Crick, F. H. C. (1953) Nature (London) 171, 964-967. 28. Bernstein, C., Bernstein, H., Mufti, S. & Strom, B. (1972) Mutat. Res. 16, 113-119. 29. Smith, M. D., Green, R. R., Ripley, L. S. & Drake, J. W. (1973) Genetics 74, 393-403. 30. Bridges, B. A., Law, J. & Munson, R. J. (1968) Mol. Gen. Genet. 103,266-273. 31. Witkin, E. M. (1976) Bacteriol. Rev. 40, 859-907.

Characterization of a mutator gene in Chinese hamster ovary cells.

Proc. Natl. Acad. Sci. USA Vol. 76, No. 12, pp. 6505-6509, December 1979 Genetics Characterization of a mutator gene in Chinese hamster ovary cells...
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