Mutation Research, 232 (1990) 171-182 Elsevier

171

MUT 04894

Molecular characterization of X - r a y - i n d u c e d m u t a t i o n s at the H P R T locus in plateau-phase Chinese h a m s t e r ovary cells Thomas L. Morgan a Earl W. Fleck b Kathy A. Poston

a

Barbara A. Denovan a

C.N. N e w m a n at Belinda J.F. Rossiter c and John H. Miller a Biology and Chemistry Department, Pacific NorthWest Laboratory, Richland, WA 99352 (U.S.A.), b Department of Biology, Whitman College, Walla Walla, WA 99362 (U.S.A.) and c Institute for Molecular Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030 (U.S.A.) (Received 21 November 1989) (Revision received 23 April 1990) (Accepted 24 April 1990)

Keywords: Hprt locus; Radiation mutagenesis; Deletions; Plateau-phase CHO cells; Hypoxanthine-guanine phosphoribosyl transferase locus

Summary CHO-K1 cells were irradiated in plateau phase to determine the effect of dose, dose fractionation, and delayed replating on the type, location and frequency of mutations induced by 250 kVp X-rays at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus. Independent HPRT-deficient cell lines were isolated from each group for Southern blot analysis using a hamster HPRT cDNA probe. When compared with irradiation with 4 Gy and immediate replating, dose fractionation (2 Gy + 24 h + 2 Gy) and delayed plating (4 Gy + 24 h) appeared to have no effect on the frequency of deletions encompassing the entire gene. Since an increase in survival was noted under these conditions, these data suggest that repair of sublethal and potentially lethal damage acts equally on all premutagenic lesions, regardless of type or location. Differences in the mutation spectrum were noted when cells were irradiated at 2 Gy and replated immediately. The location of the deletion breakpoints was determined in 15 mutants showing partial loss of the HPRT locus. In 12 of these cell lines one or both of the breakpoints appeared to be located near the center of the gene, indicating a nonrandom distribution of mutations. These results indicate that damage induced by ionizing radiation results in a nonrandom distribution of genetic damage, suggesting that certain regions of the genome may be acutely sensitive to the mutagenic effects of ionizing radiation.

In order to correlate the physical and chemical properties of radiation insult with the observed biological response, it is important to investigate

Correspondence: Thomas L. Morgan, Ph.D., Pacific Northwest Laboratory, P8-47, P.O. Box 999, Richland, WA 99352 (U.S.A.).

the molecular nature of the damage produced in the genetic material of the cell. It is apparent from a variety of studies that the majority of radiationinduced mutations result from deletions of genetic information. For example, Cox and Masson (1978) irradiated female human diploid fibroblasts and demonstrated that, depending on radiation type, up to 40% of 6-thioguanine-resistant (TG r) mu-

0027-5107/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

172

rants were associated with chromosomal aberrations (exchanges and terminal deletions) located near the mapped position of the H P R T locus. Such specific cytogenetic abnormalities have been observed, with varying frequencies, by other investigators (Fuscoe et al., 1983, 1986; Brown and Thacker, 1984; Vrieling et al., 1985). On the molecular level, recent advances in biological techniques have allowed investigators to obtain direct evidence concerning the pattern of DNA alterations responsible for these mutant phenotypes (for review, see Thacker, 1985). Using a cloned cDNA copy of mouse dihydrofolate reductase (DHFR) mRNA as a probe, Graf and Chasin (1982) demonstrated that 2 of 9 y-ray-induced mutations at the D H F R locus in Chinese hamster ovary cells (CHO) showed altered Southern blot hybridization patterns consistent with DNA deletions or rearrangements. In a larger study of radiation-induced H P R T mutants, Thacker (1986) found that 20 of 43 y-ray-induced mutants showed complete loss of coding sequences. Another 10 alterations resulted from a partial loss or rearrangement of genetic information, while the remaining 13 contained undetectable changes. Other investigators have noted similar patterns in the response of mammalian cells to the mutagenic effects of ionizing radiation (Gibbs et al., 1987; Vrieling et al., 1985). While it is well known that cellular processes associated with repair of sublethal or potentially lethal damage also act on premutational lesions (Jostes and Painter, 1980; Thacker and Stretch, 1983), no systematic study has been reported in the literature determining how the molecular structure of radiation-induced mutations is affected by radiation dose or DNA repair. The present study was conducted to explore the effect of these variables on the yield, type, and location of X-ray-induced mutations. Materials and methods

density (1000/75-cm 2 flask) prior to initiation of this study. From this level, the population was expanded until sufficient numbers were available for storage by freezing in liquid nitrogen. Individual aliquots of these cells were thawed and cultured only long enough for an individual experiment. These cells had a low spontaneous background frequency of approximately 7 H P R T mutant cells per 106 viable cells. Several treatment protocols described below required holding irradiated cell populations for varying lengths of time after irradiation. Uninhibited cell division during this holding period would have complicated calculation of surviving fraction and induced mutation frequencies. In addition, all treatment protocols required the use of cells in the same metabolic state so that meaningful comparisons of results between treatments could be made. Consequently, CHO-K1 cells were irradiated in starved plateau phase as described previously (Nelson et al., 1984). Briefly, 105 cells were seeded into 25-cm2 flasks and allowed to grow to confluence for 12 days without refeeding. Under these conditions, less than 0.1% of the cells are undergoing D N A replication (Nelson et al., 1984). Irradiated cells were subcultured into several flasks before any cell division occurred, to insure the independence of each mutant clone isolated. The flasks were maintained separately and only one mutant clone was picked from each population. Following subculture, the cells were maintained in exponential growth in non-selective medium for 9 days to permit degradation of pre-existing stocks of H P R T protein and m R N A in radiation-induced mutants. The cells were then reseeded at a density of 2 × 105/100-mm dish in Ham's F12 medium formulated without hypoxanthine (Gibco) and supplemented with 5% dialyzed fetal calf serum and 10 /~M 6-thioguanine (TG; Sigma Chemical Co., St. Louis, MO). Individual T G r clones were trypsinized using cloning cylinders and propagated in McCoy's 5a medium.

Cells and culture medium. Chinese hamster ovary cells (CHO-K1) were routinely cultured in McCoy's 5a medium (Gibco, Grand Island, New York) supplemented with 10% fetal calf serum and antibiotics. To reduce the background of spontaneous mutants, these cells were diluted to a low

X-Ray exposure. X-Irradiations were carried out using a Norelco MG-300 X-ray machine operating at 250 kVp and 10 mA (half-value layer 0.52 mm Cu). The dose rate was 1 G y / m i n . The dose rate was monitored with a Farmer model 2502/3 air ionization chamber (Nuclear Enterprises,

173 Beeham, Reading, England). Cells were irradiated at 37°C in 25-cm2 flasks containing 5 ml of medium and 5% C02/95% air.

Southern blot analysis. DNA from mutant and unirradiated cells was analyzed for mutations at the HPRT locus by the method of Southern (1975). High-molecular-weight DNA was isolated from exponentially growing cells by lysis in buffer containing 50 mM Tris, 10 mM EDTA, 100 mM NaC1, 0.5% sodium lauroyl sarcosine (pH 8). Following digestion with proteinase K (100 ~tg/ml; Sigma) at 50 °C overnight, lysates were extracted with phenol-chloroform (Maniatis et al., 1982) and treated with RNAase A (50/~g/ml; Sigma) at 37°C for 60 rain. After extraction with phenolchloroform, the purified DNA was exhaustively dialyzed against TE buffer (10 mM Tris, 1 mM EDTA, pH 8) and stored at 4°C. 20/~g of DNA from each of these samples were digested with restriction endonucleases under conditions specified by the enzyme supplier. Digested DNA was subjected to electrophoresis through 0.8% agarose gels overnight, followed by treatment with 0.4 M NaOH, 0.6 M NaC1 (30 min) and 1.5 M NaC1, 0.5 M Tris (pH 7.5; 30 min). The DNA was transferred to GeneScreen Plus hybridization membrane (Dupont-NEN, Wilmington, DE) by capillary blotting in 10 x SSC (1.5 M NaC1, 0.15 M sodium citrate, pH 7.0). After 18-24 h the membranes were removed and immersed in 0.4 M NaOH for 30-60 sec followed by 0.2 M Tris, 2 x SSC (pH 7.5). The membranes were air dried and stored at 4°C. Prior to hybridization, the membranes were pretreated at 65 °C for at least 1 h with 20-30 ml of 1 M NaC1, 1% SDS, 10% dextran sulfate, 1 × Denhardt's reagent (0.02 Ficoll, 0.02% polyvinylpyrrolidone, 0.02% BSA [fraction V]; Sigma). Following addition of 2-3 mg of denatured salmon sperm DNA and 50-100 ng of labeled probe DNA, hybridization was carried out overnight at 65 o C. The membranes were washed with 2 x SSC at room temperature (two washes of 5 min each), 1% SDS, 2 x SSC at 65 o C (two washes of 30 rain each), and 0.1 x SSC at room temperature (two washes of 30 min each). Autoradiography was carried out at -80 °C using Kodak X-omat R film and intensifying screens.

H P R T probe and labeling reaction. Blots were hybridized to the 1.3-kbp Msp I fragment of the plasmid pHPT12. This probe contained a fulllength copy of the hamster HPRT cDNA (Konecki et al., 1982). pHPT12 was provided by J.C. Fuscoe (Lawrence Livermore National Laboratory) and was isolated from E. coli HB101 by alkaline/SDS lysis and banding on CsC1 gradients as described by Maniatis et al. (1982). Probe DNA was labelled with 32p using a random primer oligolabeling reaction kit (Pharmacia, Piscataway, N J). 50 ng of DNA was denatured by heating to 95-100 °C for 10 min and annealed to 6-bp random sequence primers. This mixture was incubated overnight at room temperature with 50 /tCi of [a-32p]dCTP (3000 Ci/mmole; Dupont-NEN), unlabeled dATP, dGTP, dTTP, BSA and the Klenow fragment of DNA polymerase I. Labeled DNA was purified by NACS-52 column chromatography (Bethesda Research Labs., Gaithersburg, MD). We typically achieved a specific activity of 0.5-1.0 x 10 9 dpm//~g. Results

Response of CHO-K1 cells to X-rays The response of plateau-phase CHO-K1 cells to the cytotoxic and mutagenic effects of graded doses of 250 kVp X rays is shown in Fig. 1. The survival curve showed a characteristic shouldered response while the induced mutation frequency curve indicated a linear-quadratic response. In the case of a single dose followed by immediate replating, starved plateau-phase cells showed 3 times as many induced mutants when irradiated with 4 Gy as with 2 Gy (120 vs. 38 TG r mutants/106 viable cells). Relative to immediate replating, delaying replating of irradiated ceils by 24 h resulted in no significant change in the mutation frequency (120 vs. 113), despite a modest increase in cell survival (from 25% to 43% survival). Evidence for the repair of submutational lesions is seen in our results for the effect of dose fractionation on the frequency of radiation-induced mutations. Delivering 4 Gy in two equal fractions separated by 24 h caused a reduction in the mutation frequency to approximately two-thirds of that observed with an acute dose of 4 Gy (79 vs. 120 TGr mutants/106 surviving cells) as well as an increase in survival

174 DOSE (GY) 1

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Fig. 1. Cytotoxicity (A) and induced mutation frequency (B) as a function of X-ray dose. Platean-phase CHO-K1 cells were irradiated and plated immediately (e), given 4 Gy and held at confluence without refeeding for 24 h (B), or given two 2-Gy doses separated by 24 h (A). Each point represents the mean and standard error of at least 4 determinations.

(to 42%). The results of the delayed plating and split-dose experiments suggest that plateau-phase cells are capable of repairing a modest amount of sublethal and potentially lethal damage. However, as found by Thacker and Stretch (1983), delayed plating does not significantly reduce the number of potentially mutagenic lesions induced by a given dose of X-rays. Several studies (Thacker, 1979; Jostes and Painter, 1980; Nakamura and Okada, 1981; Radner et al., 1982; Ueno et al., 1982; Thacker and Stretch, 1983) have found that low dose rate or dose fractionation decreases the induced mutation frequency at the HPRT locus. The results presented here are consistent with these studies. Structure of the hamster H P R T gene High-molecular weight DNA was digested with the restriction endonucleases Pst I or Hind III, yielding the banding patterns depicted in Fig. 2. Pst I digestion resulted in bands of 10, 9, 6.7, 5, 3.4, 2.9 and 0.9 kbp (Fig. 2A, lane 1). Band sizes

of 14.7, 11.2 and 6.3 kbp were observed with Hind III digestion (Fig. 2B, lane 1). The 6.3-kbp Hind III and 5-kbp Pst I bands were observed in DNA from all 89 mutant cell lines. A weakly hybridizing 2.9-kbp Pst I fragment was also frequently seen, particularly following long exposures. This is consistent with the work of Fuscoe et al. (1983) which determined that these three bands belong to an unlinked pseudogene hybridizing strongly to HPRT-specific probes. Fig. 2 also indicates the exon elements which comprise each band. The hamster HPRT gene is composed of 9 exons ranging in size from 18 bp to 601 bp, spanning approximately 40 kbp (Rossiter, 1987). Exons 2, 3 and 4 are located within the 10-, 3.4-, and 6.7-kbp Pst I bands, respectively. Similarly, the 9- and 0.9-kbp Pst I fragments contain exons 6-8 and 9, respectively. In Hind III digestions, exons 2-4 are contained in the 14.7-kbp band while exons 6-9 are located within the 11.2kbp band. Although the probe we used contained exon 1, no separate fragment containing this exon was detected in the present study and was not observed by Rossiter (1987). We also failed to detect a band containing exon 5 in either the Hind III or Pst I digests. This was probably due to the size of this exon (18 bp) and the stringent washing conditions we used (65 ° C, 2 × SSC). CHO ceils contain only one X-chromosome (Deaven and Petersen, 1973) and hence, no restriction fragments from a homologous gene on an inactive chromosome were present. DNA from unirradiated V79 cells showed band sizes and migration patterns identical to CHO-K1 when either Pst I or Hind I I I was employed (data not shown). This restriction fragment migration pattern, for both the Hind III and Pst I digestions, was consistent with that observed by Fuscoe et al. (1983), Thacker (1986), Rossiter (1987) and Kaden et al. (1989). However, Fuscoe et al. (1983) observed only one large Pst I fragment, migrating at 7.8 kbp. The 5' Pst I fragment probably contains only exon 2, which is relatively small (110 bp; Rossiter, 1987) and hence, may not have been detected due to the stringent hybridization conditions employed by Fuscoe et al. (1983) (final wash, 0.1 × SSC at 50°C). The 13- and 10-kbp Hind III and 8.3- and 7.6-kbp Pst I fragments observed by Thacker et al. (1990) in the accompanying paper are somewhat

175 1

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A Fig. 2. Southern blot banding patterns of the HPRT sequences in unirradiated and mutant cell lines. DNA samples were digested with either Pst I or Hind III and electrophoresed through 0.8% agarose gels. Following transfer to GeneScreen Plus, the blots were hybridized to a cDNA copy of the HPRT gene. Molecular size markers are displayed on the left side of each panel. The exon(s) contained within each band are indicated on the right side. 'P-bands caused by hybridization to pseudogene sequences. Panel A, Pst I digestion patterns; Lane 1: CHO-K1; Lanes 2-5: mutants 2SF, 2SB, 4DA, 4PG. Panel B, Hind III digestion patterns; Lane 1: CHO-K1; Lanes 2-5: mutants 2SH, 4DD, 4DB, 4DA.

smaller t h a n the f r a g m e n t s we observed. Differences in m o l e c u l a r weight s t a n d a r d s a n d / o r subtle differences in the m o l e c u l a r structure of the C H O a n d V79 H P R T genes m i g h t a c c o u n t for these discrepancies.

Characterization of X-ray-induced mutations M u t a n t s were isolated f r o m each of the following t r e a t m e n t protocols: (i) 2 G y of X-rays, with i m m e d i a t e replating; (ii) 4 G y , with i m m e d i a t e replating; (iii) 4 G y , split into two equal fractions s e p a r a t e d b y 24 h, followed b y i m m e d i a t e subculture; a n d (iv) 4 G y , followed b y 24-h d e l a y b e f o r e

subculture. F r o m these 4 t r e a t m e n t groups we i s o l a t e d a total of 89 T G r cells fines. D N A from each cell line was d i g e s t e d with at least two restriction e n d o n u c l e a s e s . O n the basis of their S o u t h e r n b l o t b a n d i n g p a t t e r n , we sorted the observed p a t t e r n s i n t o 3 categories (for examples, see Fig. 2): (i) full d e l e t i o n s - all H P R T - s p e c i f i c b a n d s were missing (except for the u n l i n k e d pseud o g e n e b a n d s ) . M u t a n t 4 P G in lane 5 of Fig. 2A a n d m u t a n t s 4 D D a n d 4 D B in lanes 3 a n d 4 of Fig. 2B d e m o n s t r a t e the a b s e n c e of H P R T sequences; (ii) a l t e r a t i o n s - m o d i f i e d b a n d i n g p a t terns i n d i c a t i n g the p r e s e n c e o f p a r t i a l deletions,

176

insertions, rearrangements or modifications of one or more restriction-enzyme cutting sites within the H P R T locus. Mutants 2SF and 2SB in lanes 2 and 3 of Fig. 2A and mutant 4DA in lane 5 of Fig. 2B are examples of this category; (iii) no change - the banding pattern was indistinguishable from D N A isolated from untreated CHO-K1 cells. Mutants 4DA in Lane 4 of Fig. 2A and 2SH in lane 2 of Fig. 2B show unaltered banding patterns. Mutant 4DA demonstrated the importance of using at least two different enzymes: with Pst I, we observed no change, while with Hind III we noted a substantial alteration of the banding pattern. Table 1 summarizes the data collected on all the H P R T mutant cell lines. 54 mutants lacked any detectable HPRT-specific sequences. This was the predominant type of lesion observed, regardless of the treatment regime employed. Loss of or a shift in size of individual bands was the second most common pattern noted. 25 mutants were found to have altered banding patterns. 10 of the 89 mutants showed no change in the band migration pattern, which suggested the presence of small lesions, such as point mutations or small deletions. It should be emphasized here that some or all of the members of this latter class may have suffered substantial deletions of genetic information; howTABLE 1 SUMMARY OF MUTATION TYPE AS DETERMINED BY SOUTHERN BLOT ANALYSIS a Dose schedule

Mutation type b Alteration

No change

2.0 Gy

Full 9 (43%)

8 (38%)

4 (19%)

Total 21

4.0 Gy

20 (69%)

7 (24%)

2 (7%)

29

4.0 Gy (24 h delayed plating)

9 (70%)

2 (15%)

2 (15%)

13

4.0 Gy (2 Gy + 24 h + 2 Gy)

16 (62%)

8 (31%)

2 (7%)

26

Totals

54 (61%)

25 (28%)

10 (11%)

89

a Each DNA sample was digested independently with at least two restriction enzymes. b Full, no HPRT coding sequences detected. Alteration, loss of and/or change in size of one or more bands. No change, no apparent change in band size or migration pattern.

ever, because of the difficulty in resolving small changes in size of the large restriction fragments, we were unable to detect any changes. To investigate this class of mutants further, we digested each D N A with a third restriction enzyme, Ssp I, which generated 5 HPRT-specific bands ranging in size from 1.6 to 6 kbp (data not shown). Since each of these mutants again showed no apparent change in their digestion patterns we concluded that inactivation of the gene was most likely caused by small changes such as point mutations. We estimate the resolution of our experiments varied from about 1 kbp for the 14.7-kbp Hind III fragment to about 0.05 kbp for the 0.9-kbp Pst I fragment. To investigate the effect of repair on the mutation spectrum, the frequency of the various classes of lesions was determined for each treatment group. For cells irradiated with 4 G y and replated immediately, 69% (20/29) showed full deletion of the H P R T gene, while 24% (7/29) showed altered banding patterns. In cell populations irradiated with two 2-Gy doses separated by 24 h, 62% (16/26) of the mutants had suffered full deletions and 31% (8/26) showed partial deletions or rearrangements of the H P R T gene. This difference was not statistically significant. Similarly, no significant change in the mutation spectrum due to holding cells in a nonproliferative state for 24 h after irradiation was observed. These data indicate that repair does not alter the type of mutations induced by 250 kVp X-rays. We also considered the effect of dose on the mutation spectrum. Since repair had no effect on the spectrum, a statistical comparison was made between the 2-Gy treatment group and the spectrum resulting from a combination of all three 4-Gy groups. First we calculated a spontaneous mutation spectrum for each treatment group and subtracted it from the results reported in Table 1. For example, assuming that approximately one spontaneous mutant in 5 showed a full deletion or alteration-type mutation (Thacker and Ganesh, 1989), and noting that the induced mutation frequency at 2 Gy (38) was 5.4 times the background (7), we subtracted 4 mutations, one full deletion and 3 no change-type, from the 2-Gy spectrum. A similar calculation done on each 4-Gy treatment group indicated that a total of 5 no

177 change-type mutations should be omitted from the composite group. Under these conditions, only 47% (8/17) of the mutants in the 2 Gy group exhibited full deletions. In the 4-Gy group 71% (45/63) of the mutants appeared to have suffered a full deletion. This difference had a p value of 0.06 (Bevington; 1969). The principal reason for this difference was the higher fraction of alteration-type events observed in the cell population irradiated with 2 Gy (8/17) compared with the 4-Gy group (17/63). These data suggest that an increase in dose may result in the production of more large scale lesions (i.e., full deletions). However, these results are not conclusive and indicate that further exposures at higher doses and analysis of a larger number of mutants clones will be necessary before a firm conclusion can be drawn.

Location of deletion breakpoints We examined the Southern blot banding patterns from the 25 mutant cell lines showing altered or absent bands with the intention of determining the approximate size and location of each lesion. For the purposes of this analysis, we assumed that each lesion arose as the result of a single mutational event and that the structure of the CHO HPRT gene is essentially identical to that of the human locus. Both are some 40-45 kbp in length and contain 9 exons and 8 introns. While the size of the introns varies somewhat between the two species, the pattern of exon spacing is the same (Rossiter, 1987; Patel et al., 1986). 15 mutants presented banding patterns consistent with partial deletions of the HPRT locus. Fig. 3 presents representative Southern blot patterns from 7 of these mutants. With knowledge of the exon complement of each fragment, we were able to localize the deletion breakpoints to specific regions of the locus. For example, in mutant 12A the Pst I digestion pattern shows a loss of the 6.6-kbp band containing exon 4 and a new band migrating at about 8.1 kbp was noted (Fig. 3A, lane 8). This new band probably contained exon 4 since the remaining fragment migration pattern appeared to be identical to the CHO-K1 banding pattern. The Hind III digestion pattern was consistent with this interpretation since the 14.7-kbp fragment, which contains exons 2, 3 and 4, appeared to be truncated to about 11.5 kbp (Fig. 3B,

lane 8). Therefore, it appeared that this mutation probably resulted from an internal deletion which destroyed the Pst I site on the 3' end of the 6.6-kbp fragment, generating a new fragment which extended to the next available Pst I site. Because the 3' Hind III and Pst I fragments appeared undisturbed, we concluded that deletion size was equal to the amount lost from the larger Hind III fragment, about 3.2 kbp. In mutant 10E, only the 9-kbp Pst I band (containing exons 6-8) was missing (Fig. 3A, lane 6). In its place we observed a 5.5-kbp fragment. The ll.2-kbp Hind III fragment was gone and in its place a 7-kbp fragment was observed (Fig. 3B, lane 6). The 3' end of the locus appeared unmodified since the Pst I digestion pattern showed a band at 0.9 kbp, indicating the presence of exon 9. Therefore, we concluded that this mutation resulted from an internal deletion, of about 4.2 kbp, between exons 4 and 6. The remaining 13 mutants were analyzed in a similar fashion. Fig. 4 shows the approximate size and location of the lesions in these 15 cell lines. Intragenic deletions ranging in size from approximately 5 kbp to 13 kbp were observed in 9 mutants (3F3, 61, 10E, 12A, 12D, 13D, 13E, 4SF, 4PA). Two mutants suffered substantial deletions on the 3' side of the locus (8B and 8C), while 3 others were caused by loss of sequences in the 5' half (2SB, 4PC, 2SF). One mutant, 10G, resulted from a deletion extending from the 5' flanking region to a point between exons 6 and 7. Because of our inability to visualize exon 1 and lack of flanking sequence markers, we could not estimate the size of these latter 6 mutants because we could not rule out extension of the deletions into flanking regions. Cell lines from 3 of the 4 treatment groups are represented in Fig. 4. Mutants 2SB, 8B, 8C, 2SF, 13D and 13E were isolated from cells irradiated with 2 Gy. Mutants 3F3, 4SF, 12A and 12D came from cells treated with 4 Gy. From the 4-Gy split dose protocol, mutants 4PC, 61, 10E, 4PA and 10G were isolated. No intragenic or partial deletion mutants were isolated from the cells in the delayed plating experiment. However, we isolated the fewest number of mutants from this protocol. The pattern of deletions represented in Fig. 4 does not appear to be random. In this figure 24 endpoints from 15 mutants are plotted. Although the

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Fig. 3. Representative Southern blot banding patterns of parental CHO-K1 and deletion mutants 3F3, 2SB, 8B, 8C, 10E, 10G, 12A. Blots were prepared as described in Fig. 2. Informationfrom these blots was used to construct a map of deletion breakpoints for each mutant (see Fig. 4). Panel A, Pst I digestion patterns; Lanes 1-8: CHO-K1, 3F3, 2SB, 8B, 8C, 10E, 10G, 12A. Panel B, Hind III digestion patterns; Lanes 1-8: CHO-K1, 3F3, 2SB, 8B, 8C, 10E, 10G, 12A.

size of most of the deletions could not be determined precisely, there appears to be a disproportionate number of mutations in the 3' half of the gene (i.e., between exons 4 and 9). These data suggest that this region of the hamster H P R T gene is abnormally sensitive to radiation-induced events creating deletion-type mutations. The remaining 10 mutants presented banding patterns which were not consistent with deletions of the type shown in Fig. 4. In most cases, interpretation of the banding patterns from several different enzymes resulted in conflicting conclusions regarding the nature and extent of the lesion (data not shown). Two mutants (12B and 12E) showed altered Ssp I patterns indicating the presence of deletions. However, the Pst I and Hind III patterns remained unchanged. Exon assignments

for Ssp I bands have not been made. These mutants were included in the alteration-type mutation class but we could not determine where the mutation was located. 4 other mutants showed pattern changes consistent with deletions when one enzyme was used. However, when a different enzyme was used no changes were detected. These data suggested the presence of small deletions or point mutations which either removed a restriction site or generated a new site. By this analysis, 1 of these 4 mutants (6H) appeared to have a small lesion in the 3' half of the gene while the lesions in the remaining 3 mutants (2SC, 4DA, 4DH) appeared to be located in the 5' side. The approximate location of the lesions in these 4 cell lines is shown in Fig. 4. Banding patterns of the four remaining mutants (6G, 10H, 13A, 4SB3) were too complex

179 12 3

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Fig. 4. Structure of the Chinese hamster HPRT gene and position of deletion breakpoints in mutants 2SB, 3F3, 8B, 4PC, 8C, 2SF, 10E, 4SF, 13D, 12A, 12D, 13E, 4PA, 10G and 61. Exons 2-6 and 9 are located as indicated. Exons 7 and 8 have been localized to the region within the stippled box (B.J.F. Rossiter, in preparation). Black bars indicate deletions. Stippled bars represent uncertainty in the position a n d / o r extent of the deletion. The approximate location of mutations of unknown type (i.e., point mutation, insertion or deletion) are indicated by open bars containing question marks.

to interpret, suggesting the presence of chromosomal rearrangements such as translocations or inversions. Discussion We have studied the effects of dose, dose fractionation and delayed plating on the type and frequency of radiation-induced mutations in the H P R T gene of CHO-K1 cells. Starved plateauphase cells appear to be able to repair a significant amount of sublethal and potentially lethal damage, as well as damage which may be operationally defined as submutational, since our split-dose experiments showed a decreased frequency of induced mutations. This finding is consistent with several other studies (Jostes and Painter, 1980; Nakamura and Okada, 1981) and suggests that submutational damage induced by relatively low doses (i.e., 2 Gy) can be repaired to a limited

extent. However, no significant difference was demonstrated between the spectrum of mutations induced in cells irradiated with a split dose of 4 Gy and those irradiated with an acute dose of 4 Gy, suggesting such repair does not act preferentially against one specific class of mutations. Much larger samples of the spectrum of mutations induced under various exposure conditions will be required to determine if repair acts primarily as a dose-modifying factor with respect to mutation spectrum. Our failure to demonstrate any decrease in induced mutation frequency in cells held in a nonproliferative state for 24 h following irradiation is in agreement with several other investigators. No significant difference was noted by Thacker and Stretch (1983) or O'Neil and Flint (1985). In the former study a modest increase in cell survival was noted at 4 Gy. A number of investigators have reported on the molecular characterization of radiation-induced mutations. The fraction of mutant cell lines showing altered Southern blot banding patterns consistent with deletion of all or part of the gene under study varies from 16% at the APRT locus of C H O cells irradiated with 5 Gy of "y-rays (Grosovsky et al., 1986) to 100% at the D H F R locus of CHO cells irradiated with 6 Gy of y-rays (Urlab et al., 1986). At the H P R T locus deletions are the most common form of genetic lesion observed (Vrieling et al., 1985; Skulimowski et al., 1986; Thacker, 1986; Gibbs et al., 1987; Liber et al., 1987). Although our data are consistent with these reports, direct comparison is difficult due to differences in radiation dose levels, the target locus employed, and our use of plateau-phase cells. From the data presented in Table 1, we interpret the relatively high frequency of mutants lacking any detectable H P R T coding sequences as an indication that the typical radiation-induced mutation resulted from deletion of a region of the X-chromosome larger than the H P R T gene itself. Many of these deletions may have extended into one or more neighboring genes. However, because of the limited size of the C H O H P R T locus, about 40 kbp, we were unable to detect the endpoints of these deletions. Urlaub et al. (1986) investigated the induction of deletion-type mutations in the D H F R locus of C H O cells and attempted to de-

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termine their size by employing Southern blot techniques and a variety of probes from an overlapping set of cosmid clones spanning 210 kbp of DNA about this locus. 3 of 7 T-ray-induced mutants were found to have resulted from a deletion of the entire 210-kbp region. The remaining mutants were determined to have suffered deletions of 20-115 kbp. Thus it is clear that the size of radiation-induced lesions will be defined by the particular chromosomal environment in which the target gene resides. Hemizygotic loci, such as the H P R T gene, are relatively insensitive to large (i.e., multilocus) deletions; whereas heterozygotic loci, such as the T K or a 1 genes (Evans et al., 1987; Waldren et al., 1979, 1986), appear to be able to detect much larger lesions. In the case of hemizygotic loci, a multilocus deletion event is probably lethal due to loss of essential single copy genes. It is generally believed that the spatial pattern of radiation-energy deposition is random and hence, results in a similarly random distribution of genetic lesions. Our data presented in Fig. 4 indicate that this assumption is incorrect. It appears that the 3' half of the hamster H P R T gene is abnormally sensitive to the mutagenic effects of 250-kVp X-rays. The work presented here represents the largest study conducted to date on the location of radiation-induced deletion breakpoints. Previous, more limited studies, have suggested a similar conclusion. Grosovsky et al. (1986) irradiated CHO cells with y-rays and characterized 55 A P R T - mutants. Three were determined to be intragenic deletions and may have had identical breakpoints. Pastink et al. (1988) analyzed 5 X-ray-induced deletions at the white locus of Drosophila melanogaster and found that they were nonrandomly distributed; two contained the same 29-bp deletion, while 3 others were clustered. In the accompanying paper by Thacker et al. (1990) a clustering of deletion breakpoints was also observed in mutants isolated from cells treated with either T-rays or a-particles. Spontaneous mutations have also revealed an unexpected clustering of deletion breakpoints. In an extensive study of mutations in the H P R T locus of human B-lymphoblasts, Gennett and Thilly (1988) observed a disproportionate number of deletion endpoints occurred within a restricted region of the gene; 16 of 27 breakpoints were

found to lie between exons 4 and 7. In agreement with our investigations, the density of deletion breakpoints within this region of the human H P R T gene was more than twice as high as could be expected on the basis of a random distribution. These data suggest that certain regions of the mammalian genome may be uniquely sensitive to mutations occurring spontaneously or induced by ionizing radiation. Our data, as well as that presented by Thacker et al. (1990) and Gennett and Thilly (1988), suggest that one such region is located in the 3' half of the hamster H P R T locus, perhaps in the vicinity of exon 5. While the underlying mechanism responsible for this effect is unknown, it is tempting to speculate that this region may contain some unique sequence or secondary structure which renders it more sensitive to mutation induction. For example, the organization of chromatin may interfere in some way with the fidelity of D N A repair. Eukaryotic chromatin appears to be organized into looped domains of D N A constrained by sites of interaction with a structure called the nuclear matrix or scaffold (Paulson and Laemmli, 1977). Such sites have been observed within active genes. Kas and Chasin (1987) identified a region near the center of the D H F R gene in CHO cells that has affinity for attachment to the nuclear protein scaffold. Two tightly linked sites were localized within this region. Each of these scaffold-associated regions (SAR) is A + T rich and contains a cleavage consensus sequence for Drosophila topoisomerase II as well as direct and inverted repeated sequences. In the case of the H P R T locus, the region adjacent to exon 5 in RJK159 cells (a derivative of V79) has been sequenced to a limited extent (B.J.F. Rossiter, in preparation). The region immediately 5' to exon 5 has a very high A + T content ( > 70%) and contains at least one sequence which shows an 89% (16/18) homology to the cleavage consensus sequence for topoisomerase II purified from both chicken and human cells (Spitzner and Muller, 1988). The presence of SARs in the middle of an active zone of chromatin, such as the H P R T gene, might result in steric hindrance of repair enzymes and hence, sensitivity to mutagenesis. Since SARs and topoisomerase II cleavage sites are often found located within the same stretch of DNA (Kas and Chasin, 1987; Phi-Van and Str~iling, 1988), we are

181

exploring the possibility that there is a topoisomerase II binding site in the central region of the CHO HPRT gene.

Acknowledgements The authors wish to thank M.W. Cho and T.L. Curry for their excellent technical assistance on this project. This work was supported by the Office of Health and Environmental Research (OHER) U.S. Department of Energy under contract DEAC06-76RLO 1830 to Pacific Northwest Laboratory and contract DE-AM06-76-RLO2225 to the Northwest College and University Association for Science (University of Washington).

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