147

Mutation Research, 249 (1991) 147-159 © 1991 Elsevier Science Publishers B.V. 002%5107/91/$03.50 ADONIS 002751079100136D

MUT 04988

Analysis of point mutations induced by ultraviolet light in human cells Phouthone Keohavong, Vivian F. Liu i and William G. Thilly Center for Environmental Health Sciences and Division of Toxicology, Whitaker College of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139 and 1 Department of Genetics, Harvard Medical School, Boston, MA (U.S.A.) (Received 10 September 1990) (Revision received 3 December 1990) (Accepted 4 December 1990)

Keywords." Point mutation; HPRT; UV; Human lymphoblastoid cells

Summary Mutations induced in cultured human cells by 254-nm UV light were analyzed within exon 3 of the hypoxanthine guanine phosphoribosyl transferase (HPRT) gene. Five large independent cultures of human lymphoblastoid cells, line TK6, were exposed to 4 J//m 2 of 254-nm UV light and mutants at the H P R T locus were selected en masse by 6-thioguanine (6TG) resistance. Exon 3 of the H P R T gene was amplified from the mutant cells by polymerase chain reaction (PCR) using modified T7 D N A polymerase. Denaturing gradient gel electrophoresis ( D G G E ) was used to separate the mutant sequences from the wild type as m u t a n t / w i l d - t y p e heteroduplexes. Individual mutant bands were isolated from the gel and the nature of the mutations was determined by direct sequencing. Eight predominant mutations were detected in the 184-bp exon 3 sequence. Of these, 3 transitions, including 2 G-C to A-T and 1 A-T to G-C, and 2 A-T to C-G tranversions, appeared in all 5 UV-treated cultures but not in untreated cultures and were thus considered to be mutational hotspots. These observations are similar in nature to those previously reported in bacterial and rodent cells. A single G deletion, a tandem substitution of C p T for TpA, and a tandem triple substitution of G p G p A for A p A p G were also observed but in only 2, 2 and 3 of the 5 UV-treated cultures, respectively. Numerical analysis of the mutant fractions of these 8 mutations indicated that each of them was distributed as a set of non-random and independent events, i.e., a mutational hotspot.

Studies with phage and bacteria have shown that mutagens are specific with regard to the kind and position of the induced mutation (Benzer and Freese, 1958; Coulondre and Miller, 1977). A par-

Correspondence: Dr. P. Keohavong, Center for Environmental Health Sciences, E18-666, Massachusetts Institute of Technology, Cambridge, MA 02139 (U.S.A.).

ticular impediment to experimental progress has been the difficulty of the process by which information about the pattern of mutations, termed mutational spectrum, is obtained. In general, cultures of single cells have been exposed to a mutagen, independent mutants isolated one at a time and the nature of each mutant determined (Benzer and Freese, 1958). Because of the variety of possible mutations in even a short D N A sequence, a

148 large number of independent mutants must be isolated in order to obtain a reasonably reproducible spectrum among independent experiments (Coulondre and Miller, 1977). As a facilitating alternative to the clone-byclone process, we have proposed selection of mutants in mass cultures, separation of mutants by denaturing gradient gel electrophoresis ( D G G E ) (Fischer and Lerman, 1983; Lerman and Silverstein, 1987) and sequencing of mutant DNA from bands isolated from such gels (Thilly, 1985). Subsequently, the adoption of D N A amplification by polymerase chain reaction (PCR) has greatly simplified this approach (Kleppe et al., 1971; Mullis and Faloona, 1987) . Previously, we used the combination of PCR and D G G E to determine the error rates, kinds and positions of mutations induced by a series of DNA polymerases in the 104-bp low-temperature melting of exon 3 of the human H P R T gene. We found that modified T7 D N A polymerase gives the highest overall net D N A synthesis per cycle (or efficiency) and a sufficiently high degree of fidelity of amplification so that mutants representing 10 -3 of the total DNA sequences may be observed (Keohavong and Thilly, 1989). To apply this protocol to detect mutations in mixed cell populations, large cultures are used to reduce sib selection error by the weight of sample size. An artifact inherent in D N A amplification, allelic preference, can be controlled by observation of the stability of the mutational spectrum as a function of cycles of amplification (Keohavong and Thilly, 1989). Given the necessary controls, the protocol should identify hotspots in DNA samples of whole cell populations as mutations arising in all of several independent cultures. Here, we report our results using the approach to search for hotspot mutations induced by 254-nm UV light in the 184-bp third exon of the H P R T gene in cultured human TK6 cells. The data obtained are discussed in relation to those reported for bacterial and other mammalian cell systems. Materials and methods Materials

2'-Deoxynucleoside-5"-triphosphates as 100mM solutions were obtained from Pharmacia (Piscataway, N J). 6-Thioguanine (6TG) was ob-

tained from Sigma Chemical Company (St. Louis, MO). The oligonucleotides (Synthetic Genetics, CA) used as primers for PCR were: PI: 5'- CATATATTAAATATACTCAC - 3 ' P2: 5'- T¢CTGATTTTATTTCTGTAG- 3' P3: 5 ' - GACTGAACGTCTTGCTCGAG - 3' P4: 5'- TCCAGCAGGTCAGCAAA- 3' P2-GC-clamp: 5'- GCCGCCTGCAGCCCGCGCCCCCCGTGCC¢CCGCCCCGCCGCCGGCCCGGGCGCCTCCTGATTT TATTTCTGTAG- 3'.

To obtain radioactive DNA, PCR was carried out with 5'-end-labeled primers (150 C i / m m o l e ) using [y-p32]ATP (7000 Ci/mmole), T4 polynucleotide kinase, and the reagents in the 5' end D N A terminus labeling system (Bethesda Research Laboratories, Gaithersburg, MD). Modified T7 DNA polymerase (Sequenase TM) was purchased from U.S. Biochemicals (Cleveland, OH) (Tabor and Richardson, 1987). Cell culture, U V irradiation and 6 T G treatment

Human male lymphoblastoid cells, line TK6 (Skopek et al., 1978), were grown in stock spinner cultures and diluted daily to 3 x 105 cells/ml. Following 2 days' growth in anti-selective medium to reduce background mutant fractions, cultures were exposed to 254-nm UV light at 4 J / m 2 under exactly the same conditions as previously described (DeLuca et al., 1983). In order to obtain a sufficiently large number of surviving mutants, at least 30 batches of 2 x 10 7 cells each were treated and combined to create a single culture of at least 6 x 108 irradiated cells. These cells were resuspended in media (1600 ml, 4 x 105 cell/ml) and samples were taken from each culture to determine the surviving fractions. By the second day post treatment, cell concentration reached 10 6 cells/ml and the cells were subsequently diluted daily by a factor of 3 to permit continuous exponential growth. Following a 6-day expression period, the initial mutant fraction at the H P R T locus was calculated for each cell culture by determining the relative plating efficiencies in the presence and absence of the selective agent 6TG (Furth et al., 1981). The cells remaining in the bulk cultures were diluted to 8 x 105 cells/ml, treated with 6TG at 10 ~ g / m l and allowed to grow without dilution for 3 days. Two thirds of the media were then replaced with

149 fresh media to reduce debris caused by the lysis of 6TG-sensitive cells. This step was repeated until the increase in 6TG resistant (6TG R) cell number was apparent in daily cell counts. By 2 weeks after 6TG addition, all cultures were in exponential growth and were daily diluted 1 : 3 with 1 / x g / m l 6TG-containing medium. The 6TG R cells were then harvested for DNA analysis. Between 6TG addition and harvesting of cells, some 20 generations elapsed and the 6TG mutant fraction increased from 2 × 10 -5 to 1. This set of 6TG R cells representing descendants of surviving background and UV-induced mutants was designated 'mixed mutants' indicating that they are a set of different mutants within the HPRT gene.

Analysis of 6TG R mutants PCR conditions The HPRT exon 3 sequence was amplified directly from the mixed mutant cell population using modified T7 DNA polymerase under conditions which yield a sufficient degree of fidelity (Keohavong and Thilly, 1989). Pellets of 105 6TG-selected cells were first rinsed with 1 × PBS (15 mM Na2HPO 4, 1.5 mM KH2PO 4, 137 mM NaCI, 2.7 mM KC1), then resuspended in 100 /~1 of the following reaction mixture: 10 mM Tris, pH 8.0, 2.5 mM MgC12, 2.5 mM dNTP, 3 /xM each of primers P1 and P2 (see Fig. 1A). Prior to PCR, the cells were lysed by the addition of 5 #g of proteinase K (Sigma) for 30 min at 37 ° C. Each cycle consisted of boiling for 1 min (3 min for the first cycle), cooling at room temperature for 45 s and at 3 7 ° C for 15 s, adding 2 units of modified T7 DNA polymerase (Sequenase TM)(the enzyme was diluted to 2 units//~l in 20 mM KPO 4, pH 7.4, 1 mM DTT, 0.1 mM EDTA, 50% glycerol), followed by 2 min incubation at 370C. After 25 such cycles, 5 #1 of the reaction mixture was analyzed on a 6% polyacrylamide gel. The 224-bp fragment was then gel-purified from the reaction mixture and resuspended in 10 mM Tris-HC1, pH 7.5, and 1 mM EDTA. For D G G E analysis, 5 × 101°copies of the 224-bp fragment were used as template for an additional 102-fold amplification with endlabeled primers, either P1 + P3 or P4 + P2-GCClamp, to detect mutations in the 104-bp low-temperature or 80-bp high-temperature melting do-

mains of exon 3, respectively (see Fig. 1). The resultant 204-bp and 180-bp end-labeled fragments were gel-purified and analyzed by DGGE.

Denaturing gradient gel electrophoresis analysis The kinds of induced mutations were analyzed at the level of the 184-bp exon 3 sequence of the HPRT gene using the combination of high-fidelity PCR and D G G E (Keohavong and Thilly, 1989). Exon 3 is naturally composed of a 104-bp lowtemperature and an 80-bp high-temperature melting domain (see Fig. 1, bp positions 300-403 and 220-299, respectively). This structure makes the molecule a suitable template for analysis by D G G E to detect any point mutation occurring in the 104-bp low-temperature melting domain. In order to detect mutations occurring in the remaining 80-bp high-temperature melting domain, an artificial high-temperature melting domain is added during a second round of PCR using a primer carrying a G-C-rich sequence or clamp. This gives rise to a 180-bp fragment (see Fig. 1) (Sheffield et al., 1989; Cariello et al., 1990). The 204-bp or 180-bp end-labeled PCR products (specific activity = 6 x 105 cpm/pmole) were gel-purified from the reaction mixture, and samples of 105 cpm were boiled, reannealed and separated on a 12.5% polyacrylamide gel (bis/acryl = 1/37.5) containing 15-30% (for the 204-bp fragment) and 36-45% (for the 180-bp fragment) denaturant concentrations prepared as described (Cariello et al., 1988). Determination of the nature of the mutations Autoradiograms of the PCR products separated by D G G E (see Figs. 2 and 3) revealed a series of bands which putatively represented heteroduplexes each containing a strand from the mutant and the antiparallel strand from the wild type. Using the autoradiogram superimposed on the gel, each mutant band (indicated by an arrow in Figs. 2 and 3) was excised from the gel, DNA was isolated and further amplified. In subsequent D G G E analysis, the amplified product from each initial band gave rise to 2 distinct bands, one of which cofocused with the homoduplex wild-type DNA and the other represents a mutant homoduplex (see Fig. 4). DNA was then isolated from the putative mutant homoduplex and sequenced.

150

In some cases, a band contained more than 1 mutant/wild-type heteroduplex. In these cases, after further amplification and subsequent D G G E analysis, we observed a homoduplex wild-type band and 2 or more mutant homoduplexes. In experiments reported here mutant homoduplexes were all resolved from wild-type homoduplexes facilitating sequencing analysis. This is not always the case, however (Cariello et al., 1990). Results

Behavior of the untreated cultures and the cultures treated with 254-nm UV light In UV-treated cultures the average 6TG R mutant fraction of 2.4 x 10-5 was in close agreement with that found in previous work (DeLuca et al., 1983). Also, the mutant fractions in the untreated controls (1.6 x 1 0 - 6 and 3.1 x 10 -6) were within the historical control distribution for these cells. Relative cell survival in treated cultures was 0.50 as compared with the previously observed 0.9 (DeLuca et al., 1983), a difference which may be ascribed to the longer handling times inherent in obtaining larger numbers of treated cells. The number of surviving 6TG R mutants immediately after treatment of each culture was at least (6.4 x 108) (0.27) (2.4 x 10 -5) = 4150, where 0.27 represented the mean absolute surviving frac-

tion (see Table 1). Of these, (6.4 × 108) (0.27) ( 2 . 4 x 10 6 ) = 415 would be surviving background mutants, 10% of the total sample. Variance among treated cultures' mutant fractions was in accordance with expectation for the protocol employed (Leong et al., 1985). Analysis of mixed mutant cells and characterization of individual mutant sequences by DGGE Low-temperature melting domain The exon 3 sequence was first amplified from cells using flanking primers P1 and P2 which generated a 224-bp fragment (see Fig. 1A). To analyze mutations in the low-temperature melting domain, the 224-bp fragment was gel-purified and amplified an additional 102-fold using 5'-endlabeled primer P1 and internal primer P3. The resulting 204-bp radioactive D N A was boiled and reannealed to create m u t a n t / w i l d - t y p e heteroduplexes prior to analysis by D G G E . In this manner, any mutation occurring within the 104-bp low-temperature melting domain of exon 3 was expected to be detected as 2 m u t a n t / w i l d - t y p e heteroduplexes separated from the normal sequence (wild-type homoduplex) in lower denaturant concentrations of the gel. Fig. 2 shows the patterns of m u t a n t / w i l d - t y p e heteroduplexes separated by D G G E for both the

TABLE 1 EXPERIMENTAL

CELL CULTURE

PARAMETERS

Expt.

Treatment

Treated cell n u m b e r

Absolute surviving fraction

Relative surviving fraction

Mutant fraction x 106

6TG R surviving mutants a

2 3 4

None None 4 J/m 2

4.6 x 107 4.8 × 107 6.5 × 108

0.49 0.64 0.31

1.0 1.0 0.56

1.6 3.1 31

36 94 6300

5

254 n m 4 J/m 2

6.2 x 108

0.26

0.47

22

3 600

6

254 n m 4 J/m z

6.4 x 108

0.25

0.45

19

3000

7

254 nm 4 J/m 2

6.4 x 10 s

0.27

0.49

24

4 300

6.4 x 108

0.27

0.49

24

4 300

8

254 nm 4 J/m 2 254 nm

a 6 T G R s u r v i v i n g m u t a n t s : t r e a t e d cell n u m b e r × a b s o l u t e s u r v i v i n g f r a c t i o n x m u t a n t f r a c t i o n .

151

untreated (lanes 2 and 3) and UV-treated cells (lanes 4-8) for the low-temperature melting domain of exon 3. The lane numbers correspond to B

12

3 4 5 6 7 8

g2 90 8B 86

mutant/wild type heteroduplexes

84 Melting 82 Temperature 80 7e 76 74 72 70

wild type 68

homoduplex

66 64

i

i

i

i

L

220

i

i

260

i

i

300

i

i

340

i

380

i

i 420

bp - position

A

Intron2

Intron3

P2 ~

~

GC-ClamolP2

I

i

i

P4

i

i

~

~ R~

(204-bp) (224-bp)

Fig. 1. Positions of the primers used for PCR and melting map of the human HPRT exon 3 sequence. (A) Positions of the primers (P) used to amplify exon 3 of the human HPRT gene. Five primers were used: P1 and P2 are complementary to the introns (broken lines) immediately flanking the 3' end and 5' end of exon 3, respectively. Internal primer P3, adjacent to P2, is complementary to the 5' end of exon 3. Internal primer P4 is complementary to the sequence extending from bp 308 to bp 324. P2-GC-Clamp represents primer P2 extended by a 54-bp high melting sequence or clamp (see Materials and methods). The expected sizes of PCR products using P1 + P2 (224 bp), P I + P 3 (204 bp) and P4+P2-GC-Clamp (180 bp) are indicated as thick lines. (B) Melting map for exon 3 and exon 3 modified by the addition of a G-C-rich sequence. The solid line represents the melting map of the wild-type exon 3 sequence which is composed of naturally high- (positions 220299) and low- (positions 300-403) temperature melting domains. Only mutations occurring in the low-temperature melting domain of this fragment can be detected by DGGE. The dashed line represents the melting map of the 180-bp fragment which contains the naturally high-temperature melting domain (positions 220-299) extended with the clamp. The latter sequence created a new high-temperature melting domain (Sheffield et al., 1989; Cariello et al., 1990) and mutations occurring within the naturally high-temperature melting domain can now be detected.

Fig. 2. D G G E analysis of mutations occurring within the low-temperature melting domain (bp positions 300-403) of exon 3 from untreated and UV-treated cells. Two series of amplifications have been carried out: first exon 3 was amplified about 5 × 106 times from 10 s cells using flanking primers P1 + P2 which generated a 224-bp fragment (see Fig. 1A). The 224-bp fragment was gel-purified and 5 × 10 TM copies were used as templates for additional 102-fold amplification using 5'end-labeled primers P1 + P3. The resulting radioactive 204-bp fragment was gel-purified and samples of l0 s clam were boiled, reannealed and separated through a 12.5% polyacrylamide gel containing linear 18-28% denaturant concentrations (Cariello et al., 1988; Keohavong and Thilly, 1989). The gels were dried and autoradiographed. Lane 1 corresponds to D N A amplified from untreated and unselected cells. Lanes 2-8 are DNA amplified from independent experiments of 6TG-selected cells either without UV treatment (lanes 2 and 3) or after treatment with 4 J / m 2 254-nm UV light (lanes 4 to 8). Each lane shows a wild-type homoduplex band which focused at 25% of denaturant concentrations, and a pattern of bands corresponding to mutant/wild-type heteroduplexes which focused at lower denaturant concentrations of the gel. The pattern of m u t a n t / wild-type heteroduplexes seen in lane 1 represents the background of modified T7 D N A polymerase-induced mutant sequences (Keohavong and Thilly, 1989). Compared with lane 1, a faint band is observed in one of the untreated but 6TGselected cultures (indicated by an arrow in lane 3), a series of additional bands appear in each individual UV-treated culture (lanes 4-8). All additional bands visible in each individual 6TG-selected culture were excised from the gel and the DNA was isolated for further characterization. For each lane, the positions of the 2 mutant/wild-type lieteroduplexes of the predominant mutant(s) found are indicated by a pair of specific arrows or lines. The nature and frequency of each individual mutant are shown in Fig. 7.

152

the experiment numbers shown in Table 1. A separate control (lane 1) is DNA from untreated and unselected cells and represents G-C to A-T mutants known to be produced by modified T7 D N A polymerase during the PCR process (Keohavong and Thilly, 1989). Compared with lane 1, one untreated culture (lane 3) displayed an additional band (indicated by an arrow) representing about 1% of the wildtype band while the other showed no discernible band (lane 2). After UV treatment (lanes 4-8), each of the 5 cultures showed a series of bands not observed in the untreated and unselected culture (lane 1) or in the untreated but 6TG-selected cultures (lanes 2 and 3), presumably representing point mutations induced by 254-nm irradiation. Several bands appeared to occur with some variation in intensity in all 5 UV-treated cultures while others were visible in only 1 or 2 cultures.

High-temperature melting domain The 224-bp fragment was used as template for an additional 102-fold amplification using endlabeled internal primers P4 and P2-GC-Clamp (see Fig. 1A). The resultant 180-bp fragment which now contained a new high-temperature melting domain was analyzed by D G G E to detect mutations occuring within the 80-bp formerly hightemperature melting domain. Fig. 3 shows the patterns of mutant/wild-type heteroduplexes in D G G corresponding to the 80-bp high-temperature melting domain of exon 3. Compared with the control of untreated and unselected cells (lane 1), 4 additional bands were detected in each of the 2 untreated but 6TG-selected cultures as indicated by arrows in lanes 2 and 3. Five additional bands appeared in 3 UV-treated cultures (lanes 4, 5 and 6) and 3 of them were visible in the other 2 UV-treated cultures (lanes 7 and 8). These bands presumably represent background 6TG-selected point mutations (lanes 2 and 3) and UV-induced point mutations (lanes 4-8) occurring in the 80-bp high-temperature melting domain of exon 3. Isolation of mutant sequences D N A from each visible band as indicated in Figs. 2 and 3 was isolated and further amplified. The resultant D N A was gel-purified and checked

for homogeneity by boiling, reannealing and D G G E analysis before sequencing. An example of standard D G G E characteriza-

I

2

3

4

5

6

7

8

wild type homoduplex Fig. 3. D G G E analysis of mutations occurring within the high-temperature melting domain (bp positions 220-299) of exon 3 from untreated and UV-treated cells. The procedure described in Fig. 2 was carried out with 2 modifications: (1) the additional 102-fold amplification from the first round amplified 224-bp template was carried out using 5 '-end-labeled primers P4+P2-GC-Clamp and (2) the resulting 180-bp fragment was separated as mutant/wild-type heteroduplexes through a 36-45% DGG. The D N A analyzed corresponded, in lane 1, to untreated and unselected cells which represents polymerase-induced mutant sequences, in lanes 2 and 3 to 6TG-selected cells without UV treatment, and in lanes 4 - 8 to 6TG-selected cells after treatment with 4 J / m 2 254-nm UV light. Each lane shows a wild-type homoduplex band which focused at 42% denaturant concentration and a pattern of mutant/wild-type heteroduplexes at lower denaturant concentrations. Compared with the pattern of polymerase-induced mutants (lane 1), 3 additional mutant bands appeared in each of the 2 control cultures (lanes 2 and 3), 3 additional mutant bands are visible in UV-treated cultures 7 and 8, and 5 additional bands are visible in UV-treated cultures 4, 5 and 6. All additional bands indicated by an arrow were excised from each lane, and the D N A was isolated from gel slices for further characterization.

153

tion of mutant bands isolated from the gel is illustrated in Fig. 4. When the D N A isolated from the bands designated 1 and 2 in Fig. 2, lane 4 was further amplified and analyzed by subsequent D G G E (lanes - b ) , 2 major homoduplex bands, mutant and wild-type, were observed. After boiling and reannealing (lanes + b), these same DNA samples showed 2 additional bands representing the 2 respective mutant/wild-type heteroduplexes. The fact that both bands 1 and 2 gave rise to the same patterns of bands before and after boiling and reannealing (lanes - b and + b, respectively) strongly suggested that they originated from the same mutant homoduplex. Sequencing analysis of the mutant homoduplexes confirmed that they contained the same mutation, an A-T to G-C transition at bp 381 of exon 3 (see mutant D, Fig. 7, part A). In this way, all prominent bands were analyzed from all 5 UV-treated cultures and the 2 untreated control cultures. A total of 11 different 6TGselected mutants were identified. These included 6 in the 104-bp low-temperature melting domain (Fig. 5A, mutants A - F ) and 5 in the 80-bp hightemperature melting domain (Fig. 5B, mutants G - K ) . In all 11 cases the mutant homoduplex (indicated by a vertical arrow) focused at a position distinct from the wild-type homoduplex. In 3 of the 11 cases (mutants A, F and H) the 2 respective mutant/wild-type heteroduplexes did not separate from each other but were well resolved relative to the wild-type homoduplex.

Test for allelic preference in DNA amplification One important issue was the possibility of preferential amplification of mutant alleles during the PCR process which could lead to artifactually induced hotspots. In order to test this possibility, experiments were carried out to compare the efficiency of amplification for all mutants isolated to that of the wild-type. First, to test this possibility for the 6 mutants found in the low-temperature melting domain of exon 3 (Fig. 5A, mutants A - F ) , an additional 103-fold amplification was carried out from a 1/1 mixture between each individual mutant homoduplex and the wild-type, and the amplified DNA was analyzed by D G G E after boiling and reannealing (Fig. 6A). Any difference in the efficiency of amplification of a mutant

2 I

..; 1

t")

1 i

!

+b -b L -b +b

0

g

ID

Q

iD iB

tant horn~:Jupl ex

Fig. 4. Example of characterization by D G G E of individual 6 T G R mutant bands. Lane 'expt. 4' corresponds to a D N A sample identical to that shown in Fig. 2, lane 4. The D N A was isolated from bands designated 1 and 2 and further amplified. The P C R products were gel-purified and samples of 5 × 103 c p m were separated through an 18-30% D G G either without (lanes - b) or after (lanes + b) boiling and reannealing. In the former case, 2 major homoduplex bands, wild-type and mutant, were observed. After boiling and reannealing, 2 m u t a n t / w i l d type heteroduplexes appeared in addition to the m u t a n t and wild-type homoduplexes and all 4 b a n d s have equal intensity. In lanes - b , 2 faint m u t a n t / w i l d - t y p e heteroduplex b a n d s are also visible, presumably formed during the boiling and reanhealing steps of the last cycle of the P C R process (Keohavong and Thilly, 1989). The same patterns of b a n d s obtained for both D N A isolated from bands 1 and 2, with and without boiling and reannealing, suggested that these 2 b a n d s originated from the same homoduplex mutant. This was confirmed by sequencing the homoduplex mutant bands which both contained an A-T to G - C transition at the same base-pair position 381 (see m u t a n t D, Fig. 7, part A).

154

D N A relative to that of the wild-type D N A would result in a change in the ratio between the homoduplex mutant D N A and the wild-type D N A before (lanes a) and after (lanes b) additional amplification. For instance, a difference of only 5% in the efficiency of amplification from the original 90% would result in a difference in such a ratio by a factor of about 2.8 after a 103-fold amplification and would be easily detected. As shown in Fig. 6A, there was no significant difference in the efficiency of amplification for the 6 mutants found in the low-temperature melting domain of exon 3 relative to that of the wild-type sequence. In the case of the 5 mutants found in the high-temperature melting domain, a different strategy to test for allelic bias was employed. A 103-fold additional amplification of the D N A samples used for spectrum analysis (Fig. 3) was performed to observe whether any of the prominent bands changed in intensity relative to the wild-type homoduplex. D G G E analysis shows in Fig. 6B that both the 2 control mutants (indicated by arrows in lanes 2) and the 3 UV-induced mutant bands (indicated by arrows in lanes 5) did not change in relative abundance before (a) and after (b) an additional 103-fold amplification. This indicates that these mutants are amplified with the same efficiency as the wild-type allele. With regard to mutants J and K (Fig. 5B) whose respec-

A B C D E F

G H I J

O

~j

wildtype~:~pp (A)

(BI

K

tive homoduplexes and both heteroduplexes focused at the same positions of the gel, we further amplified and sequenced the homoduplex band that contained both mutants and found that their respective proportions did not change during an additional 103-fold amplification. These data

Fig. 5. Patterns in D G G of individual mutants found in the 184-bp exon 3 sequence. Each mutant homoduplex DNA was mixed with an equal amount of wild-type D N A (about 7.5 x 103 cpm each). The mixture was boiled and reannealed and the DNA was separated on either a 15-30% D G G for mutants in the low-temperature melting domain (part A) or a 36-45% D G G for mutants in the high-temperature melting domain (part B). (Part A) A total of six different 6TG-selected mutants were found in the low-temperature melting domain including 1 (mutant A) from 1 of the 2 untreated cultures and 5 (mutants B, C, D, E and F) from the 5 UV-treated cultures. Each lane shows either 4 bands (mutants B, C, D and E) including 2 homoduplexes, mutant (indicated by a vertical arrow) and wild-type, and 2 mutant/wild-type heteroduplexes or only 3 bands (mutants A and F) for which the 2 respective heteroduplexes did not resolve and focused at the same position in the gel. The 5 UV-induced mutants ( B - F ) appeared with varying frequencies among the 5 UV-treated cultures as shown in Fig. 2 (lanes 4-8). For instance, one can easily observe the predominance of mutant C in Fig. 2, lanes 7 and 6 ( *-- ), and of mutant F in Fig. 2, lanes 4 and 8 ( = ) . Mutant B is predominant in Fig. 2, lane 5 (4) and also easily visible in lane 8 (4). Although mutant D did not appear as predominant bands in any of the 5 experiments, it is easily visible in Fig. 2, lanes 4 and 7 (4) and 8 (-). Mutant E is clearly visible only in Fig. 2, lane 6 (4). To discover if the mutants found indeed existed in all 5 cultures albeit at lower frequencies in some cultures, detailed analysis was carried out. This consisted of cutting out gel slices at the positions of the expected heteroduplex bands from each of the 5 UV-treated cultures. The DNA was isolated from the gel and further amplified. Subsequent separation by D G G E and sequencing showed that mutants C, D and E were present in all 5 experiments while mutants B and F were found in only 2 experiments (Fig. 2, lanes 5 and 8 for mutant B, and lanes 4 and 8 for mutant F). (Part B) D G G E analysis of the mutants found in the high-temperature melting domain of exon 3. The bands indicated by an arrow as shown in Fig. 3 were excised from each lane of the gel (lanes 4-8), the DNA was isolated from the gel slice, further amplified and separated through a 36-45% DGG. A total of 5 different mutants were found including 2 mutants (H and I) which appeared in the 2 untreated and 6TG-selected cultures (Fig. 3, lanes 2 and 3) and 3 UV-induced mutants G, J and K. Mutants J and K were found in all 5 UV-treated cultures while mutant G was detected in only 3 of the 5 UV-treated cultures (Fig. 3, lanes 4, 5 and 6).

155 t o g e t h e r i n d i c a t e t h a t a positive bias in the amplification p r o c e s s has not a f f e c t e d the p r e s e n t esti-

Analysis of the kinds and positions of the mutations The

kind

and

position of each mutant

was

determined by direct sequencing of the mutant

mation of the frequencies of the mutants found.

a

b

'2 5' A

B

C

D

E

F

WT WT 8 0

(A)

-~- WT

(B)

Fig. 6. DGGE analysis to test the possibility of preferential amplification during the PCR process of mutant alleles corresponding to the low-temperature (part A) and high-temperature (part B) melting domains. (Part A) DNA was isolated from homoduplex mutants A - F and from the wild-type band as shown in Fig. 5A. 2.5 × 103 cpm from each mutant DNA was then mixed with an equal amount of the wild-type DNA, and a 1//102 aliquot was taken from each such mixed DNA and amplified an additional 103 times using end-labeled primers P1 and P3. The DNA was gel-purified from the reaction mixture and the increase was determined by counting the radioactivity incorporated in the amplified DNA. Samples of 5 × 103 cpm from each mixed mutant/wild-type DNA were boiled, reannealed and separated through an 18-30% DGG. The gel was dried and autoradiographed. The amount of DNA distributed among the wild-type, homoduplex mutant, and the heteroduplexes both before (lanes a) and after (lanes b) an additional 103-fold amplification was estimated by densitometry and the ratio of the recorded intensity between the mutant and wild-type homoduplex bands was then determined for each lane. (Part B) The possibility of aUelic preference for mutants found in the high-temperature melting domain of exon 3 was tested directly by using the original mixed mutant DNA similar to those shown in Fig. 3, lanes 2 and 5, which already contained the 2 control mutants and the 3 UV-induced mutants, respectively. To this end, aliquots of 5 × 102 cpm (5 × 10 s copies) of the 180-bp DNA from samples 2 and 5 were amplified an additional 103 times using end-labeled primers P4 and P2-GC-Clamp. The amplified DNA was gel-purified and 105 clam was boiled, reannealed and separated through a 36-45% DGG. The gel was dried and autoradiographed. The mutant bands before (2a and 5a) and after (2b and 5b) an additional 103-fold amplification in both the control culture (lanes 2) and the UV-induced culture (lanes 5) are indicated by arrows. Each band as indicated was excised from the gel and the amount of radioactivity was estimated by Cerenkov counting. The mutant fraction was estimated as the ratio between the radioactivity in each band and the total radiactivity in each lane.

156

homoduplex DNA. Three mutants (A, H and I) were found only in untreated cultures. Mutants A and H contained a 12-bp deletion from positions 365-376 and 2 7 7 / 2 7 8 - 2 8 8 / 2 8 9 , respectively. The mutant fractions were estimated to be 0.5% for mutant A and 2.5% for mutant H. Mutant I corresponded to an A-T to T-A transversion at position 290 and had a mutant fraction of about 2%. Out of the 8 mutants found in UV-treated cultures (Fig. 7A), 4 (mutants C, D, E and F) were substitutions at an A-T base-pair, 2 (mutants J and K) were substitutions at a G-C base-pair. Mutant B contained a 1-bp deletion of G-C at position 365 and mutant G corresponded to a tandem triple mutation at position 231-233. The 6 base-pair substitutions were: 2 G - C to A-T transitions at positions 293 and 294 (mutants J and K, respectively), an A-T to G-C transition at position 381 (mutant D), 2 A-T to C-G tranversions at positions 369 and 381 (mutants C and E, respectively) and 1 double substitution T-A to C-G, A-T to T-A at positions 399-400 (mutant F). Muta-

tions J and K occurred at 2 adjacent G - C (positions 293-294) within a run of 6 G - C base-pairs while mutations D and E occurred at the same A-T base-pair (position 381) within a run of 4 A-T base-pairs. Furthermore, mutants C, D, E, J and K were detected in all 5 cultures, while mutants B, F and G were detected in only 2, 2 and 3 of the 5 UV-treated cultures, respectively. Based on measurement of 32p incorporated in the various m u t a n t / w i l d - t y p e heteroduplex bands and in the wild-type homoduplex band as shown in Figs. 2 and 3, the mean fractions of the total 6 T G R mutants for the 5 hotspot mutations C, D, E, J and K were estimated to be 1.6%, 1.3%, 0.8%. 2.7%, and 0.8%, respectively (Fig. 7, part B). Similarly, the mean mutant fractions for mutants B, F and G, among the cultures in which they were detected, were 0.4%, 1.0%, and 1.1%, respectively. Knowledge of the fraction of 6 T G R mutants represented by each mutant band and the total 6 T G R mutant fractions in the treated or control cultures (see Table 1) allowed us to calculate mutant fractions in each culture for each specific

B. •

mutants

detected in all experiments.

mutants

detected

in o n l y

some

experiments.

3 C

o~ I

2?0 ! 5' TCTTGCT a' A~_C.Gc~

A.

the (229~ [ CCA GGT Mutants designated: ((j)

Kinds

I

~

I TT~COEI3~!

'

'

'

, ~

nlh ,4o

xk. ,~T(]~.~TATC~ACTG'I-AC;AITI'_~,TAT =

of

mutations:

A

A

T

(J) (K)

~7

G

~

(B)(C)

C

~

G

~

(D) (E)

GA

CT (F)

Fig. 7. Summary of the kind, position and percentage of each of the mutations found in the entire exon 3 sequence. Part A shows the kinds and positions of the mutations occurring in both the high-temperature (mutants G, J and K) and low-temperature (mutants B, C, D, E and F) melting domains of exon 3 (~7 = deletion). Part B shows the average percentage of each mutant shown in part A estimated as a function of the total 6TG R mutants in each of the 5 treated cultures. Those for hotspot mutants (J, K, C, D and E) are in solid bars and those for the mutants found in only 2 cultures (B and F) or 3 cultures (G) are in open bars.

157

mutant prior to 6 T G selection. This number multiplied by the number of cells surviving treatment allowed us to calculate the number of surviving specific mutants in each case. This number is the basis for analysis of the precision of the experiments (Leong et al., 1985). For the lowest fractions detected (mutants E and K) the average number of surviving mutants in each of the 5 treated cultures was at least (0.8 x 10 -2) (2.4 × 10 -5) (0.27) (6.4 x 108) = 33. The use of five independent cultures thus yielded about 165 events leading to mutants E or K. Similar calculations indicated that each treated culture would have contained an average of 54 mutants D, 64 mutants C and 112 mutants J immediately after UV treatment among surviving cells. Similarly, there would have been an average of 16 mutants B, 40 mutants F and 44 mutants G, among 5 cultures.

Discussion UV-mutational spectra in exon 3 of the H P R T gene Five different mutations (C, D, E, J and K) were detected in all 5 UV-treated cultures but in neither of the 2 untreated cultures nor in any of several other control cultures used in other studies (unpublished data). These we conclude are 254-nm UV-light hotspots induced in the 184-bp exon 3 sequence. This sequence represents about 20% of the H P R T gene in which point mutations could plausibly affect phenotype (Patel et al., 1986). These 5 hotspot mutations represented 7.2% of the total 6 T G R mutants. Two of them occurred at the level of 2 consecutive Gs within a run of 6 G - C base-pairs and resulted in G-C to A-T transitions (mutant J and K). Two others occurred in a run of 4 A-T base-pairs and resulted in 2 different base substitutions: an A-T to G-C transition (mutant D), and an A-T to C - G tranversion (mutation E). Because these 4 mutations occurred within a run of G-C or A-Ts, their appearance most probably resulted from initial formation of either (6-4) PyC or thymidine dimer photoproducts, respectively (Gordon and Haseltine, 1982; Setlow and Carrier, 1966; Tailor et al., 1988). Hotspot mutation C is more difficult to explain since the T on the opposite strand which is substituted by a G is surrounded by two purines (5'-TA_TGA-3') and therefore cannot result from intrastrand pyrimi-

dine-pyrimidine photoproduct formation. H o w ever, such UV-induced base-pair substitutions occurring at the level of a pyrimidine within purinepyrimidine-purine sequences have been previously reported (Drobetsky et al., 1987; Lebkowski et al., 1985; Vrieling et al., 1989). In the present and all 6 previously reported instances, however, there has been a purine-purine sequence 3' to the pyrimidine substitution site so that the possibility of forming an adjacent pyrimidine photoproduct existed on the opposite strand. It may be that, under certain conditions, the base-pair immediately following a photochemical lesion is at greater risk of misreplication or misrepair than the bases involved in the lesion. Unlike mutants C, D, E, J and K, mutants B, F and G were observed in 2, 2 and 3 of the 5 cultures at averages of 0.4%, 1.0%, and 1.1%, respectively. Using the method of Leong et al. (1985) to analyze the expected numerical variation of the mutant fraction for each putative hotspot, we calculated that the most prominent mutant J, with a mean mutant fraction of 6 X 10 -7, would have had an estimated standard deviation of 1.5 × 10 -7 and should have been observed in all cultures as it was. However, mutant B, with a mean mutant fraction of only 1.0 x 10 - 7 and a standard deviation calculated to be 0.8 x 10-7, would lead to an expectation that it could not be observed in some cultures consistent with our observations. Therefore, we conclude that all 8 mutations reported are non-random UV-induced mutations and that the variation among cultures is consistent with numerical expectation for mutational hotspots. UV-induced mutations have long been investigated and our results, the first using an endogenous gene in a human cell, are in general agreement with previous reports using a clone-by-clone analysis in rodent cells and bacteria. Drobetsky et al. (1987, 1989) have examined a series of 254-nm UV light (5 J / m 2) mutations induced in the A P R T gene in C H O cells both as a chromosomal gene (27 independent mutants) and as carried in a retroviral shuttle vector (34 independent mutants). The most frequent kind of mutation found in these systems was the G-C to A-T transition (17/34 and 16/27, respectively), while the set of all base-pair substitutions beginning with A-T was also found (5/34, 5/27, respectively). These data

158

were in agreement with earlier observations by Hauser et al. (1986) and Lebkowski et al. (1985) who, at 50 J / m 2, found predominance of G-C to A-T mutations in a vector carrying UV-irradiated genes replicated in monkey and human cells, respectively. Using the E. coli lacI system, Coulondre and Miller (1977) analyzed 653 UV-induced mutants and found 399 G-C to A-T transitions. This apparent predominance arose from 4 G-C to A-T hotspots accounting for 263 of the 399 G-C to A-T mutants. The large number of clones analyzed permitted identification of frequently occurring mutants or hotspots. Based on these observations it was concluded that 'ultraviolet light induces all types of base substitutions without any apparent specificity for a particular class of substitution'. Vrieling et al. (1989) have reported UV-induced mutational spectra in the H P R T gene of V79 hamster cells. Wild-type V79 cells irradiated with 12 J / m 2 of 254-nm UV light showed a variety of base-pair substitutions over the entire H P R T reading frame with the exception of the G-C to C-G transversion. Of the mutants found, one, UV-V10, contained an A-T to G-C transition which is identical to our mutant D at position 381. Two tandem double mutations (G-C to A-T, G-C to A-T) at positions 293-294 (UV-H41) and 294-295 (UVH9) were also found at the run of 6 G-C (positions 292-297) where we found 2 hotspot G-C to A-T transitions (mutants J and K). Furthermore, of the 14 single base-pair substitutions reported, 7 originated at an A-T base-pair which is comparable to the present data. On the other hand, with a UV-sensitive derivative of the V79 cell line (V-H1), single base-pair changes consisted solely of G-C to A-T transitions, while tandem and frameshift mutations were also found. In the novel procedure employed herein, 3 potential sources of bias may be noted. Firstly, the selection en masse of mutant cells requires the analysis of a set of sibling descendants of induced mutants. Sib selection may be a source of random error when each independent mutant isolated is represented in low number as is the case in most clone-by-clone studies. In our study, sibling selection was not expected to be a significant source of bias because of sufficiently high numbers of individual induced mutations found in each of 5 inde-

pendent experiments. A second possible bias is that of phenotypic selection: one kind of H P R T mutant could grow faster or slower than the average and lead to an inaccurate estimate of the induced fraction of a mutant. Early study of the 6 T G R phenotype in human B cells showed, however, that independent mutants had the same average doubling times of independent 6TG-sensitive cells (Thilly et al., 1976). Finally, allelic preference in D N A amplification could be another source of error and we have taken care to control for this form of bias. Our previous study of D N A amplification showed that certain mutant exon 3 sequences generated by T4 or Klenow fragment D N A polymerases were amplified with an efficiency significantly higher than that found for the wild-type sequence (Keohavong and Thilly, 1989). We have tested this possibility for all of the UV-induced mutants reported here and found no allelic preference in amplification in any of them. This may be because the efficiency or overall net D N A synthesis per cycle of amplification of the exon 3 wild-type sequence using modified T7 D N A polymerase was sufficiently high, approximately 90% per cycle (Keohavong and Thilly, 1989). We have not, however, eliminated the possibility that a negative bias in amplification may have caused us to miss hotspots. Further studies are required to probe this possibility. The protocol described has also been used to analyze mutational spectra induced by mutagens such as M N N G , ICR-191 (Cariello et al., 1990), benzopyrene-diol-epoxide, oxygen and hydrogen peroxide as well as in vitro spontaneous mutations (unpublished data). The method is general for any selectable marker in a single cell population and has a limit of detection of 0.1% for any point mutant isolated among a collection of mixed mutants.

Acknowledgements We thank Drs. L. Ling and A. Kat for critically reading the manuscript and R. DeMeo for typing the manuscript. This work was supported by grants from the U.S. National Institute of Environmental Health Sciences (P42-ES04675, P01-ES00597, P50ES03926-05) and the Office of Health and En-

159

vironmental Research, U.S. Department of Energy (DE-FG02-86-ER60448). References Benzer, S., and E. Freese (1958) Induction of 'specific mutations with 5-bromouracil, Proc. Natl. Acad. Sci. (U.S.A.), 44, 112-119. Cariello, N.F., J.K. Scott, A.G. Kat, W.G. Thilly and P. Keohavong (1988) Resolution of missense mutation in human genomic DNA by denaturing gradient gel electrophoresis and direct sequencing using in vitro DNA amplification: HPRT-Munich, Am. J. Hum. Genet., 42, 726-734. Cariello, N.F., P. Keohavong, A.G. Kat and W.G. Thilly (1990) Molecular analysis of complex human cell populations: mutational spectra of MNNG and ICR-191. Mutation Res. 231, 165-176. Coulondre, C., and J.H. Miller (1977) Genetic study of the lac repressor. IV. Mutagenic specificity in the lacl gene of Escherichia coli, J. Mol. Biol., 117, 577-606. DeLuca, J.G., L. Weinstein and W.G. Thilly (1983) Ultraviolet light-induced mutation of diploid human lymphoblasts, Mutation Res., 107, 347-370. Drobetsky, E.A., A.J. Grosovsky and B.W. Glickman (1987) The specificity of UV induced mutations at an endogenous locus in mammalian cells, Proc. Natl. Acad. Sci. (U.S.A.), 84, 9103-9107. Drobetsky, E.A., A.J. Grosovsky, A. Skandalis and B.W. Glickman (1989) Perspectives on UV light mutagenesis: investigation of the CIIO aprt gene carried on a retroviral shuttle vector, Somat. Cell Mol. Genet., 15, 401-409. Fischer, S.G., and L.S. Lerman (1983) DNA fragments different by single base-pair substitutions are separated in denaturing gradient gel: correspondence with melting theory, Proc. Natl. Acad. Sci. (U.S.A.), 80, 1579-1583. Furth, E., W.G. Thilly, B. Penman, H. Liber and W. Rand (1981) Quantitative assay for mutation in diploid human lymphoblasts using microtiter plates, Anal. Biochem, 110, 1-8. Gordon, L.V., and W.A. Haseltine (1982) Quantitation of cyclobutane pyrimidine dimer formation in double- and single-stranded DNA fragments of defined sequences, Radiat. Res., 89, 99-112. Hauser, J., M.M. Seidman, K. Sidur and K. Dixon (1986) Sequence specificity of point mutations induced during passage of a UV-irradiated shuttle vector plasmid in monkey cells, Mol. Cell. Biol., 6, 277-285. Keohavong, P., and W.G. Thilly (1989) Fidelity of DNA

polymerases in DNA amplification, Proc. Natl. Acad. Sci. (U.S.A.), 86, 9253-9257. Kleppe, K., E. Ohtsuka, R. Kleppe, I. Molineux and H.G. Khorana (1971) Studies on polynucleotides. XCVI. Repair replication of short synthetic DNA's as catalyzed by DNA polymerases, J. Mol. Biol., 56, 341-361. Lebkowski, J.S., S. Clancy, J.H. Miller and M.P. Calos (1985) The lacI shuttle: rapid analysis of the mutagenesis specificity of ultraviolet light in human cells, Proc. Natl. Acad. Sci. (U.S.A.), 82, 8606-8610. Leong, P.M., W.G. Thilly and S. Morgenthaler (1985) Variance estimation in single-cell assays: comparison to experimental observations in human lymphoblasts at 4 gene loci, Mutation Res., 150, 403-410. Lerman, L.S., and K. Silverstein (1987) Computational simulation of DNA melting and its application to denaturing gradient gel electrophoresis, Meth. Enzymol., 155, 482-501. Mullis, K.B., and F.A. Faloona (1987) Specific synthesis of DNA in vitro via a polymerase catalyzed chain reaction, Meth. Enzymol., 155, 335-350. Patel, P.I., P.E. Framson, C.T. Caskey and A.C. Chinault (1986) Fine structure of the human hypoxanthine phosphoribosyltransferase gene, Mol. Cell. Biol., 6, 393-403. Setlow, R.B., and W.L. Carrier (1966) Pyrimidine dimers in ultraviolet-irradiated DNA's, J. Mol. Biol., 17, 237-254. Sheffield, V.C., D.R. Cox, L.S. Lerman and R.M. Myers (1989) Attachment of a 40-base-pair G + C-rich sequence (GCclamp) to genomic DNA fragments by the polymerase chain reaction resulted in improved detection of single base changes, Proc. Natl. Acad. Sci. (U.S.A.), 86, 232-236. Skopek, T.R., H.L. Liber, P.W. Penman and W.G. Thilly (1978) Isolation of a human lymphoblastoid line heterozygous at the thymidine kinase locus: possibility for a rapid human cell mutation assay, Biochem. Biophys. Res. Commun., 84, 411-416. Tabor, S., and C.C. Richardson (1987) DNA sequence analysis with a modified T7 DNA polymerase, Proc. Natl. Acad. Sci. (U.S.A.), 84, 4767-4771. Taylor, J.S., D.S. Garrett and M.P. Cohrs (1988) Solution-state structure of the Dewar pyrimidinone photoproduct of thymidylyl-(3',5')-thymidine, Biochemistry, 27, 7206-7215. Thilly, W.G., J.G. DeLuca, H. Hoppe IV and B.W. Penman (1976) Mutation of human lymphoblasts by methyl nitrosourea, Chem.-Biol. Interact., 15, 33-50. Vrieling, H., M.L. van Rooyen, N.A. Groen, M.Z. Zdzienicka, J.W.I.M. Simons, P.H.M. Lohman and A.A. van Zeeland (1989) DNA strand specificity for UV-induced mutations in mammalian cells, Mol. Cell. Biol., 9, 1277-1283.

Analysis of point mutations induced by ultraviolet light in human cells.

Mutations induced in cultured human cells by 254-nm UV light were analyzed within exon 3 of the hypoxanthine guanine phosphoribosyl transferase (HPRT)...
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