Mutation Research, 250 (1991) 365-374

365

© 1991 Elsevier Science Publishers B.V. All rights reserved 0027-5107/91/$03.50 ADONIS 002751079100195K

MUT 02509

Reduction to homozygosity is the predominant spontaneous mutational event in cultured human lymphoblastoid cells Donna K. Klinedinst * and Norman R. Drinkwater McArdle Laboratory for Cancer Research, Universityof 14rtsconsin, Madison, I41153706(U.S.A.) (Accepted 5 April 1991)

Keywords: Mitotic recombination; aprt; Recessive mutant phenotypes; Adenine phosphoribosyltransferase locus

Summary Expression of recessive mutant phenotypes can occur by a number of different mechanisms. Inactivation of the wild-type allele by base-substitution mutations, frameshift mutations or small deletions occurs at both hemizygous and heterozygous cellular loci, while other events, such as chromosome level rearrangements, may not be detected at hemizygous loci because of inviability of the resulting mutants. In order to assess the relative contribution of each type of mutational event, we isolated a human lymphoblastoid cell line that is heterozygous at the adenine phosphoribosyltransferase (aprt) locus. The mutation rate for the expression of the mutant phenotype (aprt+/-~aprt -/-) was 1.3 × 1 0 - 5 / c e l l / generation. Molecular analysis of the DNA from 26 mutant clones revealed that 19% had undergone deletion of the entire wild-type allele. The aprt heterozygote carries a mutation in the coding sequence of the gene that results in the loss of a restriction site. Analysis of aprt-/- mutants for this restriction fragment length difference revealed that 23% of the mutants contained point mutations or small ( < 100 bp) deletions. The remainder of the mutants (58%) resulted from reduction to homozygosity of the mutant allele. We suggest that, as in tumor cells in vivo, reduction to homozygosity is a major mechanism for the expression of recessive mutant phenotypes in cultured human cells.

Correspondence: Dr. Norman R. Drinkwater, McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wl 53706 (U.S.A.). * Present address: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 (U.S.A.).

Abbreviations: aprt, adenine phosphoribosyl transferase; DAP, 2,6-diaminopurine; ENU, N-ethyl-N-nitrosourea; hprt, hypoxanthine guanine phosphoribosyl transferase; RFLP, Restriction Fragment Length Polymorphism.

Molecular genetic analyses of human and rodent tumors have demonstrated the importance of two classes of genes in the development of neoplasia. Mutations induced in cellular protooncogenes, such as members of the ras family, result in the initiation of carcinogenesis in a variety of experimental models and act dominantly to transform cells in culture (Balmain and Brown, 1988). The second class of critical mutations, those in tumor suppressor genes such as the retinoblastoma gene, are recessive at the cel-

366

lular level. Inactivation of both alleles of these genes as a consequence of gcrmline and somatic mutational events has been linked to the development or progression of a variety of cancers (rcvicwcd in Scrable et al., 1990). "l'hc cxprcssion of the mutant phenotype in a cell hetcrozygous for a mutation at a tumor suppressor genc may result from the inactivation of the remaining wild-type allele by a point mutation or deletion. However, loss of the wild-type allele by mitotic recombination and chromosomc nondisjunction arc more often responsiblc for expression of thcsc recessive mutant phenotypcs (Scrablc et al., 1990). Mutagenesis in mammalian cells has been studied predominantly at single copy gcncs such as the X-linked hypoxanthinc-guanine phosphoribosyltransferase ( h p r t ) locus or at hcmizygous autosomal loci that have a dclction of one copy of the genc, as well as (usually undetermincd amounts) of flanking DNA. At such loci, mutants can be isolated with single hit kinctics, and subscquent molecular analysis is not complicated by the presence of a second allelc. At the hprt locus point mutations, deletion mutations and insertions of genetic material havc been identified in human (Bradley et al., 1987; Monnat, 1989), hamster (Kadcn ct al., 1989; Thacker and Gancsh 1989), and murinc (Jones et al., 1987) cells. At the hcmizygous adenine phosphoribosyltransfcrase ( a p r t ) locus in Chincsc hamstcr ovary ( C H O ) cells, point mutations and deletions with one endpoint within the gene have bccn observed (Grosovsky et al., 1986; deJong ct al., 1988; Nalbantoglu c t a l . , 1983, 1986; Adair ct al., 1983; Bradley et al., 1988). Rcccnt studies (Bradley ct al., 1988; Evans ct al., 1986) have indicated that somc classes of events that result in a mutant phcnotype, such as very large deletions or chromosome loss, are not consistent with cell survival if assayed at a hemizygous locus, presumably because they simultaneously eliminate essential genes that are also hemizygous. The resulting loss of inviable mutants reduces the observed mutant frequency for a hemizygous gone relative to that seen when the same gene is heterozygous: large scale, chromosome level alterations will bc underrepresented in the mutational spectrum of the mutagenic agent or process being studied. Mitotic recombination and nondisjunctional

chromosome reduplication are alternative mcchanisms for the expression of recessive mutant phcnotypcs in normal human cells in vivo (Turner ct al., 1988; Morley ct al., 1990), in tumors (Scrable ct al., 1990; Cavenee et al., 1985) and in cultured cells (Yandell et al., lt,~90: Wasmuth and Hall, 1984; Ward ct al., 1990). By eithcr mcchanism, a preexisting recessive mutant allele, previously masked by expression of the wild-type allele on the homologous chromosome, is reduced to homozygosity. Because of the limitations imposed by the use of hemizygous loci, several laboratories have described mutational assays that utilize hetcrozygous autosomal loci as targets (Bradley ct al., 1988: Evans ct al., 1986; Yandcll ct al., 1990: Janatipour ct al., 1988), but the number of such studies is still limited, and the generality of the results obtained remains to be dctcrmincd. Our purpose was to determine which types of events give rise to recessive mutant phcnotypcs and their rclativc rates of occurrence in normal human lymphoblastoid cells. In order to insure recovery of all classes of spontaneous mutational events, we isolated a human lymphoblastoid ccll line, H8S3, hctcrozygous at the aprt [ocus (i.e., aprt " / ). This paper describes the types of spontaneous genetic alterations that give rise to the fully A P R T - p h c n o t y p e . At least three types of changes wcrc noted: (i) small intragcnic mutations; (ii) deletion events that rcsuh in loss of the entirc functional aprt allele; and (iii) mitotic recombination or chromosomc loss with reduplication which results in the presence of 2 copies of the mutant allele. Of the 3 classes of events, recombination or loss with reduplication was the most common, comprising morc than 50% of the mutants examined. These events, occurring in a normal human lymphoblastoid cell line, resemble the genetic changes seen in human tumors. Methods

Cell culture. The lymphoblastoid cell lines LCL-721 and H8S3 were routinely maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (HyClone, Logan, UT) and gentamicin (5(I p,g/mi). Murine fibroblast cell lincs (BALB 3T3, BALB 291, and AP8SI3) were grown

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in Dulbecco's modified Eagle's medium (DME) with 10% newborn calf serum and gentamicin as above. The human B-lymphoblastoid cell line, LCL721, was derived from a normal female donor by immortalization with Epstein-Barr virus (Kavathas et al., 1980). H8S3 was derived from LCL721 by mutagenesis with 1 mM N-ethyl-Nnitrosourea (ENU) following a previously described protocol (Drinkwater and Klinedinst, 1986). 10 days after mutagen treatment, cells were dispersed in 0.35% agarose with 40 /zM 2,6-diaminopurine (DAP) over a feeder layer of AP8SI3 fibroblasts to select for heterozygous aprt +/- cells. The resulting colonies were isolated and analyzed for APRT activity by enzyme assay. The structure of the aprt gene was determined by Southern blot as detailed in the results section. BALB 3T3 and BALB 291 were gifts from B. Sugden and J. Mecsas (University of Wisconsin, Madison), respectively. BALB 291 contains an integrated hygromycin phosphoryltransferase gene (Blochlinger and Diggelmann, 1984) and is resistant to the antibiotic, hygromycin. The fibroblast cell line, AP8SI3, is a derivative of BALB 3T3 ceils that was selected for DAP R after mutagenesis by treatment with 4 mM ENU in serumfree DME containing 15 mM HEPES (pH 7.4). AP8S13 had 5% of the APRT activity of the parental BALB 3T3, and has been used as a feeder layer for these experiments. A P R T enzyme assay. Cellular extracts were prepared from 1-4 × 107 exponentially growing cells. Cells were collected by centrifugation, washed twice in PBS, resuspended in 100/zl of 10 mM Tris (pH 7.4) and disrupted by 5 freeze/thaw cycles in a dry ice/acetone bath. Particulate debris was removed by centrifugation at 10000 rpm for 5 min. Protein concentration was determined by a dye-binding assay (Biorad Protein Assay, Biorad Labs, Richmond, CA). APRT activity was determined by the method of Rappaport and DcMars (1973). Southern blot analysis. Genomic DNA was prepared as described by Maniatis et al. (1982) and digested with BamHI. The DNA concentra-

tion was determined fluorimetrically (Labarca and Paigen, 1980) and 10 /zg of DNA was electrophoresed through a 0.75% agarose gel. The DNA was transferred (Southern, 1975) to Gene Screen Plus (Dupont) nylon membrane, and prehybridization, hybridization with the appropriate probe, and washes were carried out according to the manufacturer's instructions. The plasmid pHuAP (Broderick et al., 1987) contains the B a m H l fragment of the human genomic aprt clone and was kindly provided by P. Stambrook (University of Cincinnati Medical Center); pJW101 (Wilson et al., 1978) contains the human a-globin cDNA clone and was provided by Oliver Smithies (University of North Carolina); and acollagen-sis, which contains a cDNA clone of the human c-sis gene, was provided by Bill Brondyk (University of Wisconsin, Madison). Plasmid DNA or fragments corresponding to the insert of interest were radiolabelled by nick translation or by the random priming method using kits obtained from Amersham (Arlington Heights, IL). Hybridization intensities were compared by analysis of autoradiograms with a soft laser scanning densitometer (LKB, Bromma, Sweden). Fluctuation analysis. Because we were interested in comparing the mutation rates of an endogenous autosomal locus and of a shuttle vector-encoded locus in the same clones of cells, the shuttle vector, pND123 (Ingle and Drinkwater, 1989), was introduced into H8S3 by electroporation. The frequency and molecular analysis of spontaneous mutants at the shuttle vector locus will be described elsewhere (Klinedinst and Drinkwater, in preparation). Plasmid-bearing cells were suspended at cloning densities (500 cells/ml) in complete RPMI 1640 medium containing agarose (0.35%) and hygromycin (50/~g/ml), and were dispersed over a feeder layer of BALB 291 cells. The cloning efficiency, after adjustment for growth in nonselective medium, was 29%. After 3 weeks of growth, individual colonies were isolated. Eleven colonies (designated clones A, B, ... K) were grown to 2-6 × 108 cells (28-30 generations from the time of cloning). The frequency of aprt - / - mutants arising in each of the 11 clones of H853 ceils was determined by suspending 2.7 x 106 cells in complete RPMI medium

3~8

containing 100/zM DAP and 0.35% agarose at a density of 7.5 x 10 4 cells/ml over a feedcr layer of AP8SI3 fibroblasts. The cells were allowed to grow for 3 - 4 weeks with weekly addition of fresh medium and the colonies were counted with the aid of a dissecting microscope. The observed mutant frequency was adjusted to account for the cloning efficiency of the cells in nonselective medium, which ranged from 10 to 33% for individual clones.

Enzymatic

amplification

annealing (55 o C, 1 min)/extension (72 o C, 3 min) cycles were performed. Typical yields were 3-5 /zg of amplified product.

DNA sequence analysis. DNA sequences were determined by the dideoxy chain termination method (Sanger ct al., 1977), using a Sequenase DNA sequencing kit (U.S. Biochemical, Cleveland, OH), except that unlabelled dATP was substituted for dATP-a35S and the sequencing primer was labelled at its 5' end with ATP-y~2P.

of aprt sequences.

The polymerase chain reaction (Saiki et al., 1988) was carried out using Taq polymerase (Perkin Elmer Cetus, Norwalk, CT) and oligonucleotide primers located at base pairs 1874-1891 and 2855-2872 on the aprt map (Hidaka et al., 19871. Taq polymerase buffer contained 10 mM TrisHCI (pH 8.3, 25°C), 50 mM KCI, 0.9 mM MgCI~, 0.1% (w/v) gelatin, (1.2 mM each dATP, dCI'P, d G T P and dTI'P. 1 /xg of genomic DNA was provided as template. The concentration of each primer was 6 ng//zl. 35 melting (95 o C, 1.5 m i n ) /

TABI.E 1

Restriction fragment h'ngth variant analysis. The 3' half of the aprt gene (nucleotides 18742872 on the aprt map) was enzymatically amplified from genomic DNA isolated from each aprt mutant, LCL-721 and H8S3. The PCR product DNA was cleaved with NlalV and an aliquot was electrophoresed through a 6% NuSieve agarose (FMC BioProducts, Rockland, ME) gel. DNA was transferred to nylon membrane and hybridized with radiolabelled probe made from the 251 bp fragment generated by cleavage of P('R product DNA with Hphl. Results

T H E F R E Q U E N C Y OF S P O N T A N E O U S aprt MUTANTS IN I N D E P E N D E N T CLONES OF ttUMAN LYMP H O B L A S T O I D CELLS " Subclone

Frequency ( × 1 0 s)

A B C D h

2.9 16.0 9.8 5.9

E F G 11 1 J K

14.0 27.O 4.8 10.0 1.8 3.6 3.1

Mutation rate = 1.3 × 10 S / c e l l / g e n e r a t i o n " " 2 . 7 × 1 0 ~' cells from 11 independent clones ( A . B . . . K ) o f aprt" / cells that had been grown for 28-3(I generations were selected in 100 p.M DAP. Mutants were e n u m e r a t e d after 3 weeks of growth. The mutant frequency for each clone was adjusted to account for the cloning efficiency in nonselective medium ( 1 0 - 3 3 ~ ). b Clone with the median mutant frequency. ~ Calculated by the median method of Lea and Coulson ( 19491.

Isolation and characterization of the aprt heterozygous cell line, H8S3. Following mutagenesis of LCL-721 cells with ENU and selection with 411 /x M DAP, eight colonies were expanded and analyzed for their A P R T activity. One clone, designated H8, had an intermediate level of A P R T activity (60%) compared with the parental LCL721. Southern blot analysis with plasmid pHuAP demonstrated a diploid copy number at the aprt locus. H8 was subcloned and the characterization was repeated on 6 subclones. Subclone 3 (H8S3), with 50-70% of wild-type activity in numerous A P R T enzyme assays and diploid aprt copy number by Southern analysis, was chosen for subsequent mutational studies. The growth rate and cloning efficiency of H8S3 were comparable to the parental strain. The karyotypes of both H8S3 and the parental LCL-721 were determined. There was no evidence of abnormality of chromosome 16 (aprt is located at position 16q24, Reeders and Hilde-

369

brand, 1989), although H8S3 was found to have an unbalanced translocation involving chromosome 1 and chromosome 6 which results in monosomy for the region lq32 ---, lqter and trisomy of the region 6p21.3 ~ 6pter. LCL-721 cultures contained ceils with 2 karyotypes, some normal and others with the translocation exhibited by H8S3; thus, the translocation in H8S3 preexisted in the LCL-721 population. Analysis of spontaneous mutagenesis in H8S3. The frequencies of aprt - / - mutants arising in each of the eleven aprt +/- clones arc shown in Table 1. The frequencies of aprt - / mutants among the clones ranged from 2.7 x 10 -5 to 2.5 x l0 -4 with a median value of 5.9 x 10 -5. The mutation rate, as calculated by the median method of Lea and Coulson (1949), was 1.3 x 10 -5 per locus per generation. To verify that the colonies surviving in 100/.tM DAP were truly mutants with reduced A P R T activity, the rate of conversion of adenine to adenosine plus AMP was measured in 1-4 DAP R mutant colonies derived from each H8S3 clone A - K . Each DAP R colony that was analyzed (26 total) had reduced activity in comparison with the H8S3 heterozygote (range 13-56% of H8S3 or approximately 8 - 3 3 % of the wild-type LCL-721). The rate of conversion by LCL-721 was 15 + 5 pmoles adenine//.Lg p r o t e i n / h . To determine the structure and copy number of the aprt alleles in each mutant clone, Southern blot analysis was performed using the 2.2 kb BamHl genomic fragment of the human aprt locus from the plasmid p H u A P as a probe in two independent experiments. For one determination, blots were stripped and rehybridized with the probe homologous to the human c-sis gene as a control for the amount of DNA loaded per lane. Fig. 1 shows the results obtained from a subset of the 26 clones analyzed. No intragenic deletions or rearrangements were observed. DNA from 5 of the 26 clones (C1, C2, C4, GI and J4) had a band at the wild-type position that hybridized with one half the intensity as that for normal ceils, indicating that loss of the entire wild-type allele was responsible for the mutant phenotype. The remaining clones exhibited a wild-type structure and diploid copy number.

Fig. 1. Structure and allele copy n u m b e r of the aprt locus in wild-type (LCL-721 cells) and a subset of the aprt mutant clones. Genomic D N A was digested with BamHI, transferred to nylon m e m b r a n e and probed with the 2.2-kb BamHI fragment of pHuAP.

DNA from the deletion mutants C1, C4, J4, from the non-deletion mutant D1 and from LCL721 was also probed with the 265 bp BssHI Bst EII fragment from the a-globin (hbA) eDNA insert of pJW101. The hbA gene is located at chomosome 16p13.3 on the opposite arm from aprt at 16q24 (Reeders and Hildebrand, 1989). All of the mutants tested exhibited a hybridization intensity equal to that of the wild-type LCL721, indicating the loss of one copy of aprt was not a result of monosomy of chromosome 16 (data not shown). Thus, 19% of the mutants appeared to have resulted from a chromosome level event such as a large deletion encompassing the aprt locus; however, 3 of the 5 deletion mutants were derived from clone C and are likely to be identical. The remaining mutants had structures consistent with either 'point' mutations or reduction to homozygosity of the mutant allele of the heterozygote. Localization of a point mutation m the mutant allele of the H8S3 heterozygote. In order to discriminate between loss of heterozygosity at the aprt locus and point mutations that inactivated the wild-type allele, we sought to define a restriction fragment length difference at the aprt locus. As a first step, we determined the DNA sequence of the mutant allele found in the heterozygote, H8S3. Fig. 2 (top panel) shows the intron/exon structure of the aprt locus and the locations of primers used for DNA amplification and sequencing. Genomic DNA isolated from two independent deletion mutants, C1 and J4, was used to generate enzymatically amplified DNA homologous to the aprt locus. Sequence analysis of the amplified product from the 3'-end of the gene

370 Barn I'll

Barn I I1

.5 400

8t}O

12(X)

16(X)

2(XX)

24(X)

2800

I

,(0

,~-).

'(I. ,(o

,|.

t

CCATCCCCAG GA ACC AT(; AAC GCT GCC 4 Gly Thr Mct Asn Ala Ala/ ~.~. die)

~

1874 -

79

66

;g

345

; 79

6 74

102 23 104

72

44

Fig. 2. (Top panel) Depiction of the gcnome structure of the human aprt locus. Arrows with filled diamonds indicate the locations and directions of synthesis of amplification primers used fi)r the polymcrasc chain reaction. Arrows with open circles indicate locations of sequencing primers. The filled boxes represent the 5 exons of thc aprt gcnc. Thc numbering system corresponds to that of Hidaka el al. (1987). (Bottom panel) Restriction map of the portion of the aprt gene between the 3'-most amplification primers. Darkened circles represent the NlalV sites. The ()pen circle shows the location of the mutated NlalV site that results in a rcstriction fragment length difference (104 ---, 127 bp). The open square above thc line indicates thc location of the uniquc Hphl site in this region. The nucleotide sequence in the vicinity of the relevant NlalV site is shown along with thc G : (" --* A :T transition mutation at Fx*sition 2640 on the aprt map. The NlalV recognition sequence is undcrlincd and the amino acid substitution is indicated in parentheses.

(map position 1874-2872) rcvcalcd thc same base-substitution mutation in thc remaining allele of each deletion mutant (i.e. the mutant ailelc of the H8S3 heterozygote, since presumably the normal allele was involved in the deletion). A G :C --* A : T transition mutation at map position 26441 results in the substitution of isoleucine for threoninc in the second codon of exon 5 and thc loss of an NIalV recognition sitc (Fig. 2, bottom panel). Thc scquence for LCL-721 is identical to the published sequence (Hidaka et al., 1987) at that position.

Restriction fragment length l'ariant analysis of the aprt-/- mutants. The base-substitution mutation at the NlalV site produces a restriction fragment length alteration such that the two aprt alleles of the H8S3 cells can be distinguished. Thc mutant allele results in a 127-bp fragment upon NIalV cleavage while the wild-type allele produces fragments of 104 and 23 bp. D N A from each of the 26 aprt-/- clones was enzymatically amplified between map positions 1874 and 2872, cleaved with NIalV and the products were separated by agarose gel electrophoresis. After South-

ern transfer, the m e m b r a n e was probcd with thc 251-bp fragment obtained by cleaving the PCR product of LCL-721 with Itphl (the location of the Hphl site is shown as an open square above the line in the bottom panel of Fig. 2). Fig. 3 shows the results of a representative subset of the 26 aprt mutants analyzed in this way. CI, C2, C4, G I and J4, as expected for deletion mutants, exhibited only the 127-bp fragment. Of the 21 mutants with a diploid copy number at the aprt locus, only 6 of the clones maintained the hcterozygous pattern of H8S3, while 15 (71%) be-

~-127 •" - 1 0 4

Fig. 3. Restriction fragment length variant analysis of LCL-721 (aprt +/~ 1, H8S3 ( a p r t ' / - ) , and a subset of the aprt ' mutant clones. I,CL-721 contains only the wild-type 104-bp allele, while H8S3 has Ix)th the 104-bp allelc and the 127-bp allele that results from the loss of the NIalV site. Some mutants have retained both alleles, while the majority have lost the wild-type allclc.

371 TABLE 2 SUMMARY OF EVENTS GENERATING NEOUS aprt/- MUTANTS ~ Clone b A B C D E F G H I J K Total (proportion)

Reduction to homozygosity 1 1 0 2 2 3 0 2 2 0 2 15 (0.58)

SPONTA-

Deletion

Intragenic mutation

0 0 3 0 0 0 1 0 0 1 0

2 2 0 1 0 0 1 0 0 0 0

5 (0.19)

6 (0.23)

~ Assignment was made on the basis of copy number of aprt alleles in genomic D N A and restriction fragment length analysis of enzymatically amplified DNA. Intragenic mutation was inferred by diploid copy number and retention of both wild-type and mutant alleles. Deletion was inferred by haploid copy number and reduction to homozygosity was inferred by dipkfid copy n u m b e r and the presence of only the mutant allele. b 1-4 spontaneous a p r t - / - clones from each of 11 aprt ~/ clones (A, B . . . K) from the h u m a n lymphoblastoid cell line, H8S3, were analyzed.

came homozygous for the mutant (127-bp) allele (Table 2). The results of the A P R T enzyme assay were consistent with the phenotypes of the mutants. The average A P R T activity among deletion mutants was 12 + 4% of wild-type, while that of the mutants homozygous for the mutant allele was 20 + 6% of wild-type, or approximately twice that of a single mutant allele. The average A P R T activity of the point mutants was nearly identical to that of the deletions, 11 + 2% of wild-type, indicating that the point mutation at the wild-type allele generally yielded a null mutant. Discussion

We have analyzed the molecular events resulting in the DAP R phenotype in normal human lymphoblastoid cells that are heterozygous at the aprt locus as a result of a base-substitution mutation in one allele. The mutation rate for the aprt

locus we observed in this cell line (1.3 x 1 0 - 5 / cell/generation) was similar to the lower estimate reported for cultured aprt +/- human fibroblasts (2.7 x 10 -5 to 1.3 x 1 0 - 4 / c e l i / g e n e r ation) (Steglich and DeMars, 1982). It is higher than that reported for the aprt locus in C H O cells (1 x 10 -7 to 3.2 x 1 0 - 7 / c e l l / g e n e r a t i o n ) (Nalbantoglu et al., 1983; Adair et al., 1980) or in Friend erythroleukemia cells (1.3 x 10 -6) (McKenna and Ward, 1987) or for the hprt locus in human ( ~ 1-6 x 1 0 - 6 / c e l l / g e n e r a t i o n ) (Monnat, 1989; DeMars et al., 1981), or C H E F (1.1 x 10--6) cells (Kaden et al., 1989). A possible explanation for the lower mutation rates observed in the latter studies is the hemizygous nature of both the aprt locus in C H O cells and the hprt locus. Because we have used a strategy that is able to detect mitotic recombination events or nondisjunctional reduction to homozygosity, we were able to observe events that are not detected at hemizygous autosomal or X-linked loci (Rosenstraus and Chasin, 1978). Only 23% of the DAP R clones isolated in our heterozygous system result from apparent point mutations, which are the most common events detected at the hemizygous aprt locus in CHO cells (deJong et al., 1988; Nalbantoglu et al., 1983; Adair et al., 1983). In contrast, the majority of cells failing to express A P R T in our study have lost the wild-type allele while reduplicating the mutant allele, possibly duc to recombination between the homologous chomosomes. At present the available data do not allow us to distinguish between chromosome loss with reduplication and mitotic recombination with segregation, either of which would result in the presence of two mutant aprt alleles. Identification of heterozygous flanking markers on chromosome 16 will be necessary to determine the relative contribution of each type of event. The polymorphic loci analyzed to date, including the a-globin gene (Kidd et al., 1989), have been uninformative for this cell line. The results of our study are consistent with other recent studies indicating that chomosome loss with reduplication and mitotic recombination are common events both in rodent cell lines and in diploid human somatic cells in culture and in vivo. Approximately 90% of the spontaneous tkmutants derived from the heterozygous TK6 hu-

372 man lymphoblastoid cell line lost the entire wildtype allele (Yandell et al., 1986, 1990). Evaluation of flanking markers by R F L P analysis indicated that approximately 50% of spontaneous mutants also lost the linked erbAl allele. Karyotype analysis eliminated development of monosomy as a mechanism of loss of the tk gene, whilc visiblc deletions were present in only 2 of the 18 clones examined. The frequency with which spontancous mutants were recovered at the tk locus was 1.5-5 × 10 ~' which is approximately 1,/3-1/1(I of that seen in this study. This difference in mutant frequency presumably results from thc difference in the number of generations the cells were grown prior to the selection for spontaneous mutants (13 and 30 generations for the tk and aprt mutants, respectively). However, the proportion of mutants arising by loss of heterozygosity was very similar for both loci. Cultured rodent cells havc also been shown to undergo mitotic recombination. Using a PCRbased strategy to discriminate between the mutant and wild-type alleles of a C H O cell line hcterozygous at the aprt locus, Ward et al. (1990) demonstrated that approximately 40% of spontaneous A P R T - mutants had undergone loss of heterozygosity events. In another study, mitotic recombination occurring within the r R N A gcnc cluster on mouse chromosomc 6 resulted in reduction to homozygosity of the Lyl8 cell surfacc marker (Nelson et al., 1989). The rate of emcrgence of Ly18 mutants was almost identical to that we observed at the aprt locus, dcspite the fact that repetitive r D N A is involved. Although we have no data at present to suggest that repetitive D N A is involved in reduction to homozygosity at the human aprt gene, chomosome 16 has unique features that might contribute to somatic recombination including a large block of repetitive D N A located at 16q12 (Kidd el al., 1989) and 2 common fragile sites located at 16q22.1 and 16q23.2 (Moyzis ct al., 1987). Three in vivo studies also implicate mitotic recombination as a common event in normal somatic cells. Langlois et al. (1986) analyzed mature human erythrocytes from individuals hetcrozygous for the M and N alleles of glycophorin A. By fluorescent antibody staining and flow sorting M ~ , MM, N~b, and NN variants could be quanti-

tated. On avcrage, the reduplication variants were as common as the loss variants and occurred at frequencies of 1-2 × 10 -6. No molecular characterization could be carried out because the cells are anucleate. Turner ct al. (1988) dcmonstrated that 40% of human circulating T cells that failed to express one of two hetcrozygous t t L A - A alleles had lost the gene and had reduplicated the allele on the homologous chromosome. 3 8 ~ retaincd hctcrozygosity at the linked HLA-B allele, eliminating the possibility of chromosome reduplication and implicating mitotic recombination as the mechanism. A detailed analysis of several linked polymorphic markers demonstrated that mitotic recombination had occurred in approximately 30% of the H L A - A mutants (Morley ct al., 19911). Variants were isolated at a frequency of ~ 3 × 10 5, similar to that wc observed at aprt. The demonstration of reduction to homozygosity of an inherited mutant allele in rctinoblastoma (Benedict et al., 1987; Cavenee et al., 19851 provided thc first compelling cvidcncc for the importance of these chromosome-level events in the tumorigenic process. Recent studies of spontaneous mutations at several loci in a variety of cell types in vivo and in vitro demonstrate the general significance of chromosome-level events in unccwering recessive mutations. In these studies, spontaneous recombination or loss/reduplication events have been shown to occur at rates as high as or higher than base-substitution or deletion mutations at heterozygous loci. The proportions of these classes of mutational events ,observed in our studies of the aprt locus are strikingly similar to those inferred from molecular analysis of D N A isolated from rctinoblastomas; data cited in a recent review (Scrable et al., 1990) indicated that in 19 of 33 tumors, RFLP markers linked to the retinoblastoma gene showed loss of hetcrozygosity. Although our studies concentrated on spontaneous mutations, it will bc of interest to determine if carcinogens can increase the rate of this important step in the tumorigenic process. It is possible that agents that arc inactive in traditional gcnc mutation assays contribute to the risk of carcinogenesis by increasing the rate of mitotic recombination, allowing a preexisting recessive mutation to bc expressed.

373

Acknowledgements W e t h a n k Dr. G u r b a x S. S e k h o n a n d M s K a t e J. T h o m p s o n o f t h e D e p a r t m e n t o f M e d i c a l G e netics, U n i v e r s i t y o f W i s c o n s i n - M a d i s o n , for p e r f o r m i n g t h e k a r y o t y p e analysis. O l i g o n u c l e o t i d e p r i m e r s y n t h e s i s by t h e U n i v e r s i t y o f W i s c o n s i n B i o t e c h n o l o g y C e n t e r was s u p p o r t e d by N a t i o n a l S c i e n c e F o u n d a t i o n g r a n t D M B 8514305. T h i s w o r k was s u p p o r t e d by a f e l l o w s h i p p r o v i d e d by t h e E.I. D u P o n t D e N e m o u r s a n d C o m p a n y to D . K . K . a n d by P u b l i c H e a l t h S e r v i c e G r a n t s C A 37166, C A 07175 a n d C A 09135.

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