Proc. Natl. Acad. Sci. USAVol. 87, pp. 51-55, January 1990Biochemistry

Molecular dissection of mutations at the heterozygous thymidinekinase locus in mouse lymphoma cells

(carcinogenesis/mutagenesis/allele loss/somatic recombination)

M. L. APPLEGATE*, M. M. MOOREt, C. B. BRODER4§, A. BURRELL¶, G. JUHN*, K. L. KASWECK*,P.-F. LINII**, A. WADHAMSttt, AND J. C. HOZIER*t,*t*Department of Biological Science, Florida State University, Tallahassee, FL 32306; tU.S. Environmental Protection Agency, Research Triangle Park, NC27711; tDepartment of Biological Sciences, Florida Institute of Technology, Melbourne, FL 32901; 1IBM Corporation, San Jose, CA 95193; andIlDepartment of Biology, Yale University, New Haven, CT 06511

Communicated by J. Herbert Taylor, September 25, 1989

ABSTRACT The mouse lymphoma L5178Y TK+/-3.7.2C cell line allows quantitation of induced TK+/- -

TK-/- mutations at the heterozygous thymidine kinase (Tk)locus. TK-/- mutant colonies show a bimodal size distribu-tion, reflecting a difference in the growth rates of the two sizeclasses that is hypothesized to result from different degrees ofgenetic damage. The two homologous chromosomes 11 con-taining the alleles of the Tk gene in L5178Y 3.7.2C TK+/- cellsare distinguishable at the cytogenetic level. We find, in addi-tion, that the two alleles are distinguishable at the molecularlevel because of an Nco I restriction fragment length polymor-phism at the 3' end of the gene. In a set of 51 large-colony and48 small-colony TK-/- mutants induced by ionizing radiationor by chemical mutagens, we find that 78, including all exceptone of the small-colony mutants, have lost the Tk+ allele andthat some of these have two to four copies of the remaining Tk-allele. Nineteen of the large-colony TK-/- mutants that do notshow Tk+ allele loss show no other structural changes detect-able at the level of Southern blot analysis. One shows a partialdeletion. The variety of mutagenic lesions recorded at theheterozygous Tk locus may be representative ofevents observedin human malignancy and may include a wider range ofmutagenic events than can be observed at hemizygous test loci.

In humans, genetic lesions ranging from point mutationsthrough deletions of genetic material and chromosomal re-arrangements result in heritable disorders and are associatedwith neoplasia (1-4). However, the processes leading to theestablishment of such lesions are complex and poorly under-stood. During the past 20 years, well-defined in vitro assayshave been developed that measure single-locus gene inacti-vation in mammalian cells as a genotoxic response to muta-genic chemical and physical agents. Because the target geneis usually nonessential for cell survival, mutant cells can berecovered and propagated in appropriate selective medium.These assays may provide suitable models for the study ofgenetic lesions important in human disease.The mouse lymphoma L5178Y TK+/- 3.7.2C cell line is

widely used in genotoxicity assays to quantitate the potentialof chemical and physical agents to induce mutational inacti-vation of the autosomal heterozygous thymidine kinase(ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene (5-7). TK-/- mutants are selectable in medium containing trif-luorothymidine, which inhibits division in cells capable ofutilizing the pyrimidine-salvage pathway ofDNA synthesis. Astriking feature of the TK-/- colonies recovered in the mouselymphoma assay is the presence of a bimodal frequencydistribution of colony sizes. The relative frequency of the twosize classes is mutagen dependent (7, 8). Mutant clones with

slow growth kinetics, designated small colony, often havechromosomal abnormalities involving the distal end of chro-mosome 11 (9-12), to which the Tk gene has been mapped inthe mouse (13-15). In L5178Y mouse lymphoma cells, thechromosome 11 homologs are distinguishable because of acentromeric heteromorphism and are designated 11a and 11b(ref. 9; see Fig. 3). All visible chromosome 11 rearrangementsassociated with TK+/ -- TK-1- mutagenesis involve the 11bhomolog. Large-colony mutants, which have growth kineticssimilar to the TK+/- heterozygote, have chromosomes 11 thatare karyotypically indistinguishable from those of the parentalheterozygote. It has been hypothesized (8, 9, 11) that the twoclasses of mutants result from different degrees of geneticdamage. Large-colony mutants may result from mutationalevents affecting the expression of only the Tk locus. Small-colony mutants may result from event(s) that affect not onlythe Tk locus but also additional gene(s) whose inactivationcauses the cells to be slow growing.Because the mouse Tk gene has been cloned and charac-

terized (16), it is now possible to study the structure of the Tkgene in TK-1- mutants. We report here that the two alleles ofthe heterozygous Tk gene in L5178Y TK+/- 3.7.2C cells canbe distinguished by Southern blot analysis because of arestriction fragment length polymorphism. To evaluate theextent of mutagenic lesions in large- and small-colony mu-tants, we have analyzed structural changes involving the Tklocus of 99 TK-1- mutants induced by ionizing radiation orexposure to methotrexate, methyl methanesulfonate (MMS),ethyl methanesulfonate (EMS), pyrimethamine, or bleomycin.Preliminary results of this work have been published (15).

MATERIALS AND METHODSMutagen Treatment, Mutant Selection, and Mutant. Isola-

tion. L5178Y TK+/- 3.7.2C cells were treated, and TK+/-mutants were selected and isolated as described (11, 17). Thelarge-colony mutants 1826-1830, induced by methotrexate,were generously provided by D. Clive (Burroughs Well-come). Each bleomycin and EMS mutant was isolated froma separately treated culture. The threeMMS mutants and twoy-irradiation mutants are karyotypically unique, and theremaining mutants were recovered from cultures of suffi-ciently high mutant frequencies to ensure the independent

Abbreviations: MMS, methyl methanesulfonate; EMS, ethyl meth-anesulfonate.§Present address: Department of Immunology and Medical Micro-biology, College of Medicine, University of Florida, Gainesville, FL32601.**Present address: Department 207, Bristol Meyers Corp., Walling-

ford, CT 06492.ttPresent address: Department of Biophysics, University of Roch-

ester, Rochester, NY 14623.#To whom reprint requests should be addressed.

51

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

52 Biochemistry: Applegate et al.

origin of each. Cytogenetic preparations were made as soonas feasible with poly-L-lysine-coated four-chamber Lab-Tekslides (10) and/or the THC (Trypsin/Hypotonic/Colcemid)technique (18).

Isolation of DNA and RNA. DNA and total RNA wereprepared simultaneously from cultures of 50-100x 106 cells(19; R. Zakour, Biotech Research Laboratories, personalcommunication). Cells in logarithmic phase were pelleted,washed once in isotonic saline, and frozen prior to use. Asuspension of pelleted cells (5x 107 cells per 0.5 ml) wasadded dropwise to 1.1 ml of guanidine lysis buffer [6 Mguanidine hydrochloride/10 mM dithiothreitol in 0.1 MTris HCl (pH 6.5)] with Vortex mixing to produce a homo-geneous suspension. The lysate was layered onto a 0.6-mlcushion of 5.7 M CsCl/l mM EDTA, pH 7.5, and centrifugedin a TLS-55 rotor at 55,000 rpm in a Beckman TL-100ultracentrifuge. The DNA was recovered at the gradientinterface, dialyzed against 10 mM Tris HCI, pH 7.8/25 mMEDTA/150 mM NaCl (TEN buffer), and purified. Afterovernight incubation at 550C in an equal volume of lysingbuffer (3% sarkosyl/50 mM EDTA, pH 8.0, containing 1 mgof proteinase K per ml), RNase A at 150 gg/ml was added for1 hr at 55°C, and then proteinase K was added at 250,ug/mlfor 1 hr at 55°C. The solution was phenol extracted twice withgentle shaking at 4°C for 2 hr. The aqueous phase wasdialyzed extensively against 10 mM Tris HCI/1 mM EDTA,pH 8.0 (TE buffer), prior to storage at 4°C. Total cellularRNA was recovered from the guanidine hydrochloride gra-dients as a clear pellet (19).

Southern Blots. Southern blotting using GeneScreenPlus(New England Nuclear) nylon membrane was performedaccording to the protocol described by Rigand et al. (20).After transfer, the blots were hybridized at 65°C as describedin the GeneScreenPlus manual and were probed with aBamHI insert containing full-length mouse Tk cDNA fromthe plasmid pMtk4 described by Linet al. (16). The final blotwash was 0.1x SSC (lx SSC = 0.15 M NaCI/0.015 M sodiumcitrate)/0.1% NaDodSO4 for 30 min at 65°C.To quantitate gene copy number in TK+/- and mutant cell

lines, blots probed with pMtk4 insert were stripped andreprobed with mouse skeletal muscle a-actin or histone 3.2gene fragments, both expected to be present in copy numbersunaffected by events at the Tk locus in these cells. Theskeletal muscle actin and histone 3.2 sequences used bothmap to chromosome 3 in the mouse (21-23). Band intensitiesof Tk-specific and actin- or histone-specific fragments de-scribed in the legend of Fig. 4 were compared on autoradio-grams by using an Isco UA-5 monitor with a model 1312 gelscanning attachment or a Bio-Rad model 620 video densito-meter with a Hewlett-Packard model 3392A integrator. Eachband was scanned at three positions to compensate fordistortion artifacts. The intensity ratio between the Tk-specific and pseudogene bands in each lane, determined foreach mutant independently on several blots and at variousfilm exposures, was ascertained for the test blots to ensureuniformity of blotting and hybridization, as well as appropri-ate film exposures. The control probes were provided by W.Marzluff (Florida State University).

RESULTS

Identification of a Restriction Fragment Length Polymor-phism Distinguishing the Two Alleles of the Tk Gene in L5178YTK+/- 3.7.2C Cells. The cosmid pMtkll6 was recoveredfrom a mouse C57BL/1OJ liver library and contains a full-length, functional Tk gene (ref. 16; Fig. 1). DNAs fromL5178Y TK+I- 3.7.2C cells andTK-'- mutant cell lines, aswell as the cosmid pMtkll6, were restricted, blotted, andprobed with mouse Tk cDNA. A TK-deficient mouse L-cellline (LTK-), which lacks Tk coding sequences, was used to

PMtkIl6 -Kb

-- Mouse tk

R N R

R X N RIll mI

Hd N X Hd Hd N Hp

FIG. 1. Restriction map of a portion of the cosmid pMtkll6,including the regions spanning the Tk gene and part of the vectorpTCF. Part of this map and the intron/exon structure of the genomicmouse Tk gene have been published (16). Vector sequences arehatched. Exons are identified by roman numerals. Hp, Hpa I;Hd,HindIII; R, EcoRI; N, NcoI; X, XhoI.identify Tk-like sequences that may represent pseudogenes(16).Most enzymes tested, including at least HindIII and EcoRI

for all mutants, produced identical patterns of hybridiz-ing fragments from digests of TK+/+, TK+/-, orTK-/-mutant DNAs, indicating that the two alleles of the Tk geneare structurally similar in TK+/+ and TK+/- cells and thatsignificant internal deletions, insertions, or other intragenicrearrangements were not observed in this sample of TK1mutants. A single exception, one large-colony bleomycinmutant, has band patterns interpretable as a partial deletionwithin one allele. When the DNAs were digested with Nco L,however, a 6.3-kilobase (kb) band, observed in pMtkll6,TK+/+, and TK+/- DNAs (Fig. 2), was noted to be absentfrom Nco I digests of all except one of the 48 small-colonymutants tested. When DNA from 51 large-colony mutantswas tested with NcoI, the 6.3-kb fragment was absent in 31cases (Fig. 2b). Thirteen of 17 large-colony EMS mutants and5 of 28 large-colony bleomycin mutants, however, showed nostructural changes detectable by Southern blot analysis. The

+(0

-96.3_04.85

4.1

ro)-4 -i C)" I 0 0 - = l) L

+ + 0n O a, o m

6.3 SV.e *MPS.e

48 r0 Ow 4 1;

V so -YA

FIG. 2. (a) Tk-specific bands from a blot of Nco I-digested DNAisolated from L5178Y TK+/- 3.7.2C cells, from the cosmid pMtkll6,and from LTK- cells. LTK- cells have been reported to contain noTk-coding sequences, and the bands labeled 4i are probably pseudo-genes (16). The blots were probed with mouse Tk cDNA sequences(16). (b) Forty-eight small-colony and 51 large-colony TK-/- mu-tants were digested with Nco 1, and blots of the digests were probedwith pMtk4 sequences. The 6.3-kb band seen in pMtkl16 and TK+/-DNA is absent from blots of 47 of 48 of the small-colony mutants and31 of 51 large-colony mutants, as shown for the large-colony bleo-mycin-induced mutants 902, 909, 910, 911, and 915. The remainingsmall-colony mutant and 19 of the remaining large-colony mutantsshow the wild-type pattern (mutant 913). TK+/+ 3, the progenitor ofL5178Y TK+/- 3.7.2C, as well as other TK+/+ cell lines and mousespleen cells, also shows the TK+/- pattern.

Proc. Natl. Acad. Sci. USA 87 (1990)

Proc. Natl. Acad. Sci. USA 87 (1990) 53

fractions ofall large- and small-colony mutants that show lossof the 6.3-kb band are summarized in Table 1.The 6.3-kb band comigrates with a Tk-speciflc band in

pMtkll6 that spans the 3' region of the Tk gene as well as aconsiderable region of genomic DNA downstream of the Tkgene itself (Figs. 1 and 3). This 3' intergenic region may bepolymorphic for Nco I in the vicinity of the two Tk alleles ofwild-type TK+/+ and TK+1- cells. We noted that the small-colony TK-1- mutant G4 contains two copies of chromo-some 11a, believed to be the site of the Tk- allele, and noneof chromosome lib, which is the probable site of the TkVallele (14). Southern blots of G4 DNA, therefore, are ex-pected to show Tk DNA fragments specific for only onechromosome 11 homolog, presumably carrying the Tk- al-lele. Fig. 3 indicates that the Nco I digestion pattern of G4DNA shows Tk-specific bands that are also present in TK+/-DNA, with the exception of the 6.3-kb band, which is notseen in the G4 lane. One of the Tk-specific bands in TK+I-and G4 DNAs is a 4.8-kb fragment that is not seen in pMtkll6or in LTK- DNA (Figs. 2a and 3). This 4.8-kb fragment isobserved in DNA from TK+1+ cells, including the TK+1+ 3progenitor of L5178Y 3.7.2C TK+/- (Fig. 2b) and mousespleen cells, as well as in DNA recovered from all of themutant cell lines and probably results from an Nco I sitewithin the 6.3-kb Nco I fragment ofone allele. This Nco I siteis in the 3' intergenic region and probably is not associatedwith the activity of the Tk gene. Because the G4 cell line hasonly chromosomes Ha and a TK- phenotype, we believe thatthe 4.8-kb fragment is associated with the Tk- allele in TK+/3.7.2C cells. Our assignment of the Nco I sites in the vicinityof the two Tk alleles to the map positions diagrammed in Fig.3 is reinforced by blot data from Nco I/EcoRI digests of theTK+/- and G4 DNAs (Fig. 3; data not shown). We concludethat the region adjacent to the two Tk alleles of TK+/+ andTK+/- 3.7.2C cells is polymorphic for Nco I within the 3'intergenic region and that loss of the 6.3-kb Nco I fragmentin TK-/- mutant cell DNAs represents loss of the entirefunctional Tk allele in these cells.

Multiple Copies of the Tk- Allele in TK-/- Mutants. Todetermine whether the Tk- allele might be amplified in someof the cell lines that had lost the TkV allele, densities ofTk-specific bands on autoradiograms from blots of HindIII orNco I/HindIII digests of the TK+I- and TK-/- DNAs weredetermined (Fig. 4). The blots were stripped and reprobedwith a-actin- or histone 3.2-specific probes, and band inten-sities of hybridizing Tk, actin, and histone fragments werecompared. We expected that loss of the TkV allele in thesemutants would be accompanied by a 50% reduction inTk-specific band intensity relative to actin or histone bandintensity at the HindIII or Nco I/HindIII bands that wescanned, as the Tk gene is present in two copies in TK+/cells. However, some of the mutants that showed loss of theTkV allele had Tk-specific band intensities equivalent to or

Table 1. Summary of structural changes at the Tk locus ofTK+/- mutants: Fraction showing loss of the 6.3-kb Nco Ifragment observed in DNA from TK+/- cells

EMS Meth Pyr MMS y BleomycinLarge colony

31/51 4/17 5/5 1/1 - 21/28*Small colony47/48 1/1 1/1 3/3 6/6 36/37The numerator gives the number of mutants showing loss of the

6.3-kb Nco I fragment observed in TK+/- cell DNA. The denomi-nator gives the total number of mutants in each category. Meth,methotrexate; Pyr, pyrimethamine; y, y-irradiation. Total number ofcells analyzed, 99.*One large-colony bleomycin mutant shows a partial deletion outsidethe 6.3-kb band.

5' Gene 3'R X Hd Hd

N EZ 7I N1+1

AI lele6.3

1-)Al lele

4.8 N N5IS ~~~NIN

+/- G4

S40 4.8

0 * 4.1

*& 1_ K

3.7.2.C OTK -G411a | 11b 11a 111a

TK-K ]TK+ TK-1 TV

FIG. 3. TK+/- and G4 DNAs were digested with Nco 1, blotted,and hybridized to pMtk4. The cell line G4 has two chromosomes 11a,which contain the Tk- allele, and none of the chromosome 11b, asshown at the bottom of the figure. A diagram of the gene in the regionof interest is shown (Upper Left). The 6.3-kb Nco I fragment inTK+/- DNA is missing from G4 cells. The 4.8-kb band is probablya heteromorphic variant, unique to the Tk- allele, caused by anadditional Nco I site within the 6.3-kb Nco I fragment of that allele.Bands labeled /, are the Tk-like or pseudogene bands. A 4.1-kbdoublet at the 5' end of the gene (see Fig. 1) is also indicated.

higher than those in TK+I- DNA, indicating the presence oftwo or more copies ofthe remaining, Tk-, allele in these cells.

TIV allele copy number for 10 of the 78 mutants that showloss of the TkV allele has been determined. These data,together with colony size, inducing mutagen, and cytogeneticfeatures of these mutants, are summarized in Table 2. Cyto-genetic data for mutants C.516, C.540F, and C.342 have beenreported (9, 11, 18). Mutant G1 has two copies of the Tk-allele accompanying loss of the TkV sequence. In addition,this mutant is trisomic for chromosome 11, containing twonormal chromosomes 11a (the site of the Tk- allele) and one11b, from which the TkV allele has probably been deleted.The methotrexate-induced large-colony mutants 1829 and1830 are karyotypically normal and show a pattern of simpleloss of TkV sequences. One translocation mutant, C.540F,contains 3-4 copies of the Tk allele. A second translocationmutant, C.342, as well as one large-colony and three small-colony mutants with no obvious cytogenetic abnormalities,have two copies of the Tk- allele.

DISCUSSIONTo date, most analyses of the structural changes accompa-nying mutational inactivation of mammalian genes haveinvolved hemizygous loci because Southern blot analysis ofsingle copy genes is not complicated by the presence of asecond allele. Recent studies have shown that most muta-tions observed at the X chromosome-linked hypoxanthine-guanine phosphoribosyltransferase (Hgprt) gene or at a hem-izygous autosomal adenine phosphoribosyltransferase (Aprt)locus are single-base substitutions, small intragenic re-arrangements, or deletions of limited extent (reviewed in ref.24). However, few viable mutants are recovered when cellscontaining hemizygous test loci are treated with ionizing

Biochemistry: Applegate et al.

54 Biochemistry: Applegate et al.

im0E -0-aHo oo

F- LO|XAd-J o T!- J U w c c C-..E..' " ON arm

*. ::

.:3. 0, 'tat, o-actin

u0It M

4] -KTk_TTk

4*611 H3.2

FIG. 4. DNAs from TK+/- cells and from TK-1- cell linesmissing the TkV allele were digested with HindIll or with NcoI/HindIll, and the Tk-specific bands indicated by arrows werescanned densitometrically. The blots were reprobed with a 200-base-pair (bp) fragment unique to mouse skeletal a-actin (22) or witha 600-bp histone H3.2 gene fragment (23), and ratios of Tk to actin orhistone band intensities were compared. (a) HindIII digests of sevenTK-/- mutant DNAs, one of which (mutant 1829) has 50%6 of theTk-specific band intensity of TK+/- DNA (one Tk- allele). Theremainder have a Tk-specific band intensity equivalent to or higherthan that of the TK+/- band (2-4 Tk- alleles). (b) Nco I/HindIlldigest of mutant 1829 and C540 F, which have 3 or 4 Tk- alleles,respectively (see Table 2). GI, y irradiation.

radiation, with radiomimetic chemicals such as bleomycin, orwith other strong clastogens. These agents are highly muta-

Table 2. Tk- allele copy number in three large-colony (L) andseven small-colony (S) TK-/- mutants showing loss of theTkV allele

No. ofChr 11 Tk/ Tk/ Tk

Cell line Mutagen Morph H3.2* Actin* allelestTK+/- 2.0 2.0 2C.516 (L) Pyr Nor - 2.34 21829 (L) Meth Nor 1.14 1.24 11830 (L) Meth Nor 1.20 1C.540F (S) Pyr T(llq;6) 3.16 4.02 3-4C.342 (S) MMS T(2;11) 2.04 1.86 2G1 (S) y 2 11a; 2.32 1.82 2

1 11bG12 (S) y Nor 2.40 2G16 (S) y Nor - 2.50 2-3G20 (S) y Nor 1.82 2

Chr, chromosome; Morph, morphology; Nor, normal chromo-somes 11; T, translocation; Pyr, pyrimethamine; Meth, methotrex-ate; y, y-irradiation.*Relative band intensities are given as the ratio of Tk to histone oractin band intensity with values normalized to give Tk/H3.2 orTk/actin = 2 for TK+/- DNA on all blots.

tValues from the preceding two columns were averaged and roundedto the nearest whole number. The base value of 2 for TK+/- DNAincludes one TkV and one Tk- allele. Values for all mutant DNAsinclude only Tk alleles.

genic at heterozygous test loci such as the mouse L5178YTK+/- 3.7.2C cell line, and it has been suggested (8, 25-29)that hemizygous test loci may be inefficient indicators ofsome mutagenic events, such as potentially lethal multilocusdeletions or recombination processes requiring the presenceof two homologs (8, 25-29). Strong clastogens induce almostexclusively small-colony TK1- mutants, and it is possiblethat these mutants harbor mutagenic lesions not recoverable athemizygous test sites (8, 30).The data presented here show that mutation at the auto-

somal heterozygous Tk gene in mouse lymphoma cells isoften associated with loss of the entire TkV allele. Such lossmay be accompanied by duplication of the remaining Tk-allele, suggesting that somatic recombination, including mi-totic recombination or gene conversion, may account for lossofTK activity in some TK-1- mutants. Homologous somaticrecombination has recently been associated with inactivationof tumor suppressor genes and can be induced by incubationof cells in culture with DNA-damaging carcinogens (31-34).Among our small-colony mutants, 6 of 7 that were analyzedfor Tk allele copy number have lost the TkV allele and havetwo copies of the Tk- allele. One of the 6 has two chromo-somes 11a. We believe that somatic recombination resultingin Tk- allele homozygosity may be the most likely explana-tion of our observations of two Tk- alleles in the remaining5 of these mutants. Neither of 2 large-colony methotrexatemutants that had lost TkV allele sequences and were tested forTk- allele copy number has two copies of the Tk- allele.The fraction ofTK-1- mutants that has lost TkV sequences

includes all except one (47/48) of the small-colony mutants.Although most ofthe small-colony mutants reported here wereinduced by ionizing radiation or by bleomycin, both stronglyclastogenic, these studies combined with those of Little andcoworkers (refs. 25 and 26 and references therein) suggest thatthe loss of TkV sequences in small colony TK-/- mutants maybe extensive in cells treated with traditional point mutagens aswell as strong clastogens. These authors have observed, as wehave in mouse lymphomaTK-/- mutants, a strong correlationbetween the slow-growing colony phenotype and loss of Tkallele sequences in TK-/- mutants induced in ahuman cell line(TK6) that is heterozygous at the Tk locus. Slow-growing TK6TK-/- mutants show a 91-96% rate of loss of TkV allelesequences when treated with EMS, x-rays, UV light, ormitomycin C or when arising spontaneously.

In contrast to the small-colony data, structural analysis ofmutation in large-colony TK-/- mutants shows a variablemutagen-dependent rate of TkV allele loss in mouse lymphomaTK-/- mutants. Seventy-six percent of EMS-induced large-colony mutants show no apparent structural alterations at theTk locus and are probably point mutants. EMS is highlymutagenic at the hemizygous Hprt and Aprt loci and inducesmostly point mutations (reviewed in ref. 24). However, bleo-mycin, a potent radiomimetic clastogen, induces a 79%o rate ofTkV allele loss among large-colony TK-/- mutants. Bleomy-cin induces very few mutants at Hgprt or Aprt loci and asimilar low frequency of large-colony TK-/- mutants. TkVallele loss in large-colony bleomycin mutants may not involveextensive loss or rearrangement ofDNA sequences, but it mayinvolve more limited deletions similar to those recovered athemizygous loci in cells treated with ionizing radiation (30).

Fig. 5 summarizes the range of stable mutations describedin this study. Some mutants have not been classified. Be-cause the two chromosomes 11 carrying the Tk alleles aredistinguishable, we can directly identify TK-/- mutants,such as G1 and G4, that have duplications of a singlehomolog, probably as a result of mitotic nondisjunction (31),as well as mutants showing other cytogenetic abnormalities.Such information is an essential complement to molecular-level studies.

Proc. Natl. Acad Sci. USA 87 (1990)

No io .**: :qoo

Proc. Natl. Acad. Sci. USA 87 (1990) 55

I .] , -1 :_][: TL--

BI

L>-4 C)F a 3

3'V,

~~~0

o 0

3 _ to.

2: 0

;a

a'

FIG. 5. Tentative classification of TK-1- mutants by mechanismof origin. (a) Thirteen large-colony EMS mutants and six large-colony bleomycin mutants show no obvious Tk gene structural or

chromosomal aberrations. Gene inactivation in these cells is prob-ably due to single-base or small intragenic changes. One large-colonybleomycin mutant has a small intragenic deletion. (b) Two large-colony methotrexate mutants with normal chromosomes 11 have lostthe TkV allele without apparent duplication of the Tk allele. Theseare classified as simple intraband deletion mutants. (c) One small-colony y-irradiation mutant is trisomic for chromosome 11 with two11a chromosomes, one 11b, and two copies of the Tk allele. Weassume an intraband deletion of the TkV allele. A second small-colony mutant induced by y-irradiation has undergone loss of chro-mosome 11b with duplication of chromosome 11a. Chromosomes 11of these mutants have probably undergone mitotic nondisjunction.(d) Three mutants have translocations involving the distal end ofchromosome 11b. Two of these, a T(2;llb) and a T(X;llb) clone,have lost the TkV allele and have two Tk alleles (Table 2; M.L.A.,unpublished data). The third, a l(llq;6) mutant, has three or fourcopies of the Tk allele. (e and I) Two large-colony and threesmall-colony mutants with no gross karyotypic abnormalities havelost the TkV allele and duplicated the Tk allele. We postulate thatthese mutants have undergone mitotic recombination or gene con-

version, resulting in Tk allele homozygosity.

Comparisons of results obtained at different gene loci mustbe made cautiously, and few mutagenic agents are likely toact via a single mechanism. However, the following tentativepatterns emerge from these studies. Cumulative mutagenic,cytogenetic, and molecular evidence suggests that, in themouse lymphoma assay, the small-colony phenotype is as-

sociated with slow growth, clastogenicity, loss of TkV se-

quences, and mechanisms of mutation not recoverable at

hemizygous test sites. Large-colony mutants are rapidlygrowing, have normal karyotypes, show variable loss of theTkV allele, and may reflect mutations recoverable at hem-izygous test sites.Because of the significance of both single-gene mutations

and viable chromosomal aberrations in human health, chem-ical and physical agents are evaluated for their ability to

induce both types of lesions by in vitro mutagenesis assays.

The observations reported here on the extent and nature ofgenetic lesions in large- and small-colony mutants indicatethat the mouse lymphoma TK'/- -TK-/- assay may be a

useful aid in studying the range of genetic lesions importantin human disease.

The research described in this article has been supported by theU.S. Environmental Protection Agency through cooperative agree-

ments CR813969 to Florida State University and CR812163 to FloridaInstitute of Technology.

1. Gewar, R. F., Wilson, L. B., Mallaseth, F. S., Milner, P. F., Bittner, M.& Wilson, J. T. (1981) Proc. Natl. Acad. Sci. USA 78, 5081-5085.

2. Kidd, V. J., Wallace, R. B., Leakura, K. & Woo, S. L. C. (1983) Nature(London) 304, 230-234.

3. Dozy, A. M., Forman, E. N., Abuelo, D. N., Barsel-Bowers, G., Ma-honey, M. J., Forget, B. G. & Kan, Y. W. (1979) JAMA 241, 1610-1612.

4. Honey, N. K. & Shows, T. B. (1983) Cancer Genet. Cytogenet. 10,287-310.

5. Oberly, T. J., Bewsey, B. J. & Probst, G. S. (1984) Mutat. Res. 125,291-306.

6. Clive, D., Johnson, K. O., Spector, J. F. S., Batson, A. G. & Brown,M. M. M. (1979) Mutat. Res. 59, 61-108.

7. Moore,M. M., Clive, D., Howard, B. E.,Batson,A. G.&Turner,N. T.(1985) Mutat. Res. 151, 147-160.

8. Moore, M. M., Brock, K. H., DeMarini, D. M. & Doerr, C. L. (1987) inBanbury Report 28: Mammalian Cell Mutagenesis, eds. Moore, M. M.,DeMarini, D., DeSerres, F. & Tindall, K. R. (Cold Spring Harbor Lab.,Cold Spring Harbor, NY), pp. 93-108.

9. Hozier, J., Sawyer, J., Clive, D. & Moore, M. (1982) Mutat. Res. 105,451-456.

10. Hozier, J. C., Sawyer, J., Clive, D. & Moore, M. M. (1985) Mutat. Res.147, 237-242.

11. Moore, M. M., Clive, D., Hozier, J. C., Howard, B. E., Batson, A. G.,Turner, N. T. & Sawyer, J. (1985) Mutat. Res. 151, 161-174.

12. Blazak, W. F., Stewart, B. E., Galperin, I., Allen, K. L., Rudd, C. J.,Mitchell, A. D. & Caspary, W. J. (1986) Environ. Mutagen. 8, 229-240.

13. Kozak, C. A. & Ruddle, F. H. (1975) Somat. Cell Genet. 3, 121-133.14. Sawyer, J., Moore, M. M., Clive, D. & Hozier, J. C. (1986) Environ.

Mutagen. 8, Suppl. 6.15. Applegate, M. L. & Hozier, J. C. (1987) in Banbury Report 28: Mam-

malian Cell Mutagenesis, eds. Moore, M. M., DeMarini, D., DeSerres,F. & Tindall, K. R. (Cold Spring Harbor Lab., Cold Spring Harbor, NY),pp. 213-224.

16. Lin, P.-F., Lieberman, H. B., Yeh, D.-B., Xu, T., Zhao, S.-Y. & Ruddle,F. H. (1985) Mol. Cell. Biol. 5, 3149-3156.

17. Turner, N., Batson, A. G. & Clive, D. (1984) in Handbook of MutagenicityTest Procedures, eds. Killey, B. J., Ligator, M., Nichols, W. & Ramel, C.(Elsevier, New York), 2nd Ed., pp. 239-268.

18. Hozier, J. C., Sawyer, J., Moore, M., Howard, B. & Clive, D. (1981)Mutat. Res. 84, 169-181.

19. Chirgwin, J. M., Przybyla, R. J., MacDonald, R. J. & Rutter, W. J.(1979) Biochemistry 18, 5294-5299.

20. Rigand, G., Grange, T. & Pictet, R. (1987) Nucleic Acids Res. 15, 857.21. Minty, A. J., Caravatti, M., Robert, B., Cophen, A., Daubas, P.,

Weydert, A., Gros, F. & Buckingham, M. E. (1981) J. Biol. Chem. 256,1008-1014.

22. Czosnek, H., Nudee, U., Shain, M., Barker, P. E., Pravcheva, D. D.,Ruddle, F. H. & Yaffe, D. (1982) EMBO J. 1, 1299-1305.

23. Taylor, J. D., Wellman, S. E. & Marzluff, W. F. (1986) J. Mol. Evol. 23,242-249.

24. Thacker, J. (1985) Mutat. Res. 150, 431-442.25. Yandell, D. W., Dryja, T. P. & Little, J. B. (1986) Somat. Cell Mol.

Genet. 12, 255-263.26. Little, J. B., Yandell, D. W. & Liber, H. B. (1987) in Banbury Confer-

ence on Mammalian Cell Mutagenesis, eds. Moore, M. M., DeMarini,D., DeSerres, F. & Tindall, K. R. (Cold Spring Harbor Lab., Cold SpringHarbor, NY), pp. 225-236.

27. Evans, H. H., Mencl, J., Horng, M.-F., Ricanti, M., Sanchez, C. &Hozier, J. C. (1986) Proc. Natl. Acad. Sci. USA 83, 4379-4383.

28. Clive, D., Batson, A. G. & Turner, N. T. (1980) in The Predictive Valueof Short Term Screening Tests in Carcinogenicity Evaluation, eds.Williams, G. M., Droes, R., Waaijers, H. W. & van de Poll, K. W.(Elsevier, New York), pp. 103-123.

29. Carver, J. H., Carranmo, A. V. & MacGregor, J. T. (1983) Mutat. Res.113, 45-60.

30. Tindall, K. R. & Stankowski, L. F., Jr. (1987) in Banbury Report 28:Mammalian Cell Mutagenesis, eds. Moore, M. M., DeMarini, D., De-Serres, F. & Tindall, K. R. (Cold Spring Harbor Lab., Cold SpringHarbor, NY), pp. 283-292.

31. Cavenee, W. K., Dryja, T. P., Phillips, R. A., Benedict, W. F., God-bout, R., Gallie, B. L., Murphree, A. L., Strong, L. C. & White, R. L.(1983) Nature (London) 305, 779-784.

32. Knudsen, A. G., Meadows, A. T., Nichols, W. W. & Hill, R. (1976) N.Engl. J. Med. 295, 1120-1123.

33. Maher, V. M., Wang, Y., Bhattacharyya, N. P., McCormick, J. J. &Liskay, R. M. (1987) in Banbury Report 28: Mammalian Cell Mutagen-esis, eds. Moore, M. M., DeMarini, D., DeSerres, F. & Tindall, K. R.(Cold Spring Harbor Lab., Cold Spring Harbor, NY), pp. 355-363.

34. Wang, Y., Maher, V. M., Liskay, R. M. & McCormick, J. J. (1988) Mol.Cell. Biol. 8, 196-202.

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