Host cell reactivation of irradiated adenovirus in UV-sensitive Chinese hamster ovary cell mutants

In this study we have utilized the ability of rodent cells toreplicate viral DNA following semi-permissive infection by humanadenovirus (Ad) to examine the host cell reactivation (HCR)of radiation-damaged Ad in several UV-sensitive Chinese hamsterovary (CHO) cell mutants. A significant reduction in HCR ofviral DNA synthesis for UV-irradiated Ad was detected in a seriesof UV-sensitive mutants from complementation groups 1–6derived from parental CHO-AA8 cells. HCR for UV-irradiated Adin these CHO mutants varied from 18.8 to 48.0% of that in parentalAA8 cells. However, a significant reduction in HCR of viralDNA synthesis for UV-irradiated Ad could not be detected inseries of UV-sensitive PV mutants from complementation groups1, 5, 9 and 10 derived from parental CHO-K1 cells, which harbourrelatively small DNA repair deficiences. We also report a reducedHCR for gamma-irradiated Ad in UV-sensitive CHO cell mutantsfrom groups 1 and 4 derived from parental CHO-AA8 cells. ThisHCR technique for DNA synthesis of Ad can be employed to measurethe DNA repair capacity of both human and rodent cells and extendedto examine the repair of DNA damaged by a variety of differentphysical and chemical agents. ³To whom correspondence should be addressed at: Departments of Biology and Radiology, Life Sciences Building, Room 434, McMaster University, Hamilton, Ontario L8S 4K1, Canada     

Summary of complementation groups of UV-sensitive CHO cell mutants isolated by large-scale screening

A summary is given for the lineage and complementation groupassignments of 153 UV-sensitive mutants of the CHO AA8 cellline. The distribution of mutants among six complementationgroups was highly non-random, with the great majority of theisolates belonging to groups 1 and 2. This asymmetry is consistentwith the known hemizygosity of these two linked loci in CHOcells. The relative numbers of mutants induced in group 2 wasfound to depend greatly on the type of mutagen used. Mutagenesiswith UV radiation, ethyl methanesulfonate (EMS), N-methyl-N'-nitro-N-nitroso-guanidine and 7-bromomethylbenz[a]anthraceneproduced high frequencies of group 2 mutants. In contrast, ICR170and ICR191, which are thought to produce mostly frameshift mutations,yielded very few mutants in group 2. These results are of particularimportance in light of the recent finding that the human ERCC2gene, which corrects group 2 mutants, has very strong homologywith the yeast gene RAD3. RAD3 is an essential gene for viabilityin yeast, and the low recovery of group 2 mutants using theframeshift agents strongly suggests that frameshift mutationstend to be lethal in the hamster ERCC2 locus. Several mutagen-sensitivedouble mutants were isolated in two-step selections from EMS-,mitomycin C- or UV-sensitive parental cells, including the lineUVU1, the first mammalian line with two mutations that affectUV sensitivity. The first mutation inactivated excision repair,and the second mutation appears to have affected some otherrecovery process. UVU1 should be useful for studying recoveryprocesses that are separate from nucleotide excision repair. ¹To whom correspondence should be addressed     

Regional mapping of human DNA excision repair gene ERCC4 to chromosome 16pl3.13-pl3.2

Mitomycin C (MMC)-resistant interspecific somatic cell hybridsmade between human cells and the MMC-sensitive, Chinese hamsterovary (CHO) excision repair-deficient UV41 cells generally containedhuman chromosome 16, while other human chromosomes were randomlypresent. MMC-sensitive and -resistant subclones were isolatedfrom resistant clones and resistance generally segregated concordantlywith human chromosome 16 markers. UV radiation survival analysisof subclones indicated that MMC and UV resistance were correlated.Therefore the complementing gene, Excision Repair Cross Complementing4 (ERCC4) was assigned to human chromosome 16. Complementationof UV41 by human cells derived from patients with xerodermapigmentosum groups A, C, D and F excluded ERCC4 from involvementin those disease syndromes. Resistant hybrids containing onlyportions of chromosome 16 were identified by the lack of concordanceof multiple chromosome 16 markers. When such hybrids were usedas a source of probe for fluorescent in situ hybridization ontonormal human metaphases, the only region of chromosome 16 identifiedas being consistently present was 16pl3.1–pl3.3. Geneticmarker analysis of informative hybrids with mapped probes refinedthe position of ERCC4 to 16pl3.13–pl3.2 and allowed thefollowing order of markers within the region to be established:pter–(PRM1 D16S215)–D16S213–D16S53 –(D16S214,ERCC4)–D16S3–D16S96–cen ⁴To whom correspondence should be addressed     

Multiplex polymerase chain reaction-based deletion analysis of spontaneous, gamma ray- and alpha-induced hprt mutants of CHO-K1 cells

Independent Chinese hamster ovary (CHO)-K1 cell mutants at thehypoxanthine-guanine phosphoribosyltransferase (hprt) locuswere isolated from untreated, ⁶⁰Co     

An improved system of somatic cell molecular genetics for analyzing the requirements of Ig synthesis and function

A general method of relating molecular function and structureis to examine the biological and chemical effects of definedmutations. In many cases, particularly those concerned withthe rate or efficiency of gene expression, It is important toassess mutations in the normal chromosomal context. There aretwo methods of obtaining such mutants: (1) site-directed mutageneslsof the chromosomal locus, using homologous recombination totarget defined mutations to the gene of interest, and (II) phenotypicselection of mutant organisms. For most mammalian genes therarity of targeted recombinants and phenotyplcally evident mutantsimpede the use of either of these approaches. However, variousgenetic and biochemical features render the Ig heavy chain locusin B cell lines amenable to both gene targeting and phenotypicselection of mutants. We describe here a replacement-type vectorin which the selectable marker is an enhancerless gpt gene whichis particularly suitable for targeting the IgH locus. Deletionof the enhancer greatly decreased the frequency of gpt⁺ randomtransformants while still allowing property targeted transformantsto be gpt⁺ such that transformants with the predicted recombinantstructure comprised 25% of the gpt⁺ population. Thus, the laborinvolved in mutagenizing the chromosomal locus using this methodis comparable to the usual method of isolating randomly insertedtransformants, but offers the important advantages that thecopy number and integration site are the same in independenttransformants. In the hybrldoma cell lines which we have tested,the consistent copy number and integration site are sufficientto yield a uniform level of recombinant gene expression. Thesystem is therefore suitable for investigating molecular featuresencoded in the µ heavy chain locus which affect the rateand efficiency of immunoglobulin production. Other advantagesof this system derive from the fact that the gpt gene whichhas been introduced into the IgH locus renders the cells sensitiveto thioxanthine (TX). One application of this effect is thatselection for TX resistance can be used to enrich for targetedmutations introduced by a second recombination event (balt-and-switchmethod). Another application concerns the possibility of identifyingunanticipated expression enhancing elements in the IgH locusby selecting phenotypically for gpt defective mutants.     

UV-sensitive rodent mutant cell lines of complementation groups 6 and 8 differ phenotypically from their human counterparts

Rodent UV-sensitive mutant cell lines of complementation groups 6 and 8 are the genetic counterparts of human Cockayne syndrome CS-B and CS-A, respectively. The original mutant in this group, UV61, was described as defective in cyclobutane pyrimidine dimer removal after high doses of UV. We have examined the responses of several cell lines from group 6 to low doses of UV irradiation, and find that these mutants have wild-type capacity for DNA repair as indicated by incision, cyclobutane pyrimidine dimer, and (6-4) photoproduct removal. ERCC6, the product of the gene defective in CS-B and group 6 mutants, is implicated in the regulation of repair of actively transcribed genes in Cockaynesyndrome; however, this protein clearly is not required for the processing of low levels of damage in CHO cells, which occurs remarkably efficiently, 40–50% of dimers being removed in both wild-type and group 6 mutants in 5 hours following 0.1 Jm^-2 of UV. The group 8 mutant cell line US31, on the other hand, is very deficient in repair of UV damage, showing a more extreme phenotype than is seen in the corresponding human syndrome CS-A. In both complementation groups, expression of mutations in a gene involved in regulation of DNA repair takes very different forms in human and rodent cells. Environ. Mol. Mutagen. 29:152–160, 1997. © 1997 Wiley-Liss, Inc.     

Differential mutant quantitation at the mouse lymphoma tk and CHO hgprt loci

Recent reports by several laboratories indicate that not allnon-essential target loci are equally capable of detecting chromosomalmutations. The present study was undertaken to determine ifboth the tk locus in mouse lymphoma cells and the hgprt locusin Chinese hamster ovary (CHO) cells can be used to quantitatechromosomal mutations. Seven known mutagens for the tk locuswere selected. These compounds were evaluated in the mouse lymphomaassay and in a suspension adapted CHO assay for their mutagenicity.In addition to the specific locus mutagenesis analysis, mouselymphoma and CHO cells were evaluated for the frequency of grosschromosome aberrations. From these investigations, it appearsthat only those compounds [2-methoxy-6-chloro-9-(3-[ethyl-2-chloroethyl]aminopropylamino)-acridine-dihydrochloride (ICR 170), ethylmethanesulfonate (EMS) and methyl methanesulfonate (MMS)] thatinduce significant numbers of large-colony thymidine kinase(TK) mutants also induce significant numbers of hypoxanthine-guaninephospho-ribosyltransferase (HGPRT) mutants. The four acrylatesevaluated (methyl acrylate, ethyl acrylate, trimethylolpropanetriacrylate and tetraethyleneglycol diacrylate) induced almostexclusively small-colony TK mutants and very few if any HGPRTmutants. Aberration analysis revealed that both the mouse lymphomaand CHO cells responded to the clastogenicity of the compounds(except for ICR 170 which was not positive in CHO cells) andthat neither cell line was clearly more sensitive than the otherto the clastogens tested. It is significant that the four acrylatesgive little or no evidence of genotoxicity when evaluated usingselection for HGPRT-deficient mutants, yet are clearly clastogenicto the same cells in the same experiment. These results areconsistent with the hypothesis that the hgprt locus may notbe useful as a marker to evaluate the clastogenic componentof a genotoxic compound. The present study adds to the increasingnumber of studies that support the view that the hemizygousnature of the hgprt locus permits the recovery of mutationsprimarily affecting the function of a single gene; whereas theheterozygous nature of the tk locus permits the recovery ofboth single gene and chromosomal mutations. ¹To whom correspondence should be addressed     

ERCC1 mutations in UV-sensitive Chinese hamster ovary (CHO) cell lines

In mammalian nucleotide excision repair (NER), the ERCC1 protein is known to act as a complex with ERCC4 (XPF) protein, which is necessary for stability of ERCC1, and this complex introduces an incision on the 5′ side of a damaged site in DNA. ERCC1 also binds to XPA protein to make a large protein complex at the site of DNA damage. Since no human disease associated with ERCC1 has been identified, Chinese hamster ovary (CHO) cell lines defective in ERCC1 are a unique source for characterization of ERCC1 deficiency in mammalian cells. We have isolated the full length ERCC1 cDNA from a wild-type CHO cell line and analyzed mutations in two CHO cell lines which fall into complementation group 1 of UV-sensitive rodent cell lines. One cell line, 43-3B, has a missense mutation at the 98th residue (V98E). The in vitro translated mutant protein of 43-3B is unable to bind to XPA protein. Although the mutant protein is able to bind to XPF protein in vitro, the mutant protein is highly unstable in vivo. These defects presumably cause the NER deficiency of this cell line. Another mutant, UV-4, has an insertion mutation in the middle of the coding sequence, resulting in a truncated protein due to a nonsense codon arising from the frameshift. Thus, these two mutant cell lines are deficient in the function of the ERCC1 gene for NER.     

Genetic and biochemical studies with the adenosine analogs toyocamycin and tubercidin: Mutation at the adenosine kinase locus in Chinese hamster cells

The pyrrolopyrimidine nucleosides toyocamycin and tubercidin show several unique features of growth inhibition in Chinese hamster ovary (CHO) cells. Stable mutants which are more than 500-fold resistant to these drugs are obtained in CHO cells at a strikingly high frequency of approximately 10^–3,in the absence of mutagenesis. The mutants resistant to toyocamycin (Toy ^r )and tubercidin (Tub ^r )exhibit similar crossresistance patterns to the two selective drugs as well as to adenosine and 6-methyl mercaptopurine riboside, indicating that the same lesion is probably involved in all cases. The mutants examined were found to be deficient in the enzyme adenosine kinase (AK), indicating that the phosphorylation of these analogs is an essential first step in their toxic action. The above mutants (AK ^–)behaved recessively in cell hybrids, and segregation studies indicate that the AK locus is not linked to the X chromosome. The frequencies of similar Toy ^r mutants in other Chinese hamster lines, e.g., V79, CHW, M3-1, GM7, and CHO-K1, varied from similar to more than three logs less than that observed for CHO cells, indicating that various cell lines probably differ in the number of functional gene copies for this locus.     

Strand-specific mutation induction by 1,2-dibromomethane at the hypoxanthine-guanine phosphoribosyltransferase locus of Chinese hamster ovary cells

The nature of mutations induced by 1,2-dibromoethane (DBE) atthe hprt (hypoxanthine-guanine phosphoribosyltransferase) genewas analysed in Chinese hamster ovary (CHO-9) cells. Molecularcharacterization of 36 hprt mutants at the cDNA level yielded19 GCAT transitions, two ATCG transversions, three frameshiftmutations, two identical small deletions and 10 exon deletions.Further analysis of the deletion mutants by amplification ofspecific exons from genomic DNA showed two more GCAT transitionsat splice sites and an {small tilde}70 bp deletion. Assumingthat the S-[2-(N7-guanyl)ethyl]glutathione adduct is responsiblefor the GCAT transitions, 90% of the affected guanines werelocated in the non-transcribed strand of the hprt gene, suggestinga strand bias in repair of this adduct Nearest neighbour analysisof induced GCAT transitions indicates a preference for a 5'-PyPuGDNA sequence, i.e. 15/21 mutated guanines were located in eithera TGG or a CAG DNA sequence. These molecular data on DBEinducedmutations showed similar features as data from a study by Graveset al (Mutagenesis, 11, 229–233, 1996) in which they analyzed13 hprt mutants induced by DBE in CHO-K1 cells. Six of the sevenGCAT mutations were on positions mutated more than once amongthe 36 hprt mutants in the present study. The combined findingssuggest that some positions seem to be hot spots for DBE-inducedmutations. Concerning the relevance of these in vitro studiesfor germ cell mutagenesis the conclusion may be that these datalend further support to the view that mutation spectra derivedfrom in vitro systems have little predictive value for the natureof mutations induced in post–meiotic germ cells in vivo,as demonstrated for other alkylating agents in both Drosophitaand mice. ¹To whom correspondence should be addressed. Tel: +31 071 5276145; Fax: +31 071 5221615; Email: nivard{at}rullf2.medfac.leidenuniv.nl     

Calibration of the single cell gel electrophoresis assay, flow cytometry analysis and forward mutation in Chinese hamster ovary cells

The induction kinetics of genetic damage were measured in oneclone of a mammalian cell line (CHO AS52) with three genotoxicityassays, the single cell gel electrophoresis (Comet) assay, laserbeam flow cytometry and forward mutation. The first two assaysallow for the rapid analysis of genotoxic damage in individualnuclei. The alkaline Comet assay detects DNA strand breaks,alkali-labile sites and incomplete excision repair sites. Flowcytometry measures chromosome damage that results in an unequaldistribution of nuclear DNA in daughter cells. We calibratedthese assays to compare acute DNA damage and longer term clastogenicitywith forward mutation at the gpt^– locus using ethyl methanesulfonate(EMS). The EMS treatments were conducted in F12 medium for 2h. AS52 cells carry a single functional gpt gene which providesfor quantitation of gpt mutants by selecting for 6-thioguanineresistance. EMS induced a concentration-dependent response withmedian Comet tail moment values of 1.06 µm for the negativecontrol and 64.6 µm with 20 mM. The coefficient of variation(CV) of the negative control with flow cytometry was 233; theCV value increased to 4.87 in cells treated with 20 mM EMS.EMS (8 mM) induced a mutant frequency of 779.8 x10^–6 ata relative survival of 64.4%. Genetic response factors werecalculated and the data demonstrate that the induction kineticsof genetic damage as measured by the Comet assay (15.6) andflow cytometry (14.2) were more closely related than that determinedfor mutation induction (7.9). These three assays measure a widespectrum of genetic events at the level of DNA, the gene andthe chromosome and demonstrate the usefulness of the Comet assayand flow cytometry as two relatively rapid procedures to detectgenotoxic damage in mammalian cells. ³To whom correspondence should be addressed at: 364 Environmental and Agricultural Sciences Building, 1101 West Peabody Drive, Urbana, IL 61801-4178, USA. Tel: +1 217 333 3614; Fax: +1 217 333 8046; Email: m-plewa{at}uiuc.edu     

Identification of nucleotide-excision-repair genes on human chromosomes 2 and 13 by functional complementation in hamster-human hybrids

The CHO UV-sensitive mutants UV24 and UV135 (complementation groups 3 and 5, respectively) are defective in nucleotide excision repair. After fusing each mutant with human lymphocytes, resistant hybrid clones showing genetic complementation were isolated by repeated exposure to UV radiation. Using a combination of isozyme markers, DNA probes,and cytogenetic methods to analyze the primary hybrids and their subclones, correction of the repair defect was shown to be correlated with the presence of a specific human chromosome in each case. Chromosome 2 corrected UV24, and the gene responsible was designated ERCC3.Line UV135 was corrected by human chromosome 13 and the gene designated ERCC5.The UV-sensitive mouse cell line, Q31, was shown not to complement UV135 and thus appears to be mutated in the same genetic locus (homologous to ERCC5)as UV135. Breakage of complementing chromosomes with retention of the genes correcting repair defects allowed the following provisional assignments: regional localization of ERCC5to 13q14-q34, exclusion of ERCC3from the region of chromosome 2 distal to p23, and relief of the ambiguity of ACPlassignment (2p23 or 2p25) to 2p23 proximal to MDH1.     

Spontaneous mutation spectrum in the hprt gene in human lymphoblastoid TK6 cells

A spectrum of 100 mutations in the endogenous hprt gene of thehuman lymphoblastoid TK6 cell line is presented. The majorityof the mutations originates in sequences outside the codingregion of the gene. Large deletions are a major cause of inactivationof the hprt gene (57% of the mutants). Mutations in the splicesites that result in several forms of aberrantly spliced mRNAare relatively frequently recovered (16%) compared with mutantscontaining alterations in the coding region of the hprt gene(27%). The majority, but not all, of the splice mutants containan alteration in the consensus sequences of the splice sites.A spectrum of mutations in the coding region of the hprt geneenlarged to a total of 42 mutants shows that basepair substitutionspredominate (71%) and that small deletions and insertions areless frequently recovered. Basepair substitutions arise slightlymore frequently at GC basepairs than at AT basepairs. ³To whom correspondence should be addressed     

Effect of nucleotide excision repair on hprt gene mutations in rodent cells exposed to DNA ethylating agents

The role of the nucleotide excision repair (NER) pathway inremoval of DNA ethylation damage was investigated by means ofhprt mutational spectra analysis in the NER-deficient Chinesehamster ovary cell line UV5, which lacks ERCC2/XPD, and in itsparental cell line AA8. Both cell lines were exposed to ethylmethanesulfonate (EMS) or N-ethyl-N-nitrosourea (ENU). EMS gavea similar dose-dependent increase in hprt mutants in UV5 comparedwith AA8. In both cell lines EMS-induced mutations in the hprtcoding region consisted almost exclusively of GC     

Expression of an E.coli O6-alkylguanine DNA alkyltransferase gene in Chinese hamster cells protects against N-methyl and N-ethylnitrosourea induced reverse mutation at the hypoxanthine phosphoribosyl transferase locus

The spontaneous hypoxanthine phosphoribosyl transferase deficient(HPRT^– ) mutants of V79 cells (TG11 and TG15) were transfectedwith a retrovirus-based plasmid containing a truncated formof the Escherichia coli gene which codes for O⁶-alkylguanine(O⁶-AG) DNA alkyltransferase (ATase). The resultant cell linesTG11SB5 and TG15SB7 were G418 resistant and expressed high levelsof O⁶-AG ATase activity. The frequency of revertants inducedby equitoxic doses of N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea(ENU) was 10- to 50-fold higher in TG11 than in TG15. In TG11SB5and TG15SB7 induced revertant frequencies were reduced relativeto TG11 and TG15 by factors of 6–8 and 1.5–3.0,respectively, immediately after treatment. On delayed platingthe frequency of MNU-induced revertant colonies decreased ata rate inversely proportional to dose in both TG11 and TG11SB5.In contrast, after exposure of TG11SB5 to ENU (50 or 75 µg/ml)initial reversion frequencies were low compared with TG11, butthen rose to a plateau frequency by 24 h, which was maintainedfor up to 72 h. The frequency of reversion observed, the degreeof protection afforded by the E.coli O⁶-AG ATase and the kineticsof expression of revertants were thus cell line specific suggestingthat DNA sequence specific alkylation and/or preferential repairmay be responsible. The initial protection against mutagenesisis consistent with the hypothesis that MNU- and ENU-inducedreversion is the result of miscoding opposite O⁶-AG or O⁴-alkylthymineresidues. Expression of O⁶-AG ATase activity was variable whencells were continually cultured over long periods despite thepresence of the selective antibiotic G418.     

Analysis of 4-nitroquinoline-1-oxide induced mutations at the hprt locus in mammalian cells: possible involvement of preferential DNA repair

Mutation spectra induced by 4-nitroquinoline 1-oxide (4NQO)at the hprt locus for both normal (AA8) and 4NQO-sensitive (UV5)Chinese hamster ovary cells were determined to investigate theeffect of DNA repair on the nature of induced mutations. TheUV5 cell line is three times more sensitive to 4NQO than theAA8 parental cell line. In UV5 cells, the dGuo-N2-AQO adduct,which is considered to be the most toxic and mutagenic adductin Escherichia coli, is poorly repaired. The molecular natureof 30hprt mutants isolated from AA8 and 20 isolated from UV5cells was determined by sequence analysis of in vitro amplifiedhprtcDNA. Both similarities and differences emerged. In bothcell lines we found that (i) 4NQO is basically a base substitutionmutagen acting almost exclusively at G residues and (ii) G transversionsare prevalent over G transitions in both cell lines, independentlyfrom the ability to repair dGuo-N2-AQO. A high proportion (13/25)of splice mutations was observed in AA8 cells, statisticallydifferent (P < 0.04, Fisher‘s exact test) from theincidence of splice mutants in UV5 cells (4/20). In AA8 mutants,all but two of the point mutations were due to lesions localizedon the non-transcribed strand, suggesting preferential repairof the transcribed strand. Compared with AA8, the proportionof mutants due to lesions present on the transcribed strandwas higher in UV5 cells, as expected if a preferential repairmechanism was impaired in the sensitive cell line. Our dataare consistent with the molecular defect in DNA repair recentlycharacterized in UV5. ³To whom correspondence should be addressed     

Genotoxic activities of 2-nitronaphthofurans and related molecules

The genotoxic activities of 63, 2-nitronaphthofurans and relatedmolecules were examined using two bacterial short-term tests,the Salmonella mammalian microsome assay test or Mutatest, amutagenesis assay, and/or the SOS Chromotest, an assay for inductionof an SOS function in Escherichia coli. Seven compounds werealso investigated in the Chinese hamster ovary cells/hypoxanthine— guanine phosphoribosyl transferase (CHO/HGPRT) test,a mammalian gene mutation assay. Our main conclusions are thefollowing: (a) Simple empirical rules relating structure tomutagenic activity in the Mutatest can be derived for some ofthe compounds. In particular, they account for the extremelyhigh Mutagenic Potency of 7-methoxy-l-methyl-2-nitronaphtho[2,1-b]furan(R7372), {small tilde}2 x 10⁶ mutants/nmol on strain TA100.(b) There is a good quantitative correlation between the MutagenicPotency in the Salmonella/mammalian microsomes assay and theSOS-inducing potency in the SOS Chromotest. This, and previousevidence, suggests strongly that the 2-nitronaphthofurans derivativesare essentially recA and thus probably umuDC-dependent mutagens.(c) Four out of seven compounds tested hi the CHO/HGPRT assaygave responses correlated with the bacterial responses. Oneof them, 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000), is among,or is, the strongest mutagen described for mammalian cells.We briefly discuss the practical and theoretical implicationsof these results. ³To whom correspondence should be addressed     

Structural and mutational analysis of the xeroderma pigmentosum group D (XPD) gene

Individuals affected by the autosomal recessive disease xerodermapigmentosum (XP) are acutely sensitive to sunlight and predisposedto skin cancer on exposed areas. Cells cultured from XP patientsare both UV sensitive and defective in the nucleotide excisionrepair of damaged DNA. These cellular phenotypes are amenableto experimental strategies employing complementation, an approachpreviously used to demonstrate the correction of XP-D phenotypesfollowing the introduction of the XPD (ERCC2) gene. In the presentstudy, we have characterized the genomic organization of theXPD (ERCC2) gene and found it to be comprised of 23 exons. Thesedata were helpful in evaluating the functional integrity ofalleles in two XP-D cell lines. In cell line GM436 a C     

UV mutagenesis,cytotoxicity and split-dose recovery in a human—CHO cell hybrid having intermediate (6−4) photoproduct repair

Somatic cell hybrids constructed between UV-hypersensitive Chinese hamster ovary cell line UV20 and human lymphocytes were used to examine the influence of a human DNA repair gene, ERCCI, on UV photoproduct repair, mutability at several drug-resistance loci, UV cytotoxicity and UV split-dose recovery. In hybrid cell line 20HL21-4, which contains human chromosome 19, UV-induced mutagenesis at the APRT, HPRT and Na⁺/K⁺-ATPase loci was comparable to that in repair-proficient CHO AA8 cells, whereas cell line 20HL21-7, a reduced human-CHO hybrid not containing human chromosome 19, exhibited a hypermutable phenotype at all 3 loci indistinguishable from that of UV20 cells. The response of 20HL21-4 cells to UV cytotoxicity reflected substantial but incomplete restoration of wild-type UV cytotoxic response, whereas responses of UV20 and 20HL21-7 cell lines to UV cytotoxicity were essentially the same, reflecting several-fold UV hypersensitivity. Repair of UV-induced (5–6) cyclobutane dimers and (6−4) photoproducts was examined by radioimmunoassay; (6−4) photoproduct repair was deficient in UV20 and 20HL21-7 cell lines, and intermediate in 20HL21-4 cells relative to wild-type CHO AA8 cells. UV split-dose recovery in 20HL21-4 cells was also intermediate relative to AA8 cells. These results show that the human ERCCI gene on chromosome 19 is responsible for substantial restoration of UV survival and mutation responses in repair-deficient UV20 cells, but only partially restores (6−4) UV photoproduct repair and UV split-dose recovery.     

Spectrum of spontaneously occurring mutations in the HPRT gene of the Chinese hamster V79 cell mutant V-H4, which is homologous to Fanconi anemia group A

The mitomycin C (MMC)-hypersensitive Chinese hamster V79 cellmutant V-H4 has a cellular phenotype similar to Fanconi anemia(FA), and has been shown to be homologous to FA group A. Toexamine consequences of the defect in V-H4 cells on spontaneousmutagenesis, we studied the frequency and nature of spontaneousmutations at the hypoxanthine phosphoribosyltransferase (HPRT)locus in this mutant and the parental V79 cells. The mutationrates expressed as the number of mutations per cell per generationwere 8.7 X10^–7 and 3.7 X10^–7 for V-H4 and V79 cellsrespectively. The molecular spectrum of 42 spontaneous hprtmutants of V-H4 cells was determined and compared with the previouslydescribed spectrum of spontaneous mutations at the HPRT locusof Chinese hamster V79 cells. The spectra of spontaneous mutationsin the hprt gene of both cell lines are predominated by basepair substitutions and splice mutations. Among the base changes,V-H4 shows a larger frequency of transitions (13/42; 31%) thantransversions (3/42; 7%), whereas in V79 transversions are observedmore often than transitions (P < 0.001; Wilcoxon test). Thefrequency of splice mutations in V-H4 (17/42; 40%), which affectsexon 4 almost exclusively, is not significantly different fromV79. The fraction of deletions in V-H4 is low (6/42; 14%), andcomparable to the level in V79. This is in contrast with thepublished molecular spectrum of spontaneous hprt mutants inFA (group D) cells, which consists predominantly of deletions. ⁴To whom correspondence should be addressed at MGC-Department of Radiation Genetics and Chemical Mutagenesis