Determination of the spectrum of mutations induced by defined-wavelength solar UVB (313-nm) radiation in mammalian cells by use of a shuttle vector.

Mutations induced by UVB (313-nm) radiation, a wavelength in the region of peak effectiveness for sunlight-induced skin cancer in humans, have been analyzed at the sequence level in simian cells by using a plasmid shuttle vector (pZ189). We find that significant differences exist between the types of mutations induced by this solar wavelength and those induced by nonsolar UVC (254-nm) radiation. Compared with 254-nm radiation, 313-nm radiation induces more deletions and insertions in the region sequenced. In addition, although the types of base substitutions induced by the two wavelengths are broadly similar (in both cases, the majority of changes occur at G-C base pairs and the G-C to A-T transition is predominant), an analysis of the distribution of these base changes within the supF gene following irradiation at 313 nm reveals additional hot spots for mutation not seen after irradiation at 254 nm. These hot spots are shown to arise predominantly at sites of mutations involving multiple base changes, a class of mutations which arises more frequently at the longer solar wavelength. Lastly, we observed that most of the sites at which mutational hot spots arise after both UVC and UVB irradiation of the shuttle vector are also sites at which mutations arise spontaneously. Thus, a common mechanism may be involved in determining the site specificity of mutations, in which the DNA structure may be a more important determinant than the positions of DNA photoproducts.     

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency: Identification of point mutations in Japanese patients with Lesch-Nyhan syndrome and hereditary gout and their permanent expression in an HPRT-deficient mouse cell line

Two different single nucleotide transitions of hypoxanthine-guanine phosphoribosyltransferase (HPRT) were identified in a Japanese patient with Lesch-Nyhan syndrome (LNS) and a patient with hereditary gout. HPRT enzyme activities in the two patients were severely deficient, but the size and amount of mRNA were normal according to Northern analysis. Entire coding regions of HPRT cDNAs were amplified by PCR and sequenced. A G-to-A substitution at base 208 in exon 3, which predicted glycine 70 to arginine, was detected in the LNS patient (identical mutation with HPRT_Utrecht). A C-to-A substitution at base 73 in exon 2, which predicted proline 25 to threonine, was detected in the gout patient (designated HPRT_Yonago). We transfected normal HPRT cDNA, mutant cDNA with HRPT_Utrecht or mutant cDNA with HPRT_Yonago, respectively, to HPRT-deficient mouse cells and isolated permanent expression cell lines. The HPRT-deficient mouse cells had no detectable HPRT activity and a very low amount of HPRT mRNA. When the HPRT-deficient mouse cells were transfected with normal human cDNA, HPRT enzyme activity increased to 21.8% that of normal mouse cells. The mouse cells transfected with HPRT_Utrecht showed no increase in HPRT activity; however, when the mouse cells were transfected with HPRT_Yonago, the activity increased to 2.4% that of normal activity. The proliferative phenotypes of these cells in HAT medium and in medium containing 6-thioguanine were similar to those of skin fibroblasts from the patients. This series of studies confirmed that each of the two point mutations was responsible for the decreases in HPRT enzyme activity, and the proliferative phenotypes in HAT medium and medium containing 6-thioguanine.     

Screening for mutations in human HPRT cDNA using the polymerase chain reaction (PCR) in combination with constant denaturant gel electrophoresis (CDGE).

Previously, we reported the modification of denaturing gradient gel electrophoresis called constant denaturant gel electrophoresis (CDGE). CDGE separates mutant fragments in specific melting domains. CDGE seems to be a useful tool in mutation detection. Since the hypoxanthine phosphoribosyltransferase (HPRT) gene is widely used as target locus for mutation studies in vitro and in vivo, we have examined the approach of analyzing human HPRT cDNA by polymerase chain reaction (PCR) and CDGE. All nine HPRT exons are included in a 716-bp cDNA fragment obtained by PCR using HPRT cDNA as template. When the full-length cDNA fragment was examined by CDGE, it was possible to detect mutations only in the last part of exon 8 and exon 9. However, digestion of the cDNA fragment with the restriction enzyme AvaI prior to CDGE enabled us to detect point mutations in most of exon 2, the beginning of exon 3, the last part of exon 8 and exon 9. With the use of two internal primer sets, including a GC-rich clamp on one of the primers in each pair, a region containing most of exon 3 through exon 6 was amplified and we were able to resolve fragments with point mutations in this region from wild-type DNA. The approach described here allows for rapid screening of point mutations in about two thirds of the human HPRT cDNA sequence. In a test of this approach, we were able to resolve 12 of 13 known mutants. The mutant panel included one single-base deletion, one two-base deletion and 11 single-base substitutions.     

Mutations of the ATM gene detected in Japanese ataxia-telangiectasia patients: possible preponderance of the two founder mutations 4612del165 and 7883del5

The ATM (A-T, mutated) gene on human chromosome 11q22.3 has recently been identified as the gene responsible for the human recessive disease ataxia-telangiectasia (A-T). In order to define the types of disease-causing ATM mutations in Japanese A-T patients as well as to look for possible mutational hotspots, reverse-transcribed RNA derived from ten patients belonging to eight unrelated Japanese A-T families was analyzed for mutations by the restriction endonuclease fingerprinting method. As has been reported by others, mutations that lead to exon skipping or premature protein truncation were also predominant in our mutants. Six different mutations were identified on 12 of the 16 alleles examined. Four were deletions involving a loss of a single exon: exon 7, exon 16, exon 33 or exon 35. The others were minute deletions, 4649delA in exon 33 and 7883del5 in exon 55. The mutations 4612del165 and 7883del5 were found in more than two unrelated families; 44% (7 of 16) of the mutant alleles had one of the two mutations. The 4612del165 mutations in three different families were all ascribed to the same T→A substitution at the splice donor site in intron 33. Microsatellite genotyping around the ATM locus also indicated that a common haplotype was shared by the mutant alleles in both mutations. This suggests that these two founder mutations may be predominant among Japanese ATM mutant alleles. Received: 15 September 1997 / Accepted: 12 January 1998     

Characterization of HAT- and HAsT-resistant HPRT mutant clones of V79 Chinese hamster cells.

HPRT mutant clones of V79 Chinese hamster cells, isolated after 6-thioguanine (6TG) selection, normally exhibit sensitivity to growth in medium containing the folic acid inhibitor aminopterin or the glutamine analogue L-azaserine (e.g., HAT or HAsT medium). However, it has been shown that some HPRT- clones are resistant to both HAT and HAsT medium. The present study was undertaken to investigate whether any common structural gene alteration exists for such 6TGr-HATr-HAsTr clones. Four clones were studied, 1 of spontaneous origin, 2 induced by a low dose of MNU and 1 EMS-induced. In contrast to wild-type cells and a mutant clone carrying a complete deletion of the HPRT gene, these 4 investigated 6TGr-HATr-HAsTr clones all showed an enhanced incorporation of exogenous 3H-hypoxanthine in the presence of aminopterin and L-azaserine suggesting that these clones carry mutations in the structural part of the HPRT gene. Sequence analysis of PCR-amplified HPRT cDNA from these mutants showed that the spontaneous and the 2 MNU-induced mutant clones lacked exon 4, while the EMS-induced mutant had a GC to AT transition in exon 6. Southern blot analysis of genomic DNA after digestion with BglII, EcoRI and PstI showed no changes in fragment patterns as compared to the wild type. Further sequence analysis of PCR-amplified genomic DNA using exon 4-specific primers showed that all these 3 mutants had an AT to GC or GC to AT transition in exon 4, but had no alterations in the splice sites of exon 4. Based on their characteristics of hypoxanthine incorporation, the present mutant clones fit the model for the proposed functional domains of the HPRT protein.     

Molecular analysis of complex human cell populations: mutational spectra of MNNG and ICR-191

We describe a method to identify and enumerate mutants at the nucleotide level in complex cell populations. Several thousand different mutants were induced at the HPRT locus in human lymphoblastoid cultures by either MNNG, an alkylating agent, or by ICR-191, a substituted acridine. HPRT mutants were selected en masse by resistance to 6-thioguanine. The most frequent mutations (hotspots) in HPRT exon 3 were determined by a combination of denaturing gradient gel electrophoresis and polymerase chain reaction. MNNG predominantly produced GC----AT transitions at nucleotides in a GGGGGG sequence, while ICR-191 produced both +1 frameshifts in the same GGGGGG sequence and +1 frameshifts in a CCC sequence.     

Molecular analysis of five independent Japanese mutant genes responsible for hypoxanthine guanine phosphoribosyltransferase (HPRT) deficiency

Five independent mutations in the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene were identified in a partially HPRT deficient patient with gout and in four Lesch-Nyhan patients. Using the polymerase chain reaction (PCR) technique coupled with direct sequencing, the nucleotide sequences of the entire HPRT coding region amplified from the cDNA and also of each exon amplified form the genomic DNA were analyzed. Three independent point mutations in the coding region were detected in the partially HPRT deficient patient (Case 1) and in two Lesch-Nyhan patients (Case 2 and 3), resulting in single amino acid substitutions. The family study of Case 3, utilizing a PvuII restriction site created in the mutant gene, indicated that the mother was a heterozygote, and a sister and a fetal brother had inherited the normal HPRT gene from the mother. In two other mutants causing Lesch-Nyhan syndrome, a portion of the HPRT gene was deleted, and RNA splicing was missing in both mutants. A 4-bp deletion at the 5 end of exon 4 resulted in formation of three different types of abnormal mRNA (Case 4). The other mutant (Case 5) produced abnormal mRNA including 26bp of intron 8 instead of the deleted 58bp at the 5 end of exon 9, because of a 74-bp deletion from intron 8 to exon 9.     

Nonspecific interaction of Escherichia coli pyrenyl RNA polymerase holoenzyme with synthetic polynucleotides as monitored by fluorescence spectroscopy

A derivative of RNA polymerase containing approximately 2 pyrene equiv per enzyme molecule has been used to study the interaction of RNA polymerase with poly[d(A-T)].poly[d(A-T)] and poly[d-(G-C)].poly[d(G-C)]. As monitored by fluorescence spectroscopy, pyrenyl RNA polymerase displays a unique set of conformational changes with each synthetic polynucleotide as a function of temperature. An increase in the fluorescence intensity was observed for both polynucleotides at 5 degrees C. A decrease was observed in the case of poly[d(A-T)].poly[d(A-T)] at 25 and 37 degrees C, whereas no discernible perturbation was observed in the case of poly[d(G-C)].poly[d(G-C)]. Different salt dependencies were observed for the interaction of pyrenyl RNA polymerase with these polynucleotides at 5 and 25 degrees C. Further characterization of these interactions as well as correlation of the observed fluorescence changes to the corresponding open and closed complexes was carried out with heparin. The interaction between pyrenyl RNA polymerase and poly[d-(A-T)].poly[d(A-T)] at 25 degrees C was quantified by using two different methods.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specificity of mutagenesis by 4-aminobiphenyl. A possible role for N-(deoxyadenosin-8-yl)-4-aminobiphenyl as a premutational lesion

Mutagenesis by N-acetoxy-N-trifluoroacetyl-4-aminobiphenyl, a reactive form of the human bladder carcinogen 4-aminobiphenyl (ABP), was studied in Escherichia coli virus M13mp10. N-acetoxy-N-trifluoroacetyl-4-ABP-treated DNA containing 140 lesions/duplex genome, when introduced into excision repair-competent cells induced for SOS mutagenic processing, resulted in a 40-fold increase in mutation frequency over background in the lacZ alpha gene fragment. DNA sequence changes were determined for 20 independent mutants. G-C base pairs were the major targets for base pair substitution mutations, although significant mutagenic activity was also observed at certain A-T base pairs. Deletion and frameshift mutations also were found in this sample. The salient feature of this partial "mutational spectrum" was a hotspot that occurred at position 6357 (amino acid 30 of the beta-galactosidase fragment encoded by M13mp10); this A-T to T-A transversion appeared in 6 of the 20 mutants. The property of ABP to mutate A-T base pairs was consistent with the result that N-hydroxy-ABP reverted Salmonella typhimurium strain TA104, which is presumed to revert primarily due to mutations at these sites. The ability of the major carcinogen-DNA adduct formed by ABP in vivo and in vitro, N-(deoxyguanosin-8-yl)-4-aminobiphenyl, to cause base pair substitution mutations was also investigated. This adduct was positioned specifically in the minus strand at position 6270 in duplex M13mp10 DNA. In the presence of the mutagenesis-enhancing plasmid pGW16 and UV induction of SOS mutagenic processing, it was shown that fewer than 0.02% of the adducts resulted in transition or transversion mutations following transfection of DNA into excision-repair competent cells. Similar results were obtained in uvrA and uvrC backgrounds. Although the major adduct did not cause base substitution mutations under these experimental conditions, the contribution of this lesion to the entire spectrum of mutations in the lacZ alpha fragment seems likely.     

Mutations in the bacteriophage lambda pL/oL region that spontaneously occur in plasmid pRPZ126

In a previous study (Chen and Porter, 1988), we isolated spontaneous mutations in a test plasmid that had occurred under non-selective conditions and assigned them to 1 of 6 different categories or groups. The test plasmid, pRPZ126, is a pBR322 derivative containing the bacteriophage lambda immunity region with the cI857 allele so that plasmid-containing cells shifted to 42 degrees C survive only if the expression of the lambda kil gene is prevented by mutation. 75% of the total spontaneous mutations obtained fall into two of these groups where there is no readily detectable change in plasmid size. The two groups differ in that the plasmids from the group 4 mutations are missing a specific HincII site while the plasmids from the group 5 mutations had no detectable plasmid change whatsoever. In this study, we randomly selected ten group 4 mutants and ten group 5 mutants and sequenced the lambda pL/oL region of the mutant plasmid. Of the ten group 4 mutants (HincII site missing), five involved a 24- or 44-basepair deletion in the pL/oL region of the plasmid. The other five group 4 mutants and four of the ten group 5 mutants were A-T to G-C transitions in the pL/oL region. The remaining six group 5 mutants did not demonstrate any sequence change in the pL/oL region of the plasmids. 8 out of 9 of these transition mutations occurred next to the 3' end of 3 different 5'-PyGGNPuNTG-3' sequences in the lambda operator region, and this same sequence is found adjacent to the A-T to G-C transition hotspot in the lac operator region (Schaaper et al., 1986). The 9th mutation, where the A-T to G-C transition occurred one basepair away from the lambda operator, was adjacent to a very similar sequence.     

Hypersensitivity of cells from a new chromosomal-breakage syndrome to DNA-damaging agents

Cells derived from a patient with severe chromosomal breakage, immunodeficiency, and growth retardation were found to resemble those from individuals with ataxia telangiectasia (A-T) in terms of their sensitivity to cell killing and the induction of cytogenic abnormalities by X-rays. Their response to other DNA-damaging agents, including 254-nm UV light, mitomycin C, MNNG, and bleomycin was also A-T-like. In contrast to classical A-T, however, X-irradiated cells exhibited a G₁ block after release from density inhibition of growth that was not significantly different from that of normal controls.     

Z form of poly d(A-T).poly d(A-T) in solution studied by CD and UV spectroscopies

Near UV CD spectra, UV absorption spectra and their first derivatives have been recorded on poly d(A-T).poly d(A-T) solutions in presence of high NaCl concentration and various amounts of NiCl2. Comparison of the results presented here with those obtained for poly d(G-C).poly d(G-C) and poly d(A-C).poly d(G-T) in comparable conditions, and the I.R. and Raman data on poly d(A-T).poly d(A-T), allows us to assign the new spectra to the Z conformation of poly d(A-T).poly d(A-T) in solution. The mechanism by which nickel ions induce the B----Z interconversion in the presence of high NaCl concentration is discussed.     

Evaluation of acute corneal damage induced by 222-nm and 254-nm ultraviolet light in Sprague–Dawley rats

Two hundred twenty-two nanometres ultraviolet (UV) light produced by a krypton–chlorine excimer lamp is harmful to bacterial cells but not skin. However, the effects of 222-nm UV light exposure to the eye are not fully known. We evaluated acute corneal damage induced by 222- and 254-nm UV light in albino rats. Under deep anaesthesia, 6-week-old Sprague–Dawley albino rats were exposed to UV light. The exposure levels of corneal radiation were 30, 150, and 600?mJ/cm². Epithelial defects were detected by staining with fluorescein. Superficial punctate keratitis developed in corneas exposed to more than 150?mJ/cm² of UV light, and erosion was observed in corneas exposed to 600?mJ/cm² of UV light. Haematoxylin and eosin staining also showed corneal epithelial defects in eyes exposed to 254-nm UV light. However, no damage developed in corneas exposed to 222-nm UV light. Cyclobutane pyrimidine dimer-positive cells were observed only in normal corneas and those exposed to 254-nm UV light. Although some epithelial cells were stained weakly in normal corneas, squamous epithelial cells were stained moderately, and the epithelial layer that was detached from the cornea exposed to 600?mJ/cm² of light was stained intensely in corneas exposed to 254-nm UV light. In the current study, no corneal damage was induced by 222-nm UV light, which suggested that 222-nm UV light may not harm rat eyes within the energy range and may be useful for sterilising or preventing infection in the future.     

Resolution of a missense mutant in human genomic DNA by denaturing gradient gel electrophoresis and direct sequencing using in vitro DNA amplification: HPRT Munich.

The combination of denaturing gradient gel electrophoresis (DGGE) and in vitro DNA amplification has allowed us to (1) localize a DNA mutation to a given 100-bp region of the human genome and (2) rapidly sequence the DNA without cloning. DGGE showed that a mutation had occurred, but the technique revealed little about the nature or position of that mutation. The region of the genome containing the mutation was amplified by the polymerase chain-reaction technique, providing DNA of sufficient quality and quantity for direct sequencing. Amplification was performed with a 32P end-labeled primer that allowed direct Maxam-Gilbert sequencing of the amplified product without cloning. HPRTMunich was found to contain a single-base-pair substitution, a C-to-A transversion at base-pair position 397. We report the generation of a 169-bp, wild-type DNA probe that encompasses most of exon 3 of the human hypoxanthine guanine phosphoribosyltransferase (HPRT) gene and contains a low-temperature melting domain of approximately 100 bp. HPRTMunich, an HPRT mutant isolated from a patient with gout, has a single amino acid substitution; the corresponding DNA sequence alteration must lie within the low-temperature melting domain of exon 3. We report the separation of HPRTMunich from the wild-type sequence using DGGE. In addition to base-pair substitutions, DGGE is also sensitive to the methylation state of the molecule. The cDNA for HPRT was cloned into a vector and propagated in Escherichia coli dam+ and dam- strains; thus, methylated and unmethylated HPRT cDNA was obtained.(ABSTRACT TRUNCATED AT 250 WORDS)     

Allele frequencies of single nucleotide polymorphisms in the second exon of the myoglobin gene among the Japanese

The difference in the allele frequencies of two single nucleotide polymorphisms (SNPs) in the second exon of the myoglobin gene between Japanese and other populations is reported. These SNPs are the substitutions of (A79G) and (T109C), and they were investigated by a single polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis followed by direct sequencing. The substitutions were always linked and two alleles were found in the samples used: the A-T allele with no substitution at positions (79A) and (109T) and the G-C allele with substitutions of (79G) and (109C). The frequencies of these alleles were 0.755 and 0.245, respectively, and they were found to be in Hardy-Weinberg equilibrium. The distribution of alleles in the Japanese population was significantly different from that reported among whites, blacks, and Hispanics (p < 0.0001).     

Molecular analysis of hypoxanthine guanine phosphoribosyltransferase (HPRT) deficiencies: novel mutations and the spectrum of Japanese mutations

Inherited mutation of hypoxanthine guanine phosphoribosyltransferase, (HPRT) gives rise to Lesch-Nyhan syndrome or HPRT-related gout. We have identified a number of HPRT mutations in patients manifesting different clinical phenotypes, by analyzing all nine exons of the HPRT gene (HPRT1) from genomic DNA and reverse transcribed mRNA using the PCR technique coupled with direct sequencing. Recently, we detected two novel mutations: a single nucleotide substitution (430C > T) resulting in a nonsense mutation Q144X, and a deletion of HPRT1 exon 1 expressing no mRNA of HPRT. Furthermore, we summarized the spectrum of 56 Japanese HPRT mutations.     

A germ line mutation within the coding sequence for the putative 5-phosphoribosyl-1-pyrophosphate binding site of hypoxanthine-guanine phosphoribosyltransferase (HPRT) in a Lesch-Nyhan patient: missense mutations within a functionally important region probably cause disease

Lesch-Nyhan syndrome caused by a complete deficiency of hypoxanthine guanine phosphoribosyltransferase (HPRT) is the result of a heterogeneous group of germ line mutations. Identification of each mutant gene provides valuable information as to the type of mutation that occurs spontaneously. We report here a newly identified HPRT mutation in a Japanese patient with Lesch-Nyhan syndrome. This gene, designated HPRT Tokyo, had a single nucleotide change from G to A, as identified by sequencing cDNA amplified by the polymerase chain reaction. Allele specific oligonucleotide hybridization analysis using amplified genomic DNA showed that the mutant gene was transmitted from the maternal germ line. This mutation would lead to an amino acid substitution of Asp for Gly at the amino acid position 140 located within the putative 5-phosphoribosyl-1-pyrophosphate (PRPP) binding region. Missense mutations in human HPRT deficient patients thus far reported tend to accumulate in this functionally active region. However, a comparison of the data suggested that both missense and synonymous mutations can occur at any coding sequence of the human germ line HPRT gene, but that a limited percentage of all the missense mutations cause disease. The probability that a mutation will cause disease tends to be higher when the missense mutation is within a functionally important sequence.     

A 19F-NMR study of 2-fluoro-4-demethoxydaunomycin intercalation complexes with the decanucleotides d(G-C)5 and d(A-T)5

Binding configurations and equilibria of intercalation complexes formed by the novel anthracycline drug, 2-fluoro-4-demethoxydaunomycin (2FD), with the decanucleotides d(G-C)5 and d(A-T)5 have been studied by 19F-NMR spectroscopy. The 19F chemical shift of 2FD bound to d(A-T)5 was approximately 1.5 ppm downfield of that observed for 2FD bound to d(G-C)5. By mixing equimolar amounts of aqueous d(G-C)5, d(A-T)5 and 2FD, the distribution of drug between the nucleotides was followed by observing relative peak intensities and showed no G-C or A-T binding preference at room temperature. It was shown that each decanucleotide duplex bound three 2FD molecules, giving a neighbour exclusion parameter, n, of n = 3 for this drug. The stoichiometric complexes, which we denote by [d(A-T)5][2FD]3 and [d(G-C)5][2FD]3, were also purified and isolated in this study.     

Molecular Analysis of Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) Deficiencies: Novel Mutations and the Spectrum of Japanese Mutations

Inherited mutation of hypoxanthine guanine phosphoribosyltransferase, (HPRT) gives rise to Lesch-Nyhan syndrome or HPRT-related gout. We have identified a number of HPRT mutations in patients manifesting different clinical phenotypes, by analyzing all nine exons of the HPRT gene (HPRT1) from genomic DNA and reverse transcribed mRNA using the PCR technique coupled with direct sequencing. Recently, we detected two novel mutations: a single nucleotide substitution (430C > T) resulting in a nonsense mutation Q144X, and a deletion of HPRT1 exon 1 expressing no mRNA of HPRT. Furthermore, we summarized the spectrum of 56 Japanese HPRT mutations.     

Gamma-ray- and UV-sensitive strains of a radioresistant cell line: isolation and cross-sensitivity to other agents

Two gamma-ray-sensitive and two ultraviolet (UV)-sensitive variants were isolated from the gamma-ray- and UV-resistant TN-368 lepidopteran insect cell line. The isolation was performed by inducing mutations in the TN-368 cells using ethyl methanesulfonate, growing them for an expression period, irradiating with 137Cs gamma rays or 254-nm UV radiation, allowing cells to incorporate 5-bromodeoxyuridine (BrdU) in the presence of hydroxyurea (DNA repair synthesis), and finally irradiating with 365-nm UV radiation to cause DNA strand breakage at sites of BrdU incorporation with the intent of killing those cells that have undergone DNA repair synthesis and sparing those cells which, for a variety of reasons, did not. The survival of the Cs2 and Cs7 variants exposed to X rays is significantly different from the parent TN-368 line at the P less than 0.0001 level. The survival of the UV10 and UV19 variants exposed to UV radiation is different from the parent at the P less than 0.0001 and P less than 0.003 levels, respectively. In cross-sensitivity testing of the gamma-ray-sensitive variants, only Cs2 is more sensitive to 254-nm UV and only Cs7 is more sensitive to 44 degrees C heating; both are sensitive to PUVA. The UV-sensitive mutants are both sensitive to X irradiation, PUVA, and mitomycin C. However, UV10 is not sensitive to 44 degrees C heating while UV19 is, making UV19 the only variant strain sensitive to all agents examined. Despite the isolation procedure which was intended to select for DNA repair-deficient cells, the results suggest that a more general mechanism is responsible for the sensitivity of the variant cells to the agents tested.