HPRT Gene Locus Research Articles

DNMTs are required for delayed genome instability caused by radiation

The ability of ionizing radiation to initiate genomic instability has been harnessed in the clinic where the localized delivery of controlled doses of radiation is used to induce cell death in tumor cells. Though very effective as a therapy, tumor relapse can occur in vivo and its appearance has been attributed to the radio-resistance of cells with stem cell-like features. The molecular mechanisms underlying these phenomena are unclear but there is evidence suggesting an inverse correlation between radiation-induced genomic instability and global hypomethylation. To further investigate the relationship between DNA hypomethylation, radiosensitivity and genomic stability in stem-like cells we have studied mouse embryonic stem cells containing differing levels of DNA methylation due to the presence or absence of DNA methyltransferases. Unexpectedly, we found that global levels of methylation do not determine radiosensitivity. In particular, radiation-induced delayed genomic instability was observed at the Hprt gene locus only in wild-type cells. Furthermore, absence of Dnmt1 resulted in a 10-fold increase in de novo Hprt mutation rate, which was unaltered by radiation. Our data indicate that functional DNMTs are required for radiation-induced genomic instability, and that individual DNMTs play distinct roles in genome stability. We propose that DNMTS may contribute to the acquirement of radio-resistance in stem-like cells.

Assessing the Mutagenicity Potential of Multiphoton Excitation during Imaging of Intrinsic Fluorescence from Cells and Tissues

Multiphoton-excited intrinsic fluorescence emission signals from cells and tissues can be used for metabolic imaging and studies of disease morphology in chronic animal imaging experiments, and has diagnostic potential as a future in vivo clinical imaging tool. However, the signals are generally weak and require high laser intensities for imaging. Consequently, not only are viability studies important, but an investigation into the extent that multiphoton excitation is a mutagenic agent is critical. Information on the magnitude of permissible intensity levels of femtosecond pulsed near-IR light is vital to human clinical applications, yet there are currently no regulations that specifically indicate such parameters. This study is directed towards determining whether multiphoton imaging of cellular autofluorescence using 700 - 800 nm wavelength excitations causes mutations in mammalian cells. The induction of mutation by pulsed laser radiation employed for multiphoton imaging entails a risk of carcinogenicity in living tissue. The assessment of potential laser illumination toxicity was carried out by the hypoxanthine-guanine phosphoribosyl (HPRT) mammalian cell gene mutation assay, which measures mutation at the HPRT gene locus in cells, and is one of a handful of reporter loci that have been used as molecular biomarkers for both human and rodent exposure to mutagens and UV light. Experiments were performed to assess possible mutagenic effects of various intensities of 755 nm, 100 fs laser irradiation. Laser powers ranging from 20 to as high as 100 mW delivered as raster scanned excitation through a 0.7 NA objective for 20 seconds was found to be nonmutagenic to the HPRT gene locus test system, while higher laser powers initiated mutagenic responses.

Quinoid Metabolites of 4-Monochlorobiphenyl Induce Gene Mutations in Cultured Chinese Hamster V79 Cells

4-Monochlorobiphenyl (PCB3) is a component of commercial polychlorinated biphenyl (PCB) products and is an airborne environmental pollutant. Our recent study with transgenic Fischer 344 rats revealed the mutagenic potential of PCB3 in the livers of male rats. PCB3 is converted in vitro to hydroxylated metabolites, to hydroquinones (HQs, e.g., 2',5'-HQ and 3',4'-HQ), and can be further oxidized to quinones (Qs, e.g., 2',5'-Q and 3',4'-Q). This raises the question whether the mutagenic potential of PCB3 is due to the mutagenicity of PCB3 itself or of one of the metabolites. In this study, we investigated the mutagenicity of PCB3, of the monohydroxylated metabolites 2'-hydroxy (HO)-, 3'-HO-, and 4'-HO, of the HQs 3',4'-HQ and 2',5'-HQ and of the Qs 3',4'-Q and 2',5'-Q in cultured Chinese hamster V79 cells. The induction of gene mutations was determined at the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene locus by selection with 6-thioguanine. The induction of chromosome and genome mutations was assessed using the micronucleus assay and immunochemical differentiation of micronuclei containing whole chromosomes (kinetochore positive) and DNA fragments (kinetochore negative). The induction of chromosome and genome mutations, detected as micronuclei, was only observed at higher, cytotoxic concentrations of monohydroxylated, catecholic, and quinoid metabolites of PCB3. However, both PCB3-Qs induced a significant increase in the mutant frequency of the hprt gene and did so at submicromolar concentrations. Thus, the present study demonstrates for the first time the mutagenicity of PCB3 metabolites in mammalian cells and identifies quinoid metabolites of PCB3 as potential ultimate mutagens.

Ultrafine Quartz-Induced Damage in Human Lymphoblastoid Cells in vitro Using Three Genetic Damage End-Points

ABSTRACTRespirable quartz is a potential human lung carcinogen. The mechanisms involved in this carcinogenesis, however, remain unclear, especially for the ultrafine particles (diameter <100 nm). The aim of the present study was to investigate the effects caused by ultrafine quartz (UF-quartz) in a human cell culture model. Genotoxicity and cytotoxicity induced by UF-quartz were investigated through the cytokinesis block micronucleus assay (CBMN), the Comet assay, the HPRT assay, the population growth assay, and the 3-(4, 5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. WIL2-NS cells were incubated for 10h with 0, 60, and 120 μg/mL UF-quartz. Significant decreases in percent of cell survival in the MTT assay were seen at higher doses, for example, 83%, and 64% relative survival at 60 μg/mL and 120 μg/mL, respectively. Only slight population regrowth was observed, with the population sizes recovering slightly by day 4 after quartz particles were removed. A significant increase in the frequency of micronucleated binucleated cells (MNed BNCs) was seen with 120 μg/mL quartz, from approximately 5 in 1000 BNCs in controls to 12 in 1000 BNCs. A significant reduction in the nuclear division index was observed by the CBMN assay, indicating inhibition of cell division by high-dose UF-quartz. A dose-dependent increase in induced HPRT-gene locus mutant frequency with increasing dose of UF-quartz was observed by the HPRT assay. No significant difference was found in DNA strand breakage as detected by the Comet assay. Collective findings suggest that UF-quartz can cause cytotoxicity and genotoxicity to human lymphoblasts in this model system.

Further examination of the effects of nitrosylation on Alternaria alternata mycotoxin mutagenicity in vitro

Previously, Alternaria extract and metabolite mutagenicities ± nitrosylation were characterized using Ames Salmonella strains TA98 and TA100, which are both reverted at GC sites. To examine other targets for mutation, the metabolites Altertoxin I (ATX I), Altenuene (ALT), Alternariol (AOH), Alternariol monomethyl ether (AME), Tentoxin (TENT), Tenuazonic acid (TA) and Radicinin (RAD) were reexamined ± nitrosylation, using Ames Salmonella strain TA97, sensitive to frameshift mutations at a run of C's, as well as strains TA102 and TA104, reverted by base pair mutations at AT sites and more sensitive to oxidative damage. ATX I was also assessed for mammalian mutagenicity at the Hprt gene locus in Chinese hamster V79 lung fibroblasts and rat hepatoma H4IIE cells. When tested from 1 to 100 μg/plate without nitrosylation, ATX I was mutagenic in TA102 ± rat liver S9 for activation and weakly mutagenic in TA104 ± S9, demonstrating direct-acting AT base pair mutagenicity. AOH was also directly mutagenic at AT sites in TA102 ± S9 while AME was weakly mutagenic in TA102 ± S9 and TA104 + S9. Nitrosylation of ATX I enhanced mutagenicity at AT sites in TA104 ± S9 but produced little change in TA102 ± S9 compared to native ATX I. However, nitrosylated ATX I generated a potent direct-acting frameshift mutagen at C sites in TA97 ± S9. While ATX I was not mutagenic in either V79 cells or H4IIE cells, 5 and 10 μg/ml nitrosylated ATX I produced a doubling of 6-thioguanine resistant V79 colonies and 0.5 and 1 μg/ml were mutagenic to H4IIE cells, becoming toxic at higher concentrations. These results suggest ATX I, AME and AOH induce mutations at AT sites, possibly through oxidative damage, with nitrosylation enhancing ATX I frameshift mutagenicity at runs of C's. Nitrosylated ATX I was also directly mutagenic in mammalian test systems.

Molecular, biochemical, and genetic characterization of a female patient with Lesch–Nyhan disease

Lesch–Nyhan disease (LND) is a rare X-linked recessive disorder caused by virtually complete deficiency of activity of the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8). Human HPRT is encoded by a single structural gene located on the long arm of the X-chromosome (Xq26). The classical LND phenotype occurs almost exclusively in males, manifested in excessive purine production and characteristic neurological manifestations, including compulsive self-mutilation, choreoathetosis, spasticity, and occasionally developmental delay. Heterozygous females are usually phenotypically normal, due to the random inactivation of the X chromosome (Lyonization mechanism). However, six females were reported to be affected with the full biochemical and clinical manifestations of LND. All these cases were heterozygous for an HPRT mutation. Absence of transcription of the normal HPRT allele was attributed in all of them to non-random inactivation of the X chromosome carrying the normal allele. Here we describe an additional LND female, who presented with acute renal failure at the age of two months, in whom absence of transcription of the two HPRT alleles occurred due to as yet undescribed mechanism in LND females: the transcription of one HPRT allele was blocked due to a de novo X chromosome–autosome translocation 46,XX,t(X:2)(q26:p25), with a breaking point encompassing the HPRT gene locus, whereas the transcription of the normal allele was inhibited due to non-random inactivation of the second X-chromosome. Cultured fibroblasts from this patient exhibited the biochemical alterations in purine nucleotide metabolism characteristic of male LND fibroblasts.

Differential regulation of the von Willebrand factor and Flt-1 promoters in the endothelium of hypoxanthine phosphoribosyltransferase–targeted mice

An important limitation of standard transgenic assays is that multiple copies of the transgene are inserted randomly into the mouse genome, resulting in line-to-line variation in expression. One way to control for these variables is to target a single copy of the transgene to a defined locus of the mouse genome by homologous recombination. In the present study, we have used such an approach to target the promoters of 2 different genes, namely von Willebrand factor (VWF) and Flt-1, to the hypoxanthine phosphoribosyltransferase (Hprt) gene locus. Consistent with previous findings in standard transgenic animals, we report that the VWF promoter contains information for expression in a subset of endothelial cells in the heart, skeletal muscle, and brain. In contrast, the Flt-1 promoter directs expression in all vascular beds except for the liver. The Flt-1 transgene was active in the endothelium of tumor xenografts, whereas the VWF promoter was not. Under in vitro conditions, conditioned medium from tumor cells resulted in a significant up-regulation of Flt-1 mRNA and promoter activity, but no change in VWF levels. Taken together, these results suggest that (1) Hprt locus targeting is a valuable tool for studying vascular bed-specific gene regulation, (2) the VWF and Flt-1 promoters are regulated by distinct transcriptional mechanisms in the intact endothelium, and (3) tumor angiogenesis results in the differential activation of endothelial cell-specific promoters.

Measuring gene-specific nucleotide excision repair in human cells using quantitative amplification of long targets from nanogram quantities of DNA

We have been developing a rapid and convenient assay for the measurement of DNA damage and repair in specific genes using quantitative polymerase chain reaction (QPCR) methodology. Since the sensitivity of this assay is limited to the size of the DNA amplification fragment, conditions have been found for the quantitative generation of PCR fragments from human genomic DNA in the range of 6–24 kb in length. These fragments include: (1) a 16.2 kb product from the mitochondrial genome; (2) 6.2, 10.4 kb, and 15.4 kb products from the hprt gene, and (3) 13.5, 17.7, 24.2 kb products from the human β-globin gene cluster. Exposure of SV40 transformed human fibroblasts to increasing fluences of ultraviolet light (UV) resulted in the linear production of photoproducts with 10 J/m2 of UVC producing 0.085 and 0.079 lesions/kb in the hprt gene and the β-globin gene cluster, respectively. Kinetic analysis of repair following 10 J/m2 of UVC exposure indicated that the time necessary for the removal of 50% of the photoproducts, in the hprt gene and β-globin gene cluster was 7.8 and 24.2 h, respectively. Studies using lymphoblastoid cell lines show very little repair in XPA cells in both the hprt gene and β-globin locus. Preferential repair in the hprt gene was detected in XPC cells. Cisplatin lesions were also detected using this method and showed slower rates of repair than UV-induced photoproducts. These data indicate that the use of long targets in the gene-specific QPCR assay allows the measurement of biologically relevant lesion frequencies in 5–30 ng of genomic DNA. This assay will be useful for the measurement of human exposure to genotoxic agents and the determination of human repair capacity.

Targeting the Hprt locus in mice reveals differential regulation of Tie2 gene expression in the endothelium.

To study the in vivo expression of the murine Tie2 gene, we have targeted the hypoxanthine phosphoribosyltransferase (Hprt) gene locus to generate two single-copy transgenic mice: T1, containing the 2,100-bp Tie2 promoter upstream from the beta-galactosidase (LacZ) gene, and T5, which also included an enhancing element originating from the first intron of the Tie2 gene. Comparing T1 and T5 embryos at day E10.5 revealed differential endothelial cell-specific expression of LacZ, whereas colocalization analyses showed that the expression was confined to endothelial cells. Moderate reporter gene activity was observed in the brain and kidney of T1 adults, whereas extensive LacZ gene expression was seen in the vasculature of most organs of the T5 adults. This study demonstrates the feasibility of targeting the Hprt locus with endothelial cell-specific sequences to analyze the spatial-temporal expression of transgenes. Of particular importance is the observation that the analysis of a single transgene copy in a defined locus allows for an accurate and rapid comparison of transcriptional activity among regulatory DNA sequences.

Genetic Instability and the Etiology of Somatic PIG-A Mutations in Paroxysmal Nocturnal Hemoglobinuria

ABSTRACT: Paroxysmal nocturnal hemoglobinuria (PNH) is a hematologic disorder characterized by acquired PIG-A gene mutations that lead to defective bioassembly of glycosylphosphatidylinositol (GPI) anchors and the absence of GPI-linked surface proteins. As the etiology of these acquired PIG-A gene mutations is unknown, we hypothesized that patients with PNH have overall genetic instability and acquire somatic mutations throughout their genome. We first analyzed microsatellite sequences and found equivalent size variation using DNA from GPI-negative granulocytes compared with the DNA of paired GPI-positive B cell lines or normal granulocytes. We next quantitated the frequency of mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) gene locus, and found 1 PNH patient with a large increase in hprt- mutant frequency (256.7 x 10-6 vs. 27.8 ± 19.9 x 10-6 for normal adults) that was confirmed on 4 independent blood samples. We also quantitated “illegitimate” VDJ genetic recombination events between the T cell receptor Vγ and J β gene loci, and found a second PNH patient with a large increase (43.5 events per μg of DNA vs. 1.3 ± 0.8 events per μg of DNA for normal adults), confirmed on 4 independent DNA samples. Both of these PNH patients are young females with no history of aplastic anemia. Our data show that PNH patients can have increased numbers of acquired somatic mutations in gene loci distinct from PIG-A. These data suggest that genetic instability may be associated with the development of PIG-A mutations that lead to the clinical picture of PNH.

Studies on the genotoxicity of the mammalian lignans enterolactone and enterodiol and their metabolic precursors at various endpoints in vitro

The mammalian lignans enterolactone (ENL) and enterodiol (END) are formed by intestinal bacteria from the plant lignans matairesinol (MAT) and secoisolariciresinol (SEC), respectively, which are ingested with different types of food. ENL and END are weak estrogens. According to epidemiological and biochemical studies, lignans may act as anticarcinogens, but little is known about their genotoxic potential. We have therefore investigated the effects of ENL, END, MAT and SEC on cell-free microtubule assembly and at the following genetic endpoints in cultured male Chinese hamster V79 cells: disruption of the cytoplasmic microtubule complex, induction of mitotic arrest, induction of micronuclei and their characterization by CREST staining, and mutagenicity at the HPRT gene locus. The lignans were tested at concentrations of 200 μM in the cell-free system and 100 μM in cultured cells, which represents the limit of solubility in each assay. The established aneuploidogen diethylstilbestrol and the clastogen 4-nitroquinoline- N-oxide were used as positive reference compounds. As none of the four lignans had any activity at the endpoints studied, we conclude that ENL, END, MAT and SEC are devoid of aneuploidogenic and clastogenic potential under the experimental conditions used in this study.

Prenatal diagnosis of HPRT mutant genes in Lesch-Nyhan syndrome.

Lesch-Nyhan syndrome is an X-linked recessive disorder characterized by hyperuricemia, physical and mental retardation, choreoathetosis, and compulsive self-mutilation.1 This disease is associated with the complete absence of activity of an enzyme involved in purine metabolism, hypoxanthine guanine phosphoribosyltransferase (HPRT, EC 2.4.2.8).2 Partial HPRT deficiency generally presents as gout and uric acid overproduction in early adulthood.3 The marked genetic heterogeneity of HPRT deficiency is well known. As reviewed previously by Sculley et al.,4 many different mutations at the HPRT gene locus (deletions, insertions, duplications, abnormal splicing, and point mutations at different sites of the coding region from exon 1 to 9) have been reported. We have identified Japanese and Korean HPRT mutants,5–12 including two rare cases in female subjects,5,9 by polymerase chain reaction of reverse-transcribed mRNA (RT-PCR) and the multiplex amplification technique of all nine HPRT exons from the genomic DNA coupled with direct sequencing. In this study, we report prenatal diagnoses of HPRT mutant genes which have been carried out in five Lesch-Nyhan families.

In vivo antioxidant status, DNA damage, mutation and DNA repair capacity in cultured lymphocytes from healthy 75- to 80-year-old humans

The accumulation of damage to cellular biomolecules, including DNA, over time may play a significant role in the aetiology of the ageing process. We have previously quantified DNA damage and mutation within cultured lymphocytes from healthy human male subjects in three different age groups (35–39, 50–54 and 65–69 years). The results of that study showed an age-related increase in DNA damage and mutations in lymphocytes. In addition, an age-related decrease in the capacity of the lymphocytes to repair H 2O 2-induced DNA damage was found. In this article, we report the findings of an extension to the earlier study. Thirty-one generally healthy male and female subjects between the ages of 75 and 80 years were recruited. Using a number of bioassays, we were able to determine; basal levels of DNA damage (for 18 subjects) and mutant frequency at the hypoxanthine phosphoribosyltransferase ( hprt) gene locus (for 16 subjects) within cultured lymphocytes. In addition, in vivo antioxidant status (for all study subjects) and the capacity of lymphocytes to repair H 2O 2-induced DNA damage (for 18 subjects) were also assessed. The results obtained showed: that the mean basal level of DNA damage in lymphocytes from subjects in the 75- to 80-year age group (12.6±4.7%) was similar to that of the 35- to 39-year age group (13.3±3.3%), p=0.42 (Mann–Whitney); there was no significant difference between log mean mutant frequency at the hprt gene locus in lymphocytes from the 75- to 80-year age group (0.31±0.33) compared to that observed in the 35- to 39-year age group (0.24±0.21; Student's t-test, t=0.68, p>0.05). Levels of the antioxidants glutathione peroxidase (GPx EC 1.11.1.9), catalase (CAT; EC 1.11.1.6) and caeruloplasmin (CPL; EC 1.16.3.1) were significantly elevated in the 75- to 80-year age group, compared to the 35- to 39-, 50- to 54- and 65- to 69-year age groups. Levels of bilirubin (BR) were reduced in the 75- to 80-year age group, the decrease being contributed by the female subjects. No differences in levels of superoxide dismutase (SOD; EC 1.15.1.1) or uric acid (UA) were found between the 4 age groups. Following treatment of lymphocytes with H 2O 2, we did not find any difference in the susceptibility of lymphocytes to DNA damage in the 75- to 80-year age group, compared to the other age groups. The DNA repair capacity in lymphocytes from individuals in the 75- to 80-year age group was similar to that of the 35- to 39-year age group, for all time points assessed. These results highlight the importance of DNA repair processes and antioxidant defence systems for maintaining genomic stability in vivo.

Efficient combination of large DNA in vitro: in gel site specific recombination (IGSSR) of PAC fragments containing alpha satellite DNA and the human HPRT gene locus.

In an attempt to combine a cloned genomic copy of a selectable gene with different cloned centromeric sequences to develop mammalian artificial chromosomes (MAC) we used site specific recombination mediated by purified Cre recombinase acting on the loxP sequence in PAC vector DNA. A new method was required to purify highly concentrated, virtually 100% intact PAC DNA which could be stored for a long period. Here we show the efficient linking of linearized PACs containing alpha satellite DNA from chromosomes X and 17 with sizes of 125 and 140 kb, respectively, to a 95 kb restriction fragment derived from a 175 kb PAC containing the intact human HPRT gene locus.

Prenatal diagnosis of HPRT mutant genes in Lesch-Nyhan syndrome

Lesch-Nyhan syndrome is an X-linked recessive disorder characterized by hyperuricemia, physical and mental retardation, choreoathetosis, and compulsive self-mutilation.1 This disease is associated with the complete absence of activity of an enzyme involved in purine metabolism, hypoxanthine guanine phosphoribosyltransferase (HPRT, EC 2.4.2.8).2 Partial HPRT deficiency generally presents as gout and uric acid overproduction in early adulthood.3 The marked genetic heterogeneity of HPRT deficiency is well known. As reviewed previously by Sculley et al.,4 many different mutations at the HPRT gene locus (deletions, insertions, duplications, abnormal splicing, and point mutations at different sites of the coding region from exon 1 to 9) have been reported. We have identified Japanese and Korean HPRT mutants,5–12 including two rare cases in female subjects,5,9 by polymerase chain reaction of reverse-transcribed mRNA (RT-PCR) and the multiplex amplification technique of all nine HPRT exons from the genomic DNA coupled with direct sequencing. In this study, we report prenatal diagnoses of HPRT mutant genes which have been carried out in five Lesch-Nyhan families.

Mutation induction in γ-irradiated primary human bronchial epithelial cells and molecular analysis of the HPRT − mutants

We have examined various radiobiological parameters using commercially-available primary normal human bronchial epithelial (NHBE) cells, which can be subcultured more than 20 population doublings, and have established the mutation system in order to characterize the molecular changes in γ-irradiated primary cells. The survival curve, obtained after irradiation of cells with 137Cs γ-rays, indicates that the D 0, D q, and n values are 1.34 Gy, 1.12 Gy, and 2.3, respectively. The induction of HPRT − mutation was dose-dependent and the mutant fraction increased in a non-linear fashion. Since the doubling number of NHBE cells is limited, DNA was extracted directly from the single mutant colonies and alteration in the HPRT gene locus was analyzed using multiplex PCR technique. Among spontaneous mutants, the proportion with total and partial deletions of the gene was 10.0% ( 2 20 ) and 60.0% ( 12 20 ), respectively, while 30.0% ( 6 20 ) did not have any detectable changes in the nine exons examined. On the other hand, the fraction of total deletion increased by more than 2-fold among mutants induced by γ-rays in that 26.3% ( 10 38 ) of them showed the total gene deletions. Twenty-five out of 38 γ-induced mutants (65.8%) had partial deletions and 3 mutants (7.9%) had no detectable alteration. The present results showed that γ-irradiation efficiently induced HPRT gene mutation in primary human epithelial cells and that most of the induced mutants suffered larger deletions compared to that observed in spontaneous mutants. This system provides an useful tool for determination of mutagenicity and understanding the molecular mechanisms of environmental carcinogens in primary human bronchial cells.

Sequencing reactions for ALF (EMBL) automated DNA sequencer. European Molecular Biology Laboratory.

Automated fluorescent DNA sequencing (, , ) has turned into a subject of great interest during the last few years, and has successfully been applied to large-scale sequencing projects, like sequencing of the human HPRT gene locus (). Most actual genome sequencing efforts, such as sequencing the C. elegans genome, are performed on automated devices.

Mutation screenig of bleomycin-induced V79 Chinese hamster hprt mutants using multiplex polymerse chain reaction

We have shown by the filter hybridization technique that bleomycin (BLM) induces different types of mutations at the hprt gene locus of V79 Chinese hamster cells. DNA of mutants identified by Southern blots as partial deletions was subjected to further analysis using multiplex polymerase chain reaction (PCR) to localize the endpoints of the deletions over the hprt gene. The PCR analysis revealed that deletions occur in all parts of the hprt gene but are distributed non-randomly. Deletions occurred most frequently at the 3'-end of the hprt gene suggesting a possible existence of a hot spot for deletions in this region; exons 1, 2 and 3 appeared to be less affected by deletions. As PCR can detect microdeletions which are below the limit of resolution of Southern blot hybridization we analysed 25 HPRT- mutants with Southern wild-type pattern to distinguish between point mutations and small deletions. Of these HPRT- mutants, all except five showed PCR amplification products identical to that of V79 wild-type cells. These results are consistent with previous Southern analyses indicating that a large portion of BLM-induced HPRT- mutants are real point mutations. Five mutants, however, showed differences in fragment sizes of single PCR products or did not yield one single exon fragment and thus are probably the result of deletions which were not to be detected by Southern analyses.