Strand specificity for mutations induced by (+)-anti BPDE in the hprt gene in human T-lymphocytes.

Mutations in the hprt gene in T-lymphocyte clones isolated from primary cultures treated with the (+)-anti enantiomer of 7,8-dihydroxy-9,10-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene (BPDE) in vitro, and from untreated control cultures, were characterized using polymerase chain reaction and direct sequencing of hprt cDNA and genomic fragments. The spectrum of BPDE-induced mutations was very specific and clearly different from the background spectrum, which comprised many different types of mutations. Of the BPDE-induced mutations, 20/22 were transversions of GC base pairs and 18/22 were GC greater than TA transversions, which is in agreement with what has been found in other mammalian systems. While no particular 'hotspot' was observed for BPDE in the hprt gene, a sequence context specificity was detected. Ten of the 14 BPDE-induced mutations in the coding region were located in the sequence context AGG, and 2 in AG dinucleotides, which indicates that such sequences are sensitive to BPDE mutagenesis. Nine of the 22 BPDE-induced mutations and 2/12 background point mutations caused mRNA splicing errors. Six of the BPDE-induced splicing errors were caused by GC greater than TA transversions in the AG dinucleotide of different splice acceptor sites, which indicates that these sites may be frequent targets of BPDE mutagenesis. All mutated GC base pairs in the BPDE-induced spectrum were oriented so that the guanine was located on the non-transcribed strand. Assuming that the premutagenic lesion in these cases was covalent binding of BPDE to guanine and that BPDE bound randomly to both strands, the strand specificity of the BPDE-induced mutations indicates that preferential excision repair of BPDE adducts on the transcribed strand occurs in the hprt gene in human T-cells.     

Use of T-cell receptor gene probes to quantify the in vivo hprt mutations in human T-lymphocytes

T-cell receptor (Ti) gene restriction fragment patterns (RFPs) were determined by Southern blots of genomic DNA obtained from T-lymphocyte colonies isolated from a single normal individual. 4 wild-type colonies and 11 in vivo derived 6-thioguanine-resistant mutant colonies with previously characterized hprt gene structural alterations were studied. Among the hprt mutants, 10 of the 11 showed unique Ti RFPs indicating their origins in different in vivo progenitors. Unique Ti RFPs were also seen among the wild-type T-cell colonies. One, however, shared its Ti RFP with a mutant. These results suggest that mutation in vivo of the hprt gene in human T-lymphocytes occurred after thymic maturation and that the 11 recovered hprt mutants probably resulted from 11 independent mutational events.     

Southern-blot analyses of human T-lymphocyte mutants induced in vitro by gamma-irradiation

G0 phase cultures of human peripheral blood T-lymphocytes from a single individual were exposed to 300 rad of gamma-irradiation from a 137Cs source and cultured in vitro for 8 days to allow phenotypic expression. Thioguanine-resistant (TGr) mutants were isolated by a cell cloning assay in microtiter plates. These mutants were studied by Southern blot analysis to define the gross structural alterations in the hypoxanthine-guanine phosphoribosyl transferase (hprt) gene by use of an hprt cDNA probe. A similar analysis of the T-cell receptor (TCR) gene rearrangement patterns was employed to define the independent nature of each mutant colony by use of TCR beta and gamma cDNA probes. 74 mutants were isolated in 5 separate experiments. TCR gene rearrangement analysis showed these to represent 24 independent mutations, of which 18 contained hprt structural alterations. These alterations included simple deletions (10/18) as well as more complex rearrangements resulting in molecular weight changes of restriction fragments representing both the 5' and 3' regions of the hprt gene (4/18 and 4/18, respectively). These results demonstrate that gamma-irradiation primarily induces TGr mutations through gross structural alterations in the hprt gene and that these alterations are randomly distributed across the gene. This approach to mutation analysis will provide information on the types of alterations induced by this irradiation, especially the extent of deletions involving the hprt gene. These results also demonstrate the feasibility of employing in vitro exposure of human T-lymphocytes to a single mutagenic agent as an aid to understanding the mechanisms of mutations occurring in vivo in humans.     

Analysis of X-ray-induced HPRT mutations in CHO cells: insertion and deletions.

Molecular alterations were examined in the hypoxanthine guanine phosphoribosyltransferase (hprt) gene of 41 independent X-ray-induced thioguanine-resistant (TGR) Chinese hamster ovary (CHO) cell clones. Rapid screening of the clones by multiplex polymerase chain reaction (PCR) for the presence or absence of exons revealed that the causes of the mutant phenotype were total gene deletion (26/41), partial gene deletion (4/41), and an insertion (1/41). No alterations of exon number or sizes were apparent in 10 of the mutants. Southern blot analysis confirmed the deletion data and revealed an additional class of mutants that had a gene disruption but retained all hprt exons (2/41). Therefore, at least 80% of the ionizing radiation-induced mutations were due to mechanisms involving DNA breakage and rejoining. The distribution of deletion sizes suggests that the two DNA breaks required for a deletion are not independent events. A possible mechanism is presented. In addition, the DNA sequence of the insertion mutation was determined. The insertion (229 bp) is coupled with a deletion (31 bp). An imperfect inverted repeat with flanking hprt DNA was identified and may be involved in the insertion event.     

Molecular analysis of spontaneous and ethyl methanesulphonate-induced mutations of the hprt gene in hamster cells

Independent spontaneous or ethyl methanesulphonate (EMS)-induced mutants lacking HPRT enzyme activity were analysed for changes in hprt gene structure. Of 21 spontaneous mutants, 6 had total gene deletions, 2 had partial gene deletions, and 13 were indistinguishable from wild-type by Southern analysis. In contrast a sample of 23 EMS-induced mutants, each of which showed potentially interesting characteristics (e.g. high reversion frequency, X-chromosome rearrangement), showed no detectable hprt gene changes. RNA isolated from 59 mutants with presumptive point mutations (13 spontaneous, 46 EMS-induced) was analysed on dot blots for changes in the amount of hprt mRNA. A wide range of mRNA levels was found, from mutants with undetectable amounts to those with more than wild-type amounts. However, Northern blots of all these mutant RNAs revealed only one (EMS-induced) mutation with a change in hprt mRNA size. Taken with our previously-published data on these mutants, it is argued that they represent a broad range of mutational types, and that the hprt gene mutation system provides a sensitive means of distinguishing mutational spectra of different DNA-damaging agents.     

Mutational spectra in human B-cells. Spontaneous, oxygen and hydrogen peroxide-induced mutations at the hprt gene.

To test the hypothesis that reactive species in the oxygen cascade are responsible for spontaneous mutation, we examined the spectra of oxygen and hydrogen peroxide-induced mutations at the hprt locus in a human B-lymphoblastoid cell line. We compared these spectra with the spontaneous mutational spectrum. Large gene alterations were studied by Southern analysis of individual TGR clones. A combination of high fidelity polymerase chain reaction, denaturing gradient gel electrophoresis and direct DNA sequencing were used to detect and identify point mutations in exon 3 of hprt. With regard to spontaneous mutations, a previous study showed that 39% of the spontaneous TGR clones had large gene alterations. In the present study, the analysis of spontaneous point mutations within exon 3 revealed two hotspots. A one base-pair deletion (-A) at base-pair 256 or 257 and a two base-pair deletion (-GG) at base-pair 237 and 238, were detected in triplicate cultures. Each of the hotspots comprised about 1% of the TGR mutants. The analysis of individual oxygen-induced TGR clones (48 h, 910 microM-O2) showed 43% had large gene alterations similar to the spontaneous TGR clones. However, none of the spontaneous point mutation hotspots was found among triplicate oxygen-treated cultures. Two point mutations in common with H2O2-treated cultures were found in one of the three oxygen-treated cultures. Hydrogen peroxide-induced mutations (1 h, 20 microM) also differed from spontaneous mutations. Only 24% of the hydrogen peroxide-induced TGR clones had large gene alterations. The analysis of point mutations showed three hotspots within exon 3 of hprt. An AT to TA transversion at base-pair 259 had an average frequency of 3% of all TGR mutants (present in all of 3 H2O2-treated cultures). Two GC to CG transversions at base-pairs 243 and 202 were present at a frequency of 0.6% and 0.4%, respectively. A five base-pair deletion (base-pair 274 to 278) was present at an average frequency of 0.3%. The latter three mutations were detected in two of three H2O2-treated cultures. Thus, the point mutation spectra of both oxygen and hydrogen peroxide were significantly different from the spontaneous spectrum. The oxygen and hydrogen peroxide-induced spectra shared some features, suggesting that oxygen and hydrogen peroxide share some but not all pathways for induction of mutations within the DNA sequence studied here.(ABSTRACT TRUNCATED AT 400 WORDS)     

Molecular characterisation of camptothecin-induced mutations at the hprt locus in Chinese hamster cells

The capacity of the topoisomerase I inhibitor camptothecin (CPT) to induce single locus mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene and the DNA changes underlying induced mutations were analysed in Chinese hamster ovary cells. Camptothecin treatments increased hprt mutations up to 50-fold over the spontaneous levels at highly cytotoxic doses. Genomic DNA was isolated from 6-thioguanine resistant clones and subjected to multiplex PCR to screen for gross alterations in the gene structure. The molecular analysis revealed that deletion mutants represented 80% of the analysed clones, including total hprt deletion, multiple and single exon deletions. Furthermore, a fraction of the analysed clones showed deletions of more than one exon that were characterised by the absence of non-contiguous exons. These data show that single locus mutations induced by camptothecin are characterised by large deletions or complex rearrangements rather than single base substitutions and suggest that the recombinational repair of camptothecin-induced strand breaks at replication fork may be involved in the generations of these alterations at the chromatin structure level.     

Induction of mutations by bismuth-212 alpha particles at two genetic loci in human B-lymphoblasts.

The human lymphoblast cell line TK6 was exposed to the alpha-particle-emitting radon daughter 212Bi by adding DTPA-chelated 212Bi directly to the cell suspension. Cytotoxicity and mutagenicity at two genetic loci were measured, and the molecular nature of mutant clones was studied by Southern blot analysis. Induced mutant fractions were 2.5 x 10(-5)/Gy at the hprt locus and 3.75 x 10(-5)/Gy at the tk locus. Molecular analysis of HPRT- mutant DNAs showed a high frequency (69%) of clones with partial or full deletions of the hprt gene among radiation-induced mutants compared with spontaneous mutants (31%). Chi-squared analyses of mutational spectra show a significant difference (P < or = 0.005) between spontaneous mutants and alpha-particle-induced mutants. Comparison with published studies of accelerator-produced heavy-ion exposures of TK6 cells indicates that the induction of mutations at the hprt locus, and perhaps a subset of mutations at the tk locus, is a simple linear function of particle fluence regardless of the ion species or its LET.     

Fine structure mapping of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene region of the human X chromosome (Xq26).

The Xq26-q27 region of the X chromosome is interesting, as an unusually large number of genes and anonymous RFLP probes have been mapped in this area. A number of studies have used classical linkage analysis in families to map this region. Here, we use mutant human T-lymphocyte clones known to be deleted for all or part of the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene, to order anonymous probes known to map to Xq26. Fifty-seven T-cell clones were studied, including 44 derived from in vivo mutation and 13 from in vitro irradiated T-lymphocyte cultures. Twenty anonymous probes (DXS10, DXS11, DXS19, DXS37, DXS42, DXS51, DXS53, DXS59, DXS79, DXS86, DXS92, DXS99, DXS100d, DXS102, DXS107, DXS144, DXS172, DXS174, DXS177, and DNF1) were tested for codeletion with the hprt gene by Southern blotting methods. Five of these probes (DXS10, DXS53, DXS79, DXS86 and DXS177) showed codeletion with hprt in some mutants. The mutants established the following unambiguous ordering of the probes relative to the hprt gene: DXS53-DXS79-5'hprt3'-DXS86-DXS10-DXS177 . The centromere appears to map proximal to DXS53. These mappings order several closely linked but previously unordered probes. In addition, these studies indicate that rather large deletions of the functionally haploid X chromosome can occur while still retaining T-cell viability.     

Formaldehyde-induced and spontaneous alterations in human hprt DNA sequence and mRNA expression

Human lymphoblast mutants at the X-linked hprt locus have been examined by Southern blot, Northern blot and DNA sequence analysis. A previous study had shown that approximately a third of the spontaneously-arising mutants and half those induced by formaldehyde showed no alteration in restriction fragment pattern and thus were classified as point mutations. In this report, Northern blot analysis was used to show that these point mutants fall into 4 categories: normal size and amount of RNA, normal size but reduced amounts, reduced size of RNA or no RNA. Sequence analyses of cDNAs prepared from hprt mRNAs were performed on 1 spontaneous and 7 formaldehyde-induced mutants with normal Northern blots. The spontaneous mutant was caused by an AT----GC transition. 6 of the formaldehyde-induced mutants were base substitutions, all of which occurred at AT base-pairs. There was an apparent hot spot, in that 4/6 independent mutants were AT----CG transversions at one specific site. The remaining mutant had lost exon 8.     

Molecular analysis of spontaneous mutations at the gpt locus in Chinese hamster ovary (AS52) cells

AS52 cells are Chinese hamster ovary (CHO) cells that carry a single functional copy of the bacterial gpt gene and allow the isolation of 6-thioguanine-resistant (6TGr)mutants arising from mutation at the chromosally integrated gpt locus. The gpt locus in AS52 cells is extremely stable, giving rise to 6TGr mutants at frequencies comparable to the endogenous CHO hprt locus. In this study, we describe the spectrum of spontaneous mutations observed in AS52 cells by Southern blot and DNA sequence analyses. Using the polymerase chain reaction (PCR) and the Thermus aquaticus (Taq) polymerase, we have enzymatically amplified 6TGr mutant gpt sequences in vitro. The PCR product was then sequenced without further cloning manipulations to directly identify gpt structural gene mutations. Deletions predominant among the 62 spontaneous 6TGr-AS52 mutant clones analyzed in this study. Of these, 79% (49/62) of the mutations were identified as deletions either by Southern blotting, PCR amplification or DNA sequence analysis. Among these deletions is a predominant 3-base deletion that was observed in 31% (19/62) of the mutants. These data provide a basis for future comparisons of induced point mutational spectra derived in the AS52 cell line, and demonstrate the utility of PCR in the generation of DNA sequence spectra derived from chromosomally integrated mammalian loci.     

Molecular analysis of hprt mutants induced by 2-cyanoethylene oxide in human lymphoblastoid cells

The mutagenic epoxide metabolite of acrylonitrile, 2-cyanoethylene oxide (ANO), was used to treat human TK6 lymphoblasts (150 microM x 2 h ANO). A collection of hypoxanthine-phosphoribosyltransferase (hprt) mutants was isolated and characterized by dideoxy sequencing of cloned hprt cDNA. Base-pair substitution mutations in the hprt coding region were observed in 19/39 of hprt mutants: 11 occurred at AT base pairs and 8 at GC base pairs. Two -1 frameshift mutations involving GC bases were also observed. Approximately half (17/39) of the hprt mutants displayed the complete loss of single and multiple exons from hprt cDNA, as well as small deletions, some extending from exon/exon junctions. Southern blot analysis of 5 mutants with single exon losses revealed no visible alterations. Analysis of 1 mutant missing exons 3-6 in its hprt mRNA revealed a visible deletion in the corresponding region in its genomic DNA. The missing exon regions of 4 mutants (one each with exons 6, 7 and 8 loss and one mutant with a 17-base deletion of the 5' region of exon 9) were PCR amplified from genomic DNA and analyzed by Southern blot using exon-specific probes. The exons missing from the hprt mRNA were present in the genomic hprt sequence. DNA sequencing of the appropriate intron/exon regions of hprt genomic DNA from a mutant with exon 8 loss and a mutant exhibiting aberrant splicing in exon 9 revealed point mutations in the splice acceptor site of exon 8 (T----A) and exon 9 (A----G), respectively.     

Herpes simplex virus type 2 mutagenesis: characterization of mutants induced at the hprt locus of nonpermissive XC cells.

In a previous report, herpes simplex virus type 2 (HSV-2) was shown to increase the frequency of mutation at the hypoxanthine phosphoribosyltransferase (hprt) locus of nonpermissive rat XC cells (L. Pilon, A. Royal, and Y. Langelier, J. Gen. Virol. 66:259-265, 1985). A series of 17 independent mutants were isolated after viral infection together with 12 spontaneous noninfected mutants to characterize the nature of the mutations induced by the virus at the molecular level. The DNA of the mutants isolated after viral infection was probed with cloned HSV-2 fragments representing the entire genome. In these mutants, no authentic HSV-2 hybridization could be detected. This was indicative of a mechanism of mutagenesis which did not require the permanent integration of viral sequences in the host genome. The structure of the hprt gene was determined by the method of Southern (J. Mol. Biol. 98:503-517, 1975), and the level of hprt mRNA was analyzed by Northern blots. Except for the identification of one deletion mutant in each of the two groups, the HPRT- clones showed no evidence of alteration in their hprt gene. A total of 7 of 12 spontaneous mutants and 11 of 15 mutants isolated from the infected population transcribed an hprt mRNA of the same size and abundance as did the wild-type cells. Thus, the majority of the mutants seemed to have a point mutation in their hprt structural gene. Interestingly, the proportion of the different types of mutations was similar in the two groups of mutants. This analysis revealed that HSV-2 infection did not increase the frequency of rearrangements but rather that it probably induced a general increase of the level of mutations in the cells. This type of response is thought to be compatible with the biology of the virus, and the possible mechanisms by which HSV-2 induces somatic mutations in mammalian cells are discussed.     

Mutations affecting RNA splicing in man are detected more frequently in somatic than in germ cells

The spectrum of DNA sequence alterations in the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene of HPRTase-deficient T-lymphocytes isolated from the blood of healthy male donors was determined and compared with the spectrum found in patients suffering from genetic diseases (Lesch-Nyhan syndrome or gouty arthritis) associated with a mutation in the same gene. Most of the T-cell mutants still produced hprt mRNA which was converted into cDNA and used for DNA sequence analysis after amplification using the polymerase chain reaction (PCR). In 39% of the 31 analyzed T-cell mutants of normal donors 1 or 2 exons were completely or partially deleted from hprt mRNA, probably because of a mutation in a splice acceptor site. Among patients suffering from the Lesch-Nyhan syndrome or gouty arthritis, the class of splice mutations amounts only to 7%. These data suggest that carriers of splice mutations often do not show the characteristics of HPRTase deficiency associated with these genetic diseases, because correctly spliced hprt mRNA is still produced at a low level.     

Molecular characterization of hprt mutants induced by low- and hgh-LET radiations in human cells

Southern blotting techniques were employed to examine the spectrum of molecular alterations in DNA induced by internally emitting iodine isotopes and X-rays at and around the hprt locus in a human lymphoblastoid cell line. We analyzed 165 mutant clones using a cDNA probe for the human hprt locus, and 3 anonymous sequenceprobes for regions of the X-chromosome which are linked to hrpt. The results were compared with those for 35 spontaneously arising mutant clones. The majority of ionizing radiation-induced mutants showed changes in the normal restriction patterns at the hprt locus, whereas very few alterations were seen at linked markers along the X chromosome. Total hprt coding sequence deletions comprised 30–48% of the changes observed at this locus, while partial deletions and rearrangements comprised 14–54% of the observed changes. In the case of mutants induced by [^125I]dUrd, a densely ionizing radiation, the spectrum of alterations was dose-dependent; at low doses it was not significantly different from that seen after sparsely ionizing X-ray exposure, whereas a higher proportion of gene deletions and rearrangements occurred after high doses of this incorporated isotope. Changes were rarely observed in the 3 linked markers examined. Overall, these results indicate that the distribution of mutational events at the hprt locus in irradiated human cells may not only be LET-dependent but dose-dependent, and that deletions involving large regions of the X chromosome surrounding the hprt locus are rae events.     

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.     

The nature of mutants induced by ionising radiation in cultured hamster cells. III. Molecular characterization of HPRT-deficient mutants induced by gamma-rays or alpha-particles showing that the majority have deletions of all or part of the hprt gene

DNA from 58 independent HPRT-deficient mutants of V79 hamster cells induced by ionising radiation was analysed by Southern blot hybridization to a full-length hamster hprt cDNA. About half of the gamma-ray-induced mutants (20/43) were apparently total gene deletions, because they lacked all functional hprt gene sequences hybridizing to the cDNA probe. Another 10 mutants showed various partial deletions and/or rearrangements of the hprt gene. The remaining 13 mutants showed no detectable change in comparison to the structure of the normal gene, which correlated well with previous characterization of these mutants indicating that most carry point mutations in the hprt gene. However, it is probable that some of these point mutations occurred spontaneously rather than being radiation-induced. A smaller number of alpha-particle induced mutants gave similar results: out of a total of 15 mutants, 6 appeared to be total gene deletions, 5 had partial deletions and/or rearrangements, and 4 had no detectable changes. Thus, 70% or more of radiation-induced HPRT-deficient mutants arise through large genetic changes, especially deletions of all or part of the hprt gene. This result is to be contrasted with data published previously by ourselves and others indicating that the majority of spontaneous and ethyl methanesulphonate-induced mutations of hprt and similar genes arise by point mutation.     

Molecular analyses of in vivo hprt mutations in human T-lymphocytes. III. Longitudinal study of hprt gene structural alterations and T-cell clonal origins

The hprt clonal assay detects mutations occurring in vivo in the hypoxanthine-guanine phosphpribosyltransferase (hprt) gene of human T-lymphocytes. Analysis of 94 wild-type and 326 hprt mutant clones from 3 normal males was performed using Southern blotting with hprt and T-cell receptor (TCR) gene probes. Gross structural alterations of the hprt gene occurred in 14% of the in vivo derived mutants. Breakpoints were randomly distributed across the gene with one possible mutational “hot spot” observed. Most hprt mutants were independent as judge by TCR gene rearrangement patterns indicating that the measured hprt mutant frequency is a good measure of the actual hprt mutation frequency. However, sibling mutants (generally doublets and triplets except for one nonamer) were detected. Information on the timing in vivo of the hprt mutational events and the persistence in vivo of sibling mutants was also obtained.     

Molecular basis of X-ray-induced mutation at the HPRT locus in human lymphocytes

Human lymphocytes lacking functional HPRT enzyme after a dose of 300 rad X-radiation were cloned and the monoclonal populations expanded so that sufficient genomic DNA was obtained for Southern analysis. A total of 33 mutant clones were analysed. Wild-type clones showed no evidence of changes to the HPRT gene resolvable by Southern banding patterns whereas 17 of 33 mutant clones showed changes. The alterations observed included total gene deletions (3 clones) and partial gene deletions with or without the appearance of novel bands (12 clones). Two clones showed the appearance of novel bands only. There were no changes observed in 16 of the 33 mutant clones. Three clones showed changes inconsistent with deletion of portions of the gene. In these clones inversion seems to have been the most likely cause of the mutation. The spectrum of gene alterations following ionizing radiation appears different to that previously observed for spontaneous mutations. Consequently, ionizing radiation or radiomimetic agents would appear to be aetiologic, at the most, for only a minor proportion of in vivo somatic mutations.     

Hprt mutations and karyotype abnormalities in T-cell clones from healthy subjects and melphalan-treated ovarian carcinoma patients

In vivo mutations at the locus for hypoxanthine phosphoribosyl transferase (hprt) were studied in 6-thioguanine (TG)-resistant T-lymphocytes clones from healthy male and female subjects and ovarian carcinoma patients treated with melphalan. Southern blot analysis of 108 clones showed alterations in 14% (4/29) of the colnes from healthy males, 4.3% (2/47) of the clones from healthy females and 3.1% (1/32) of the clones from melphalan-treated patients. 2 of the 7 abnormal clones had a total deletion of the hprt gene; the others had partial deletions. Karyotype analysis of 82 clones revealed 1 clonal abnormality in 29 mutant clones from healthy males (3.6%). Loss or structural aberration of 1 X-chromosome occurred in 6% of the clones from healthy females. The frequency of karyotypic abnormalities (excluding those affecting one of the X-chromosomes) was significantly higher in clones from patients (37%) as compared to healthy females (5.9%). No aberration was found to affect the hprt locus at Xq27 in any of the 82 clones studied.