The efficiency of restriction endonuclease digests determined by PCR.
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Base Sequence↗
Biomedical subjects
J Thacker
Publications and source records attributed to J Thacker.
The genetic basis of resistance to ionising radiation damage in cultured mammalian cells.
To test the genetic similarity of independently-isolated hamster cell mutants sensitive to ionising radiation, these were fused in pairs and the hybrids exposed to X-rays. Some mutants (irs1, irs3, xrs-1, XR-1, BLM2) were found to complement all others tested for radiosensitivity in hybrids, and are therefore in separate genetic groups. The mutants irs2 and V-E5, both isolated from V79 cells, did not complement and therefore belong to the same group. Another pair, EM7 and irs1SF, formed hybrids with intermediate levels of survival between mutant and wild-type. However, the parental cells fused to irs1SF also showed intermediate sensitivity, suggesting a semi-dominant mutant phenotype rather than a lack of complementation. Crosses of some of these hamster mutants to the radiosensitive mouse mutant M10 showed clear complementation (irs1 x M10, irs2 x M10) but for others the complementation did not greatly exceed the sensitivity of one (irs3 x M10) or both mutants (XR-1 x M10). Taken with our previously-published data, these results show that there are at least 8 genetic groups determining resistance to ionising radiation damage in rodent cells.
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The rejoining of double-strand breaks in DNA by human cell extracts.
A double-strand DNA break was introduced at a specific site within the lacZ gene of plasmid pUC18 using one of several restriction enzymes, and the plasmid exposed to nuclear extracts from human cell lines. Physical rejoining of DNA was monitored by Southern analysis after gel separation, and the fidelity of rejoining by expression of the lacZ gene after bacterial transformation with the treated plasmid. Breaks at the SalI and EcoRI sites were rejoined by extracts to form circular monomers, but the efficiency of rejoining was much higher at the SalI site. Measurement of rejoining at several adjacent sites having different types of termini, consistently showed a range of efficiencies with 5' 4-base greater than 3' 4-base overhangs and 4-base greater than 2-base greater than no overhang. Similar efficiencies were found for nuclear extracts from transformed cell lines, both from a 'normal' individual and an ataxia-telangiectasia (A-T) patient, and from a non-transformed normal cell culture. In contrast at some sites, especially those with a low rejoin efficiency, the fidelity of rejoining was very much lower for the A-T extracts than for normal cell extracts. Mis-rejoining was, however, unrelated to rejoin efficiency at other sites, suggesting that factors such as the exact sequence at the break site on the molecule may also influence the fidelity of rejoining.
Base Sequence↗
Localization of deletion breakpoints in radiation-induced mutants of the hprt gene in hamster cells.
DNA was analysed from a large set of hamster hprt gene mutants, some induced by ionising radiations and others occurring naturally, to identify those with large alterations in part of the gene. DNA from these mutants was restricted further with different endonucleases and probed to establish the patterns of restriction fragments remaining. Of 15 mutants characterized, one showed a duplication of part of the 5' end of the gene, and the remainder showed deletions of various sizes. It was possible to approximately locate the breakpoints of the deletions by comparison of fragment patterns to a recently-established map of the hamster gene. The relatively small number of mutants examined precludes rigorous analysis of the distribution of breakpoints in the hprt gene, but taken with other recent studies of deletion mutagenesis it is suggested that non-random induction or selection of this type of mutation may occur.
Alpha Particles↗
DNA-break repair, radioresistance of DNA synthesis, and camptothecin sensitivity in the radiation-sensitive irs mutants: comparisons to ataxia-telangiectasia cells.
Induction and rejoining of DNA single-strand breaks (ssb) and double-strand breaks (dsb) after gamma-irradiation were measured, respectively, by alkaline and neutral sucrose gradient sedimentation methods. The radiosensitive mutants irs1, irs2, and irs3 showed no significant difference from wild-type V79 hamster cells in ability to rejoin either ssb or dsb, while the previously-described xrs-1 mutant showed the expected defect in rejoining dsb. The resistance of DNA synthesis to gamma-irradiation was measured in the 3 irs mutants and, for comparative purposes, in transformed human cell lines from normal and ataxia-telangiectasia (A-T) individuals. The irs2 mutant was found to be very similar in response to the A-T lines, showing a marked decrease in inhibition of DNA synthesis, compared to V79 cells, in both time-course and dose-response experiments. However, irs1 also had some decrease in inhibition at the higher doses used, while irs3 was similar to the wild-type V79 cells. Both irs1 and irs2 were found to be considerably more sensitive to the DNA topoisomerase I-inhibitor camptothecin, while irs3 was only slightly more sensitive than the parent V79 line. These data place the irs mutants in a similar category of radiosensitive phenotype to A-T cells, but we view this as only the beginning of a useful classification of this type of mutant. The irs2 mutant has the strongest links to A-T cells, through its sensitivity profile to DNA-damaging agents and radioresistant DNA synthesis, but irs1 in particular has other similarities to A-T.
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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.
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Fingerprinting cell lines: use of human hypervariable DNA probes to characterize mammalian cell cultures.
Hypervariable DNA sequences may be used as probes to derive DNA "finger-prints" for individuals. To assess the use of the human 33.15 and 33.6 probes (isolated by Jeffreys and coworkers) for characterizing cell lines of nonhuman origin, DNA from different stocks of Chinese hamster (CH) cells was screened. All CHO (ovary) sublines could be readily distinguished from CH-V79 sublines by their fingerprints, but where two stocks had been derived recently from the same line, their fingerprints were nearly identical. Similarly fingerprints of HPRT-deficient mutants derived from one cell stock were identical. A V79 x CHO fusion hybrid showed equal fingerprint band-sharing with each parent line, while early-passage diploid CH cells had a fingerprint closer to CHO than to V79. Thus these data introduce a simple means of typing cell lines to avoid cross-contamination, of checking cell hybrids, and of assessing the divergence of cell stocks from one another.
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Inactivation of DNA-mediated transformation of hamster cells by gamma-rays and deoxyribonuclease I.
DNA damage has been induced in the mammalian expression vector pSV2-gpt by irradiation with X-rays or treatment with deoxyribonuclease I (DNAase I) under controlled conditions in vitro. The biological effect of such treatment was assessed by stable gene expression in Chinese hamster ovary (CHO) cells using DNA-mediated gene transfer. Induction of DNA double-strand breaks (dsbs), resulting from the interaction of independently-induced single-strand breaks (ssbs) under the present conditions, was measured by agarose gel electrophoresis of the treated vector. The correlation between radiation-induced gene inactivation and dsb induction mediated by OH radicals suggests that a dsb in the gene is a major inactivating lesion in this system. Individual radiation-induced ssbs and nucleotide damage are produced much more frequently than dsbs under these conditions, but the majority of these lesions do not appear to inactivate the gpt gene. DNAase I treatment, giving only simple 5' P + 3' OH breaks in the vector DNA, gave a correlation of approximately 1.5 dsb in the gpt gene per inactivating event, confirming little repair of dsbs in this system. Inactivation of the gpt gene without appreciable formation of dsbs was found, however, when the vector was irradiated at high dose rate in the presence of the OH-radical scavenger KBr. The nature of non-break damage causing inactivation requires further study.
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Examination of vectors with two dominant, selectable genes for DNA repair and mutation studies in mammalian cells.
A series of vectors with two dominant selectable genes was constructed for repair and mutation studies following transfer into mammalian cells. The recombinant genes (SV-gpt and HSVtk-neo) were placed in different relative orientations and positions in the vectors. These variables were shown to affect transformation frequency of cells by the vectors especially where one of the genes had a relatively weak expression, modelled by truncating the promoter of the HSVtk-neo gene. The use of two-gene vectors to assess DNA repair was investigated by cutting the SV-gpt gene with a restriction endonuclease and monitoring correct rejoining by selecting for gene activity after transfer into various cell types. In such experiments, selection was first applied for the undamaged HSVtk-neo gene to eliminate transfer artefacts, followed by counterselection for the activity of the damaged SV-gpt gene. The measured frequency of correct rejoining of the damaged gene was found to vary both with the vector construct and with the recipient cell species (Chinese hamster V79 or human transformed fibroblasts). Despite this variation, correct rejoining was found to be consistently lower in radiosensitive (ataxia telangiectasia) human cells than in wild-type human cells, irrespective of the vector construct. In these experiments, some of the transformed cell colonies showed 'sectoring' on exposure to the counterselection, suggesting a slow determination of the fate of transferred DNA. For mutation studies a V79 cell clone carrying a single copy of one of these two-gene vectors was identified and shown to be stably integrated. Mutations of the SV-gpt gene in these cells were isolated while maintaining selection for the HSVtk-neo gene, to attempt to limit mutational loss of the total integrated sequence and provide at least one identifiable junction for analysis of deletion events. Spontaneous and X-ray-induced mutants were identified with a variety of genetic changes, as shown by Southern analysis, from presumed point mutations to deletions and rearrangements of the vector sequence. Rescue of integrated two-gene vector sequences from transformed cells, by recloning in E. coli, was shown to be feasible; thus alterations in transferred DNA can be analysed in detail.
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Six complementation groups for ionising-radiation sensitivity in Chinese hamster cells.
The ionising radiation-sensitive mutants irs 1, irs 2, irs 3, xrs-1 (or xrs-7), EM7 and XR-1 were fused to wild-type cells or to each other in pairs to create hybrid cells. These hybrids were checked chromosomally and their X-ray sensitivity tested. Each mutant was found to be recessive to wild-type and to complement the X-ray sensitivity of the other mutants. Thus there appear to be at least 6 complementation groups for ionising radiation sensitivity in Chinese hamster cells.
Animals↗
Reaching towards 2000: development and evaluation of a community education program on diet and cancer prevention.
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Attitude to Health↗
Isolation and cross-sensitivity of X-ray-sensitive mutants of V79-4 hamster cells.
The V79-4 Chinese hamster line was mutagenized and surviving clones screened for X-ray sensitivity using a replica microwell technique. One slightly sensitive clone and 3 clearly sensitive clones were isolated from approximately 5000 screened, and designated irs 1 to irs 4. The 3 more sensitive clones showed different responses to the genotoxic agents mitomycin C (MMC), ethyl methanesulphonate (EMS) and ultraviolet light (UV). irs 1 showed considerable sensitivity to all the agents tested, in the order MMC much greater than EMS greater than UV. irs 2 and irs 3 had similar sensitivities to EMS and to UV (EMS greater than UV) but irs 3 was more sensitive than irs 2 to MMC. None of these mutants is identical in phenotype to previously published mutants.
Animals↗
Gene recombination in X-ray-sensitive hamster cells.
Recombination was measured in Chinese hamster ovary (CHO-K1) cells and in the X-ray-sensitive mutants xrs1 and xrs7, which show a defect in DNA double-strand break repair. To assay recombination, pairs of derivatives of the plasmid pSV2gpt were constructed with nonoverlapping deletions in the gpt gene region and cotransferred into the different cell types. Recombination efficiencies, measured as the transformation frequency with a pair of deletion plasmids relative to that with the complete pSV2gpt plasmid, were about 6% in both CHO-K1 and the xrs mutants for plasmids linearized at a site outside the gpt gene. However, these efficiencies were substantially enhanced by the introduction of a double-strand break into the homologous region of the gpt gene in one of a pair of deletion plasmids before cotransfer. This enhancement was apparently only about half as great for the xrs cells as for CHO-K1, but variation in the data was considerable. A much larger difference between CHO-K1 and the xrs mutants was found when the DNA concentration dependence of transformation was explored. While the transformation frequency of CHO-K1 increased linearly with DNA concentration, no such increase occurred with the xrs mutants irrespective of whether complete plasmids or pairs of deletion plasmids were transferred. The fraction of cells taking up DNA, assayed autoradiographically, was similar in all cell types. Therefore we suggest that while homologous recombination of plasmid molecules may not be substantially reduced in the xrs mutants,processes involved in the stable integration of plasmid DNA into genomic DNA are significantly impaired.
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The nature of mutants induced by ionizing radiation in cultured hamster cells. II. Antigenic response and reverse mutation of HPRT-deficient mutants induced by gamma-rays or ethyl methanesulphonate.
A large series of independent mutants deficient in HPRT enzyme activity, isolated from V79-4 hamster cells (Brown and Thacker, 1984), were assessed for properties which reflect the nature of the genetic changes induced. A total of 88 mutants were screened, 43 isolated from gamma-ray-treated cultures and 45 induced by ethyl methanesulphonate (EMS). Firstly, each mutant was assayed for the presence of protein with the antigenic response of HPRT (cross-reacting material, CRM), using an antibody raised against partially purified V79-4 HPRT enzyme. In a competitive inhibition assay, 31% of EMS-induced mutants were CRM-positive compared to 7% of the gamma-ray series. Secondly, each mutant was tested for ability to revert to HPRT proficiency, either spontaneously or after treatment with the powerful mutagen ethyl nitrosourea (ENU). All except 2 of the EMS-induced mutants reverted with ENU, and many reverted spontaneously, under the given conditions. However reversion was not detected in about 80% of gamma-ray-induced mutants, suggesting that the types of forward mutation caused by ionizing radiation differ qualitatively from those caused by EMS. The EMS-induced mutations are likely to be mostly point mutations, with at least 40% of the missense type, while gamma-ray-induced mutations may arise mostly through larger genetic changes.
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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.
Alpha Particles↗
The induction of chromosome exchange aberrations by carbon ultrasoft X-rays in V79 hamster cells.
V79 hamster cells in plateau (extended G1) phase were irradiated with either 250 kV ('hard') X-rays or carbon K characteristic ultrasoft X-rays under conditions minimizing cell overlap. These cells were killed most effectively by the carbon X-rays, by a factor of about 3 relative to hard X-rays, in agreement with our previous findings with cells in exponential growth. Chromosome-type aberrations were measured at 3 fixation times within the first division cycle after irradiation, and an approximately uniform sensitivity to aberration induction was found for both radiations. The combined aberration data show that carbon X-rays are 2 or more times as effective as hard X-rays, depending on dose and/or data fit. Exchange aberrations require recombination between two separate chromosomes, but they are induced efficiently by carbon X-rays with a substantial linear component to the dose-response despite the very short electron tracks (approximately less than 7 nm) that they produce in the cell. This implies either that the participating DNA helices must be lying extremely close together at the time of radiation damage, so that one track can effectively damage both helices, or that only one radiation-damaged chromosome is needed to promote an exchange event.
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The use of recombinant DNA techniques to study radiation-induced damage, repair and genetic change in mammalian cells.
A brief Introduction is given to appropriate elements of recombinant DNA techniques and applications to problems in radiobiology are reviewed with illustrative detail. Examples are included of studies with both 254 nm ultraviolet light (u.v.) and ionizing radiation (i.r.) and the review progresses from the molecular analysis of DNA damage in vitro through to the nature of consequent cellular responses. The section on the Molecular distribution of DNA damage (section 2) focuses on the use of defined DNA molecules to assess the nature, sites and frequency of radiation damage. Recombinant DNA techniques have also been used in the study of enzyme-DNA interactions, to comment upon the rôle of specific types and sites of damage in producing cellular responses. The use of DNA-mediated gene transfer to assess damage and repair (section 3) indicates that recombinant DNA molecules can be used to implicate (or reject) specific types of DNA damage in gene inactivation. Some gene-transfer assays may also be able to confirm the presence of specific repair functions in mammalian cells. Restriction endonucleases are essential for the construction of recombinant DNA molecules, but their ability to cut DNA at specific sequences is also being exploited to implicate the double-strand break as an important type of damage leading to the well-characterized responses of irradiated cells. The DNA double strand break: use of restriction endonucleases to model radiation damage (section 4) documents experiments showing that blunt-ended cuts introduced into cellular DNA are able to produce chromosome aberrations and cell death. Assays based upon the introduction of restriction endonuclease-cut plasmids into radiosensitive and normal cells suggest that sensitivity is in some instances, e.g. the radiosensitive disorder ataxia-telangiectasia, a result of excessive degradation of DNA around broken ends. Identification and cloning of DNA repair genes (section 5) reviews the successful cloning of one human repair gene and the putative identification of others, as well as the lack of success in identifying genes complementing radiosensitive human disorders. Analysis of radiation-induced genetic change (section 6) links the types of DNA damage observed in defined DNA molecules with the types of mutations occurring in irradiated prokaryotes. In mammalian cells recombinant DNA techniques have allowed the nature of mutational changes to be determined for the first time: to date it seems that u.v. produces mainly small (point) mutations while i.r. produces mainly large changes (deletions/rearrangements).(ABSTRACT TRUNCATED AT 400 WORDS)
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Responses of 4 X-ray-sensitive CHO cell mutants to different radiations and to irradiation conditions promoting cellular recovery.
Four X-ray-sensitive mutants of CHO cells, described previously by Jeggo and Kemp (1983), showed enhanced sensitivity to both 60Co gamma-rays and 238Pu alpha-particles relative to the responses of the parent line. The enhanced response to a densely ionising radiation (alpha-particles) was less than that to X- or gamma-rays, suggesting that these mutants are deficient mainly in the repair of damage from relatively sparsely ionising radiation tracks. Plateau-phase cultures of the parental CHO cells showed considerable recovery upon irradiation with low-dose-rate gamma-rays, compared to irradiation at 'high' dose rates, but little or no recovery was seen for the mutants. Similarly, preliminary data on recovery during post-irradiation holding of plateau-phase cultures show that this process is also absent in the mutants. These responses have several similarities to those of cells from patients with the radiosensitive disorder ataxia telangiectasia (AT), and are discussed with reference to AT cells and other radiosensitive mutants.
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