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J Thacker

Publications and source records attributed to J Thacker.

At least 37 records · Page 2Linked to original sources
PubMed1996

Mutations induced by DNA double-strand breaks: the influence of genomic site.

The transgenic CHO cell line PL61, carrying a recombinant SV40-gpt gene, was treated with restriction endonucleases to assess mutagenesis from defined DNA double-strand breaks. Mutations in gpt were measured under two conditions: a stringent condition where selection ensured that the closely-linked neo gene was retained functionally intact, or a relaxed condition without the requirement for neo gene function. Despite testing 18 different restriction endonucleases with various numbers of potential break-sites within the transgene, mutations were only found under relaxed selection conditions. These mutations commonly led to complete loss of the transgene, suggesting that large deletions predominate when selection is relaxed. It is argued, in comparison to mutation data for other genomic sites in CHO cells, that variations in the 'effective target size' for mutagenesis may explain the response of the transgene under different conditions.

Animals↗
PubMed1995

Localization to chromosome 7q36.1 of the human XRCC2 gene, determining sensitivity to DNA-damaging agents.

The identification of genes controlling cellular response to DNA damage is of considerable importance, and cell lines showing hypersensitivity to DNA-damaging agents can be used as vehicles to map and clone these genes. In this study the hamster cell line irs1, showing hypersensitivity to a number of different DNA-damaging agents, was fused to normal human cells to complement the defect. The resultant hybrids were analysed by Alu-PCR, chromosome painting, and with DNA markers to map the complementing gene (named XRCC2) to a specific chromosomal region. These hybrids showed correction of sensitivity to both X-rays and to mitomycin-C, and contained human chromosome 7, often as their only human component. Hybrids showing unstable retention of human chromosomes were sub-cloned to show that loss of chromosome 7 and loss of resistance to mitomycin-C occurred concordantly. Two separate hybrids were found to have a smaller piece of chromosome 7, and specific DNA probes and microsatellite markers defined this as a contiguous region at 7q35-36. Hybrid irradiation-fusion methods were used to further reduce the size of the complementing genomic region and to localize the gene to an approximately 3-5 Mb region at 7q36.1.

Animals↗
PubMed1995

The genetic basis of cellular recovery from radiation damage: response of the radiosensitive irs lines to low-dose-rate irradiation.

Recovery from the lethal effects of irradiation is commonly found when cultured mammalian cells are irradiated at low dose rates when compared to the same cells irradiated at higher dose rates. However, this cellular recovery process is severely reduced or absent in a number of radiosensitive cell lines, including those derived from the human disorder ataxia telangiectasia (AT). The genetic and molecular basis of such recovery processes is not understood, despite their importance. The responses of cells of three further radiosensitive lines, irs1, irs2 and irs3, shown previously to be mutated in different genes, to low-dose-rate radiation are now presented. Plateau-phase cultures of cells of the irs2 line were found to have little or no cellular recovery, while irs1 and irs3 had considerable recovery potential. In comparing the known properties of the radiosensitive lines lacking cellular recovery, including xrs, XR-1 and scid as well as AT and irs2, it is argued that the gene products lacking in these lines normally act coordinately in a specific damage-recognition pathway. The recovery pathway is likely to be associated with the rejoining of DNA double-strand breaks, since several of these recovery-defective lines have a measurable deficiency in break repair.

Animals↗
PubMed1994

The study of responses to 'model' DNA breaks induced by restriction endonucleases in cells and cell-free systems: achievements and difficulties.

The use of restriction endonucleases (RE) as a means of implicating DNA double-strand breaks (dsb) in cellular responses is reviewed. The introduction of RE into cells leads to many of the responses known to be characteristic of radiation damage--cell killing, chromosomal aberration, oncogenic transformation, gene mutation and amplification. Additionally, radiosensitive cell lines are hypersensitive to RE, including those from the human disorder ataxia-telangiectasia. However, quantitation of response and comparisons of the effectiveness of different RE are difficult, partly because of unknown activity and lifetime of RE in the cell. RE-induced dsb have also been used to reveal molecular mechanisms of repair and misrepair at specific sites in DNA. Dsb have been implicated in recombination processes including those leading to illegitimate rejoining (formation of deletions and rearrangements) at short sequence features in DNA. Also model dsb act as a signal to activate other cellular processes, which may influence or indirectly cause some responses, including cell death. In these signalling responses the detailed chemistry at the break site may not be very important, perhaps explaining why there is considerable overlap in responses to RE and to ionizing radiations.

Animals↗
PubMed1994

Gene mutation and V(D)J recombination in the radiosensitive irs lines.

The lines irs1, irs2 and irs3, derived from V79-4 hamster cells, are sensitive to DNA-damaging agents including ionizing radiations. However, unlike some other radiosensitive lines, the irs lines show no apparent defect in the repair of DNA strand breaks. We have now assessed the mis-repair of DNA damage in the irs lines by measuring spontaneous and X-ray induced frequencies of mutation in the HPRT gene. irs1 was found to be hypermutable, showing instability in spontaneous mutant frequency and an elevation of the radiation-induced frequency relative to the parental line. In contrast, irs2 and irs3 showed similar mutational responses to the parental line. The results support other lines of evidence suggesting that irs1 has a mis-repair phenotype. The irs2 line has previously been shown to have a phenotype similar to cells from the human disorder ataxia-telangiectasia and this similarity is maintained in their mutational response to X-rays. The irs lines were also tested for ability to undergo V(D)J recombination, since this process has recently been found to be defective in some radiosensitive lines with impaired double-strand break repair. Using an extrachromosomal vector containing a V(D)J rearrangement cassette, correct recombination was shown to occur at similar frequencies to parental V79-4 cells in each of the three irs lines. Thus the irs lines indicate that processes other than DNA double-strand break repair also control radiosensitivity, in particular those processes which may affect the regulation of DNA repair.

Animals↗
PubMed1994

Cellular radiosensitivity in ataxia-telangiectasia.

Hypersensitivity to both the cell-killing and chromosome-damaging effects of ionizing radiations, and other agents causing DNA breakage, is a consistent feature of cells from individuals with the cancer-prone disorder ataxia-telangiectasia (A-T). Evidence for a defect in DNA strand break rejoining is slight, but a higher-than-normal level of chromosomal breaks persists in irradiated A-T cells. There is also evidence for elevated frequencies of DNA recombination and deletion mutation in A-T cells; these responses may be linked through a loss of fidelity in rejoining DNA breaks through recombination mechanisms. Additionally the regulation of cell-cycle responses is altered in A-T cells: in all phases of the cycle there is some loss of 'checkpoint' function shortly after irradiation, allowing cells to continue cycling despite extensive DNA damage. However, on present evidence, radiation hypersensitivity cannot be explained simply by this loss of regulatory function. It is suggested that the A-T gene product acts in the early stages of a DNA damage-recognition pathway, normally interacting with regulatory proteins such as p53, but also with proteins involved in the processing of DNA breaks. Reduced efficiency in this type of signalling function could well explain the link between radiosensitivity and cancer proneness.

Ataxia Telangiectasia↗
PubMed1993

Formation of large deletions by illegitimate recombination in the HPRT gene of primary human fibroblasts.

HPRT gene mutants were isolated from untreated and x-irradiated cultures of primary human fibroblasts, and mutants carrying large deletions were identified. The breakpoints of the deletions were mapped by methods based on the polymerase chain reaction, and the deletion junctions of four different mutants were sequenced. Alu repeats were associated with one end of three of these junctions, but in each case repeat sequences were not found at the other end. Sequence features found at the deletion breakpoints included in particular short direct and inverted repeats, which may mispair to promote illegitimate recombination. One mutant had additional bases inserted at the deletion junction; these bases formed a direct repeat with a sequence immediately adjacent to the junction, suggesting a mechanism of templated repair of broken DNA in deletion formation.

Base Sequence↗
PubMed1993

The effectiveness of restriction endonucleases in cell killing and mutation.

The use of restriction endonucleases (RE) to study the importance of DNA break end structures in differential cellular response has proved controversial. The number of DNA cut sites and the accessibility of RE are recognized examples of confounding factors. We have eliminated these factors by comparing the effectiveness of isoschizomers. Additionally, we considered for the first time the tolerance of the enzymes to cellular conditions. Cell killing and mutation were compared to the overall cutting ability of the enzymes in an "intracellular" buffer. We found that the activity of each RE combined with its lifetime, under simulated cellular conditions, were the overriding factors in determining effectiveness.

Animals↗
PubMed1993

Analysis of large deletions in the HPRT gene of primary human fibroblasts using the polymerase chain reaction.

Spontaneous and X-ray-induced mutants of the HPRT gene were isolated from two primary human fibroblast lines. The limited life-span of the mutants restricted the use of methods requiring large quantities of DNA, and the polymerase chain reaction (PCR) was used in particular to check for the presence of multiple genomic sites in mutant analysis. Robust PCR primers were designed to amplify sites of up to 1 kb, mostly with 1-kb spacings between sites, over the entire 56-kb HPRT gene region. Using PCR, large deletions were found in 43% of independent X-ray-induced mutants, and their breakpoints were localized where these fell within the gene. Anonymous DNA sites in the Xq26 chromosomal region containing HPRT (covering > or = 1.5 Mb) were also amplified by PCR to assess codeletion with HPRT; sites up to 1 Mb distal to the gene (DXS86, DXS10) were codeleted in some mutants, but no mutant was found with loss of a proximal site (DXS79).

Base Sequence↗
PubMed1993

Repair and misrepair of site-specific DNA double-strand breaks by human cell extracts.

The rejoining by human cell extracts of a double-strand break induced by endonuclease treatment at one of several sites within a small DNA molecule was studied. Rejoining was found at each of 8 sites tested, but the rejoin efficiency varied with the nature of the break (e.g., breaks with cohesive ends were rejoined more efficiently than blunt-ended breaks). Extracts from primary and immortalized cell lines, as well as those from individuals with ataxia telangiectasia (A-T), showed the same pattern of relative rejoin efficiencies. However, mis-rejoining varied with the cell extract used, and was particularly elevated with two immortalized A-T cell lines. Mixing experiments showed that the mis-rejoining property of extracts could act in a semi-dominant fashion, depending on the individual efficiencies of the component extracts. The mis-rejoin mechanism involved deletion at sites of short direct repeats at various distances from the initial break site. A model of deletion formation (the strand-exposure and repair model) is restated to explain the sequence repeat dependence found, and is compared to models of homologous DNA recombination.

Ataxia Telangiectasia↗
PubMed1993

Weiss Lecture. Effects of radiations of different qualities on cells: molecular mechanisms of damage and repair.

Studies of ionizing radiations of different quality are discussed with particular emphasis on damage to DNA of mammalian cells. Three related themes are followed. Firstly, inactivation and mutation experiments with ultrasoft X-rays and slow heavy ions, coupled with theoretical analyses of the structures of the radiation tracks, have emphasized the biological importance of localized track features over nanometre dimensions. This led to the suggestion that the critical physical features of the tracks are the stochastic clusterings of ionizations, directly in or very near to DNA, resulting in clustered initial molecular damage including various combinations of breaks, base damages, cross-links, etc. in the DNA. The quantitative hypotheses imply that final cellular effects from high-LET radiations are dominated by their more severe, and therefore less repairable, clustered damage, and that these are qualitatively different from the dominant low-LET damage. Second, relative effectiveness of different types of radiation led to questions on the mechanisms of induction of chromosome exchanges. The high efficiency of ultrasoft X-rays, despite their very short track lengths, suggested that single sites of DNA damage may lead to exchanges by a molecular process involving interaction with undamaged DNA. Also it is shown that a single site-specific DNA break, introduced by restriction enzymes, sometimes leads to a large deletion when misrepaired by cell extracts. These deletions occur between short DNA repeats, and are therefore a form of 'illegitimate' recombination, but clearly do not involve the interaction of two damage sites. Third, it was shown that cells from patients with the radiosensitive disorder ataxia-telangiectasia (AT) lack a post-irradiation recovery process. The sensitivity of AT cells to high LET radiations was found to be reduced relative to that for normal cells, reinforcing the concept that high LET damage is less easy to repair. AT patients are prone to lymphoreticular cancers, and their cells show characteristic chromosomal rearrangements, which may be associated with misrepair at specific genomic sequences. Similarly, studies of radiation-induced leukaemia in the mouse have implicated rearrangement at specific interstitial chromosome sites, which are rich in telomere-like repeat sequences.

Animals↗
PubMed1993

Response of radiation-sensitive human cells to defined DNA breaks.

We have investigated the response of four human cell lines, representing a range of sensitivities to ionizing radiation, to enzymes which induce defined DNA double-strand breaks (dsbs). Cell lines were derived from a normal individual, from the cancer-prone disorders ataxia-telangiectasia (AT) and Bloom's syndrome (BS), and from an immunodeficient individual (46BR). The molecular defects in AT and BS are unknown, while 46BR is known to be DNA ligase I deficient. We assayed the clonogenic survival of the cell lines following in vivo scission of the DNA by the restriction endonucleases PvuII and BanI. These two enzymes differ in their action; PvuII gives rise to dsbs with blunt termini, while BanI generates staggered ends with a 4 bp overhang. We found a correlation between the sensitivity of the cell lines to X-rays and to the blunt-end cutter PvuII, but not to the cohesive-end cutter BanI.

Acquired Immunodeficiency Syndrome↗
PubMed1993

High-resolution cytogenetic analysis of X-ray induced mutations of the HPRT gene of primary human fibroblasts.

We report cytogenetic analysis of X-ray induced mutants of the HPRT gene isolated from a primary human fibroblast line. The mutants were shown previously by molecular analysis to have total or partial HPRT gene deletions. Detailed analysis of the chromosomal region containing HPRT (the cytogenetic band Xq26) identified Xq26 aberrations in five of five total HPRT gene deletions but failed to detect any changes in two of two partial gene deletions. Four microdeletions were verified and quantified by a method termed the band ratio, which compares the distance between bands encompassing Xq26 and an adjacent X-chromosome region in elongated chromosomes. These measurements were supported by the presence or absence of the sub-band Xq26.2. One total HPRT gene deletion was also associated with an X;11 translocation involving band Xq26. The data strengthen earlier findings on the tolerance of the Xq26 region to large genetic changes (> 1 Mb), but also indicate that some mutations are more complex than simple deletion of DNA sequence.

Cells, Cultured↗
PubMed1992

A mechanism for deletion formation in DNA by human cell extracts: the involvement of short sequence repeats.

DNA molecules carrying a site-specific double-strand break were exposed to nuclear extracts from human cell lines. It was shown previously that breaks could be rejoined correctly by human extracts, but that a proportion of the rejoined molecules had suffered deletions and insertions. The 'mis-rejoined' proportion was higher with cell extracts from an individual with the disorder ataxia-telangiectasia than with normal cell extracts. We now show by sequence analysis that deletions in extract-treated molecules occur exclusively between short direct repeats (2-6 base pairs). A mis-rejoined molecule containing an insertion of 300 bp also had a repeat-based deletion at the same site. A number of different direct repeats are involved; however, some clustering of these occurs especially on the upstream side of the initial breakpoint. These data are most simply interpreted in terms of a model of deletion formation involving single-strand exposure and repair, perhaps with the action of other DNA-metabolising enzymes influencing the frequency with which some repeats are involved.

Ataxia Telangiectasia↗
PubMed1992

Multiple components are involved in the efficient joining of double stranded DNA breaks in human cell extracts.

We describe a rapid and efficient in vitro system for the rejoining of double stranded breaks in DNA based on extracts of human 293 cells. Using this system as an assay, we have separated the nuclear extract into several components involved in break rejoining. The unfractionated system can convert approx. 100% of the input DNA, linearized with a restriction enzyme, to high molecular weight material at low temperature (17 degrees C), and at the physiological temperature of 37 degrees C we have shown that competing activities in the extract can also act on the DNA template. We present the fractionation of the extract and the partial purification of a novel factor which will stimulate a crude rejoin activity and in addition increases the activity of purified DNA ligase I. We have also partially purified the break joining activity and show that the chromatographic properties do not directly correspond with the three DNA ligases previously described, indicating that the activity observed may not be due to a single enzyme species. By studying the rejoining of double stranded DNA breaks as a biochemical process, we have demonstrated that the efficient joining of such breaks requires factors in addition to DNA ligases.

Cell Extracts↗
PubMed1992

Age and dialysis.

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Age Factors↗
PubMed1992

Rapid screening for deletion mutations in the hprt gene using the polymerase chain reaction: X-ray and alpha-particle mutant spectra.

Conditions were devised for the isolation of DNA from single-mutant colonies on dishes, to give reproducible results in the polymerase chain reaction (PCR). Primers for 3 exons of the hamster hprt gene were used in a multiplex reaction to show rapidly whether the mutants carried deletions at these sites. 138 independent mutants were screened in total, some spontaneous and others induced by X-rays or by alpha-particles from plutonium-238. Few deletions were found among the spontaneous set, while 'total' gene deletions formed about half the mutants found after irradiation. At equitoxic doses, little difference in mutant spectrum was found for the X-ray set compared to the alpha-particle set. This rapid technique should be applicable to many instances of comparative mutagenesis.

Alpha Particles↗