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J Thacker

Publications and source records attributed to J Thacker.

At least 73 records · Page 4Linked to original sources
PubMed1984

The nature of mutants induced by ionising radiation in cultured hamster cells. I. Isolation and initial characterisation of spontaneous, ionising radiation-induced, and ethyl methanesulphonate-induced mutants resistant to 6-thioguanine.

A large number of thioguanine (TG)-resistant mutants of V79-4 Chinese hamster cells was isolated from untreated cultures and from cultures exposed to gamma-rays, alpha particles or ethyl methanesulphonate (EMS). Selection conditions were chosen to optimise the survival of all types of TG-resistant mutant, and the isolation procedure ensured that each mutant originated independently of any other. Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) enzyme activity was measured in cell-free extracts of each mutant, and compared with repeat measurements made on the parental V79-4 cells and on a series of non-mutant clones. Ionising radiation-induced mutants were found to be mostly (or perhaps entirely) of the 'zero HGPRT activity' type, but about 20% of EMS-induced mutants and 50% of spontaneously occurring mutants had significant HGPRT activity. However, none of the TG-resistant mutants were found to lack activity of another X-chromosome-linked enzyme, glucose-6-phosphate dehydrogenase. Few mutants were found with visible X-chromosome changes, but the incidence of hyperploidy was higher among spontaneous mutants than in the parental line and the induced mutants. Isoelectric focussing of cell extracts from those mutants which retained some HGPRT activity revealed several with shifts in the isoelectric points for HGPRT enzyme activity.

Animals↗
PubMed1983

The use of a cloned bacterial gene to study mutation in mammalian cells.

The recombinant DNA molecule pSV2-gpt, which contains the bacterial gene coding for xanthine-guanine phosphoribosyl transferase (XGPRT) activity, was introduced into a hamster cell line lacking the equivalent mammalian enzyme (HGPRT). Hamster cell sublines were found with stable expression of XGPRT activity and were used to study mutation of the integrated pSV2-gpt DNA sequence. Mutants were selected by their resistance to 6-thioguanine (TG) under optimal conditions which were found to be very similar to those for selection of HGPRT-deficient mutants of mammalian cells. The frequency of XGPRT-deficient mutants was increased by treatment with X-rays, ethyl methanesulphonate and ethyl nitrosourea. X-Ray induction of mutants increased approximately linearly with dose up to about 500 rad, but the frequency of mutants per rad was very much higher than that usually found for 'native' mammalian genes. However, still higher frequencies of mutation were found for the hamster HGPRT gene when it had been stably transferred into the same hamster cell line. It is suggested, therefore, that transferred DNA may integrate in sequences which are more 'reactive' than most of the genome. Cell-free extracts of 10 TG-resistant mutants of XGPRT-proficient sublines showed no measurable XGPRT activity. High molecular weight DNA from XGPRT-proficient sublines used in the mutation studies hybridized with nick-translated pSV2-gpt DNA, showing two distinct bands when cut with the restriction enzyme Eco R1. This suggests that a single copy of pSV2-gpt DNA was integrated in these sublines. DNA from most spontaneous and mutagen-induced TG-resistant mutants had lost these two hybridizing bands, but one spontaneous mutant was found with rearranged pSV2-gpt sequence.

Animals↗
PubMed1983

Recovery from lethal and mutagenic damage during postirradiation holding and low-dose-rate irradiations of cultured hamster cells.

Survival and mutation to thioguanine resistance were measured in V79-4 hamster cells grown to plateau phase without refeeding and irradiated with 60Co gamma rays. The effects of low-dose-rate irradiation and of postirradiation holding on recovery from gamma-ray damage leading to these two responses were also studied. The responses of these plateau (extended G1)-phase cells to acute irradiation were similar to those we previously found for exponentially growing cells, including the linear relationship between induced mutant frequency and (log) surviving fraction. Irradiation at low dose rate (0.34 rad/min) considerably reduced both the lethal and mutagenic effects of given doses of gamma rays, but the linear mutation-survival relationship was approximately the same as for acute irradiation. In contrast, cells given a 5-hr holding period after acute irradiation showed the anticipated recovery from potentially lethal damage but no recovery from damage leading to mutation. These results are discussed in terms of previously proposed cellular repair processes (sublethal damage repair and potentially lethal damage repair) and the possibility that the radiation damage leading to lethality is different from mutagenic damage.

Animals↗
PubMed1982

A simple autoradiographic method for checking HGPRT-deficiency in colonies of mammalian cells.

HGPRT enzyme activity in mutant colonies selected in 6-thioguanine can be assessed directly in petri dishes using an autoradiographic method. The application of this method to large-scale quantitative mutation experiments verifies that: (a) the maximum induced frequency of mutation of HGPRT-deficiency can be measured at short expression times, such as 3 days after treatment, when cells are respread into relatively low thioguanine concentrations, and (b) ionizing radiation induces predominantly mutants with zero HGPRT activity. Other potential applications of this method are discussed.

Animals↗
PubMed1981

Is selective absorption of ultrasoft x-rays biologically important in mammalian cells?

This paper tests whether photon absorption processes in particular atomic element(s) may be responsible for the observed high relative biological effectiveness (RBE) of ultrasoft X-rays. The effectiveness of titanium K characteristic X-rays (4.55 keV) is compared with previous observations for aluminium (1.5 keV) and carbon (0.28 keV) K ultrasoft X-rays. For a given absorbed dose, five times more Ti K than Al K photons are absorbed in phosphorus; since Al K X-rays are observed to be more effective in killing human and hamster cells it is concluded that absorption in phosphorus does not play a dominant lethal role. This is supported by the observation that the absolute number of Al K photons absorbed in phosphorus of DNA of human fibroblasts is less than 1 per lethal event. For no element is the relative number of absorbed photons of the three X-ray energies even approximately proportional to their observed RBEs. The effectiveness of ultrasoft X-rays is apparently not due to selective absorption but rather to the secondary electrons; consequently the mechanism of action should be common to the large numbers of low energy secondary electrons produced by most other ionising radiations, including gamma-rays.

Aluminum↗
PubMed1981

The chromosomes of a V79 Chinese hamster line and a mutant subline lacking HPRT activity.

Cells of the V79-4 Chinese hamster line were found to have a consistent set of 20 chromosomes. G- and C-band analysis showed that, compared to the standard 22 chromosome set of freshly isolated Chinese hamster cells, the V79-4 chromosomes had various characteristic deletions and rearrangements. However, it is probable that at least one copy of each autosome was still present, and three pairs of autosomes appeared unchanged from those in the freshly isolated cells. The establishment of this karyotype for V79-4 allows mutant sublines to be screened for chromosomal changes associated with the altered phenotype, and an example is given of a radiation-induced mutant lacking HPRT activity that had a clear X-chromosome rearrangement.

Alpha Particles↗
PubMed1980

Resistance to methyl mercaptopurine riboside in cultured hamster cells. Preliminary characterization of resistant cells and conditions affecting their selection in quantitative mutation studies.

Cells resistant to high concentrations of methyl mercaptopurine riboside (MMPR), an anlogue of adenosine, were found at a frequency of about 6 x 10(-6) per viable cell in untreated cultures of V79 Chinese hamster cells. Resistant cells were selected less efficiently if purines were added to the MMPR-medium, but high cell densities had little effect upon selection. 6 independently-isolated spontaneous MMPR-resistant sublines were characterized by their resistance to the toxicity of different purines, rate of purine excretion, incorporation of radioactive adenosine, electrophoresis of cell extracts, and expression of resistance in hybrids to an MMPR-sensitive line. 5 of these sublines showed recessive expression of MMPR-resistance in hybrids and had characteristics consistent with loss of adenosine kinase activity, while the remaining subline was much less resistant to MMPR and showed semi-dominant expression of resistance without loss of adenosine kinase activity. Cells with high resistance to MMPR were not found in a "tetraploid" (hybrid) V79 line or in freshly-isolated human cell cultures, but occurred at a comparable frequency to V79 in another commonly-used aneuploid hamster line, CHO-K1. The frequency of MMPR-resistant cells in V79 cultures was increased to a similar extent by treatment with gamma-rays or with ethyl methanesulphonate, providing a suitable post-treatment interval was allowed for the expression of resistance. A genetic interpretation of these data is given in which it is proposed that resistance most usually arises through mutation of an autosomally-linked gene of which one copy has been inactivated or lost in V79 and in CHO-K1 cells. In comparison to published data on the selection of "mutants" resistant to 6-thioguanine, it is argued that MMPR could be as useful a selective agent as thioguanine and may select a different range of types of mutagenic event.

Adenosine Kinase↗
PubMed1980

Mutation and inactivation of cultured mammalian cells exposed to beams of accelerated heavy ions. IV. Biophysical interpretation.

A biophysical analysis is made of the results of recent experiments which used accelerated heavy ions of 20 to 470 keV micron-1 to induce inactivation and mutation (resistance to 6-thioguanine) in cultured V79 Chinese hamster cells and HF19 human diploid fibroblasts. It is shown that the discrete nature of the primary ions must be explicity taken into account before the numbers of induced lethal and mutagenic lesions can be deduced from the observed radiosensitivities. The measured numbers of lesions produced by the radiations of different LET are compared with the relative numbers predicted by various models of radiation action. The observations can be explained on the hypothesis that each lethal lesion is produced by a deposition of small energy (small number of ionizations) in a distance of about 3 nm. Two different lesions appear to be involved, one of which requires greater than or equal to 100 eV and is dominant with low-LET radiations, and the other requires greater than or equal to 300 eV and is dominant at high-LET. Similar conclusions may apply to mutagenic lesions except that the mechanism which dominates at high-LET requires significantly more than 300 eV. More precise assessments of the hypothesis and these numerical values must await detailed track structure calculations of the radiation on the nanometre scale. Alternative models which invoke 'accumulation of sublethal damage' or 'interaction between sublesions', over distances of the order of microns, do not provide a consistent explanation of the observations. This suggests that the frequently observed curvature of low-LET dose-responses is not due to interaction between sublesions but rather to some other mechanism such as a dose-dependent repair process. It is also shown that low velocity, high-LET ions produce an average of appreciably less than one lethal lesion in traversing the nucleus of the above mammalian cells; 90 keV micron-1 helium ions produce about 0.03-0.06 lethal lesions micron-1 of track through the nucleus of the cells of thickness about 7 microns. Some estimates are also made of the size of the nuclear region which is sensitive to the induction of mutation to 6-thioguanine-resistance; it is concluded that this region extends beyond the DNA of the structural gene itself.

Animals↗
PubMed1980

Chromosome aberrations induced in human lymphocytes by ultrasoft Al (K) and C (K) X-rays.

Induction of structural chromosome aberrations was studied in human peripheral blood lymphocytes irradiated in the unstimulated state with ultrasoft X-rays. Aluminium K X-rays (1.49 ke V) and carbon K X-rays (0.28 ke V) were used. The frequencies of dicentric aberrations and of excess acentric fragments were found to increase approximately linearly with absorbed dose of both radiations. Carbon X-rays were more effective than aluminium X-rays. The corresponding linear yield coefficients for dicentrics are (3.7 X 10(-1)) Gy-1 and (2.2 X 10(-1)) Gy-1 respectively. AT low doses both these radiations are more effective than hard X-rays in producing dicentric aberrations, but at high doses aluminium X-rays are less effective than hard X-rays because of the large positive curvature of the hard X-ray dose-response. The most significant observation of these experiments is that electrons of less than 280 eV, produced by carbon X-rays, are efficient in producing dicentric aberrations. This implies that single local energy events of about 14 ionizations with in less than 7 nm are able to produce exchange aberrations.

Cell Division↗
PubMed1979

Effectiveness of 0.3 keV carbon ultrasoft X-rays for the inactivation and mutation of cultured mammalian cells.

Carbon K characteristic ultrasoft X-rays of energy 0.278 keV were found to be effective in inducing inactivation and mutation to thioguanine resistance in cultured V79 Chinese hamster cells and human diploid fibroblasts. These X-rays act as a probe of the sensitive sites within the cells since they produce low-energy photoelectron tracks of range about 7 nm; this is an order of magnitude smaller than those produced by the 1.5 keV aluminium X-rays used in previous studies. A detailed interpretation of the results requires assumptions to be made about the positions of the sensitive sites within the cells but, for any reasonable set of assumptions, the carbon X-rays are found to be more effective than gamma-rays and are probably at least as effective as long tracks of helium ions of similar LET. These observations extend the conclusions previously drawn from the observed effectiveness of aluminium X-rays regarding the sizes of the subcellular sites involved in inactivation and mutation. They imply that the sensitive sites smaller than about 7 nm, and that highly localized energy depositions consisting of less than or approximately 14 ionizations are sufficient to produce biological effects. These results are also in contradiction to models of radiation action which require relatively large sites, such as the usual form of the 'theory of dual radiation action'.

Aluminum↗
PubMed1979

Mutation and inactivation of cultured mammalian cells exposed to beams of accelerated heavy ions. II. Chinese hamster V79 cells.

Inactivation and mutation to thioguanine-resistance of V79 hamster cells were studied after irradiation with accelerated helium, boron or nitrogen ions covering a range of linear energy transfer from 28 to 470 keV micrometers-1. For all radiation qualities a dose-dependent increase in mutant frequency was found for doses giving surviving fractions greater than about 0.20. The effectiveness per unit dose for both inactivation and mutation induction increased with the linear energy transfer of the radiation to a maximum in the range 90-200 keV micrometer-1. However, the maximum mutagenic effectiveness relative to gamma-rays was about two or more times that for inactivation. It is suggested that a proportion of the radiation-induced mutants suffer extensive genetic damage, and that some forms of this damage may be induced with high efficiency by radiations of high linear energy transfer.

Animals↗
PubMed1978

Mutation to ouabain-resistance in Chinese hamster cells: induction by ethyl methanesulphonate and lack of induction by ionising radiation.

The spontaneous frequency of mutants resistant to growth inhibition by ouabain (OUAR mutants) was found to be about 5:10(-5) per viable cell in uncloned cultures of Chinese hamster V79-4 cells. In freshly-isolated clones or cultures started from a few cells this frequency was initially reduced to about 1.10(-6) in 1 mM ouabain. No increase in the frequency of OUAR mutants was found in cultures treated with gamma-rays despite exploration of such variables as radiation dose, ouabain concentration, post-treatment interval before selection, cell density in selective medium, and clonal state of the cells at the time of adding ouabain (in situ vs. respreading method). A similar negative result was found for accelerated helium ions, for which the mutagenic effectiveness per unit dose has been shown to be about 10 times higher than gamma-rays for the induction of thioguanine-resistant mutants in these cells. Some evidence was found for an interaction between cellular radiation damage and ouabain-resistance, which may lead to a reduction in the survival of OUAR mutants in irradiated populations, but this damage seemed insufficient to account for inability to detect radiation-induced OUAR mutants. Reproducibly large increases in the frequency of OUAR mutants were found in cultures treated with various concentrations of ethyl methanesulphonate (EMS) by respreading cells in 1 mM ouabain for up to 8 days after EMS treatment. The concentration-OUAR mutant induction curve was approximately linear with low EMS concentrations. Recent evidence is reviewed in support of the suggestion, made in earlier studies, that ionising radiation is unable to induce OUAR mutants because of the severity of the genetic damage it causes.

Cell Line↗
PubMed1977

Th induction of thioguanine-resistant mutants of Chinese hamster cells by gamma-rays.

The induction of mutation to purine analogue resistance was assessed in Chinese hamster V79-4 cells exposed to gamma-radiation. After irradiation, the cells were grown in non-selective medium for different time intervals before respreading into medium containing 0.5-0.7 mug/ml thioguanine. In some experiments colonies arising in thioguanine-medium were counter-selected in medium containing the glutamine analogue azaserine, which distinguishes mutants with very little activity of the enzyme hypoxanthine-guanine phosphoribosyl transferase. Only these mutants were increased in frequency by radiation, the maximum measured frequencies occuring in cells respread after 2 days growth in non-selective medium. With longer intervals of post-irradiation growth in non-selective medium a fraction of the induced mutants was lost, and after large doses of radiation it is doubtful if the maximum frequency observed after 2 days post-irradiation growth represents the true induced frequency. The detection of freshly-induced mutants seemed to depend upon the dilution and decay of products formed from the genes prior to their mutation by radiation, since (a) selection of mutants in a higher concentration of thioguanine (2 mug/ml) increased the post-irradiation growth interval required to detect the maximum frequency of induced mutants, and (b) with increasing duration of post-irradiation growth in the non-selective medium, induced mutant cells were able progressively to overcome the growth-limiting effects of the analogue, to give large colonies when respread in thioguanine-medium. The radiosensitivities of 7 isolated mutant lines were indistinguishable from that of wild type cells, but the mutants were at a slight disadvantage when grown in competition with wild type cells. This disadvantage was consistent with the expected fitness of mutants relative to wild type cells calculated from estimates of the spontaneous mutation rate and the mean spontaneous mutation frequency. The induction of mutation to thioguanine resistance was non-linear with dose, yielding induced frequencies per rad of 5-10(-8) to 3-10(-7), but a plot of induced mutation frequency against log surviving fraction gave an approximately linear relationship. The same linear relationship holds for recently-published data on human and mouse cell cultures, so that all three mammalian cell types exhibit the same fixed probability of mutation induction relative to the extent of inactivation caused by ionising radiation.

Cell Division↗