Margaret Fox

Molecular Analysis of X-ray-induced Mutants at the HPRT Locus in V79 Chinese Hamster Cells

Spontaneous and X-ray-induced mutants at the hypoxanthine phosphoribosyl transferase (HPRT) locus have been isolated from V79 Chinese hamster cells and characterized at the biochemical and cytogenetic levels. Fourteen spontaneous and 24 X-ray-induced clones were azaguanine and thioguanine resistant, did not grow in HAT medium (AZRTGRHATS) and failed to incorporate significant levels of [14C]hypoxyanthine. Cytogenetic analysis of two spontaneous and eight X-ray-induced mutants revealed no major X chromosome rearrangements. In two induced mutants, one of which was hypotetraploid (mode 35-39) with 2 X chromosomes, the short arm of the chromosome (Xp) was slightly shorter than normal. A third mutant was hyperdiploid (mode 22-23) compared with the parental clone (mode 21). When compared with wild-type clones, no other cytogenetic changes were evident in the remaining mutants. Analysis at the DNA level using a Chinese hamster HPRT cDNA probe showed major deletion of HPRT sequences in two and partial deletion in another two induced mutants. In two of the mutants with deletions of HPRT sequences there was a visible shortening of the Xp arm. In the other six mutants two spontaneous and four induced) no karyotypic changes or alterations in restriction fragment patterns were detected suggesting that they carry small deletions or point mutations at the HPRT locus.

Continuous Irradiation of a Murine Lymphoma Line P388F in Vitro

SummaryThe response of the P388F murine lymphoma in vitro to continuous irradiation at dose-rates between 60 and 210 rad/day from tritiated water in the culture medium has been studied. The population doubling time of the cultures increases with increasing dose-rate. Labelling experiments with 3H thymidine indicated some increase in cell-cycle time and also the existence of non-dividing cells. Cloning techniques demonstrated that not all cells capable of synthesizing DNA have the capacity for indefinite reproduction. A model which accounts for the results is discussed. It proposes that, as a result of the continuous irradiation, at each cell division a proportion of the daughter cells lose their reproductive integrity but may nevertheless undergo several divisions before becoming completely sterile.

Effect of 14 MeV Monoenergetic Neutrons on HeLa and P388F Cells in Vitro

SummaryThe effects of 14 meV monoenergetic neutrons were compared with those of 300 kV x-rays. Cell survival data were used to compute dose-response curves from which values of oxygen enhancement ratio (OER) and relative biological effectiveness (RBE) have been derived. The OER for HeLa cells was 1·51 with neutrons and 2·39 with x-rays; giving a gain factor of 1·58. The RBE for HeLa cells varied between a value of 4·3 at the 90 per cent level of cell survival to 2·3 at the 3 per cent level. The RBE can be assumed to fall to a constant value of 1·95 at lower levels of survival. A constant value of 1·9 was determined for the RBE for P388F mouse lymphoma cells.

Repair Replication after U.V.-irradiation in Rodent Cell-lines of Different Sensitivity

SummaryRepair replication and unscheduled DNA synthesis have been demonstrated in a number of cultured tumour cell-lines of rodent origin. The amount of repair replication observed was less than that observed in HeLa cells when the same labelling procedures were used. u.v.-sensitive mutants of Yoshida cells which showed reduced D0 value and an extrapolation number of 1 showed reduced repair replication. The u.v.-sensitive mutant of L5178Y cells which differed only in its extrapolation number showed the same amount of repair replication as its more resistant mutant. The data show that (1) the amount of repair replication in rodent cells is less than that in HeLa cells and (2) that differences in amount of repair replication between two closely related cell lines are associated with changes in D0 value but not with changes in shoulder size and extrapolation number.

Repair replication in x-irradiated lymphoma cells in vitro.

SummaryRepair replication using isopycnic CsCl density—gradient techniques has been studied in P388 cells and in two lines of L5178Y lymphoblasts showing a large difference in radiosensitivity. The results indicate that in all three cell-lines only small numbers of bases < 10 are inserted/damaged site. The radiosensitive L5178Y cell line consistently showed greater base insertion/damaged site than the radioresistant line and P388 cells. DNA of the two L5178Y cell-lines was equally sensitive to breakage by x-irradiation, as shown by alkaline sucrose gradient sedimentation techniques. A component of DNA associated with a lipid containing material has been identified which contaminates native DNA after all extraction procedures used. Characterization of this material suggests that it represents DNA attached to a membrane fraction. Such material when present may be mistaken for ‘repair’ incorporation. The present data fit the generalization that only small numbers of bases are inserted/break and are in close ...

TOXICITY OF TRITIATED THYMIDINE IN P388F LYMPHOMA CELLS. I. INCORPORATION INTO DNA AND CELL SURVIVAL.

SummaryThe incorporation of thymidine into the newly-synthesized DNA of P388F lymphoma cells, increases non-linearly with respect to an increasing concentration of 3H-thymidine (TdR) of constant specific activity. As the external TdR concentration rises, in the presence of a constant low level of tritium a progressively greater proportion of the incorporation is hydroxyurea-resistant. These effects are attributed to negative-feedback actions of TdR metabolites on nucleotide biosynthesis.Thymidine incorporation is stimulated at high specific activities of 3H-TdR, largely owing to enhanced hydroxyurea-sensitive DNA synthesis, resulting from DNA damage produced by the incorporated tritium. For concentrations of 3H-TdR up to 8·4 mµM, reduced survival of P388F cells is solely the result of effects of tritium incorporated into newly-synthesized DNA.

The Implication of Repair Processes in the Mechanism of DNA Integration by Lymphoma Cells

SummaryThe uptake and integration of exogenous 3H-IUdR labelled DNA by irradiated cultures of an IUdR resistant mutant of P338F cells has been studied by preparative CsCl density gradient centrifugation. 10−2 M hydroxyurea was found to inhibit DNA synthesis as measured by uptake of 14C cytidine, but did not inhibit the integration of the exogenous DNA. This implies that enzymes involved in integration and repair are different from those concerned with DNA synthesis. ‘Repair’ synthesis of DNA has been demonstrated after 150 and 400 rads of x-rays. The amount of DNA uptake and integration was higher in irradiated cells and increased with increasing dose. Density-gradient centrifugation of DNA integrated into irradiated cells suggests the possibility of the release of nucleases from irradiated cells. The survival of the irradiated cells was unaffected by the integration of exogenous DNA.

CHARACTERISTICS OF REPAIR SYNTHESIS IN X-IRRADIATED P388F LYMPHOMA CELLS.

SummaryPreparative CsCl density-gradient centrifugation has been used to demonstrate ‘repair synthesis’ in P388F cells after 300 rads x-rays. Repair synthesis was stimulated two-fold when cells were grown in 5-iodo-2-deoxyuridine before irradiation. A time course has been determined by autoradiography and after DNA isolation in cultures treated with hydroxyrea (HU) to inhibit normal DNA synthesis. The rate of HU-insensitive DNA synthesis in irradiated cultures was found to exceed that in control cultures by a factor of two from 0–60 min after irradiation. Autoradiographic studies have shown that the radiation stimulated thymidine incorporation is restricted to S cells.

Interference in hybrid clone selection caused by Mycoplasma hyorhinis infection

Abstract Mycoplasma hyorhinis strains were isolated from Chinese hamster DON cells which lacked the ability to produce hybrid colonies in HAT medium. The mycoplasma isolates were virtually devoid of HGPRT activity in vivo and in vitro in the presence of excess co-enzyme, phosphoribosylpyrophosphate. Deliberate infection of mycoplasma-free cells caused no alterations in the HGPRT − and TK − phenotypes of the cells. Heterokaryon formation with infected cells was normal and the failure to produce hybrid colonies resulted from depletion, by nucleoside phosphorylase activity, of exogenous thymidine required for rescue of hybrid cells in HAT medium. Increasing the thymidine concentration and repeatedly replenishing HAT medium permitted hybrid clone formation.

Post-irradiation DNA degradation and response to fractionated doses in Micrococcus radiodurans.

SummaryThe response of M. radiodurans to fractionated doses given during the post-irradiation division delay has been studied. When incubated at 30°c under growth conditions, bacteria were most resistant when the second dose was given 1 or 2 hours after the first, and most sensitive between 3 and 4 hours. At 4°c smaller changes in sensitivity were observed. Incubation in PO4 buffer at 30°c resulted in greater sensitivity to divided doses than to the single dose at all times studied, i.e. no recovery. Loss of activity from 125IUdR-labelled cells incubated in broth after irradiation was much less at 4°c than at 30°c. Post-irradiation incubation of pre-labelled cells in PO4 buffer at 30°c resulted in significant but not progressive loss of activity. DNA degradation and fractionation response may be associated for cells held in broth, but when cells were held in buffer DNA degradation occurred in the absence of recovery. Two-dose response curves at 1, 2 and 3·5 hours demonstrated that the fractionation patter...