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T G Rossman

Publications and source records attributed to T G Rossman.

At least 55 records · Page 3Linked to original sources
PubMed1985

HGPRT- mutants of V79 cells that revert specifically by base pair substitution and frameshift mutations.

In order to determine the mutagenic specificity of mutagenic and carcinogenic agents in mammalian cells, a reversion system capable of distinguishing between frameshift mutations and various kinds of base pair substitutions would be useful. We report here a method for the isolation and characterization of HGPRT- Chinese hamster V79 cell mutants that might form the basis for such a system. Two mutants of different specificity have been partially characterized. DEW-1, isolated following N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment, is revertible by the base pair substitution mutagens MNNG and ethyl methanesulfonate (EMS), but not by frameshift mutagens. DSW-3, isolated following ICR-191 treatment, is specifically reverted by frameshift mutagens, but not by EMS or MNNG. With the further characterization of these and other mutants, it should be feasible to determine not only whether an agent is mutagenic in V79 cells, but also to determine the type(s) of mutation(s) it produces.

Animals↗
PubMed1984

The genetic toxicology of metal compounds: I. Induction of lambda prophage in E coli WP2s(lambda).

A number of metal compounds have been shown to be human carcinogens. Others, while not proven human carcinogens, are able to cause tumors in laboratory animals. Short-term bacterial assays for genotoxic effects have not been successful in predicting the carcinogenicity of metal compounds. We report here the ability of some metal compounds to cause the induction of lambda prophage in E coli WP2s(lambda). By far the strongest inducing ability was observed with K2CrO4, followed by Pb(NO3)2 greater than MnCl2 greater than Ni(OOCCH3)2 greater than CrCl2 greater than NaWO4 greater than Na2MoO4 greater than KMnO4. With the exception of chromate, long-term exposures in a narrow, subtoxic dose range were required in order to demonstrate phage induction. A new microtiter assay for lambda prophage induction, which incorporates these features, is described. This system also was able to detect very small amounts of organic carcinogens.

Bacteriophage lambda↗
PubMed1983

Effects of bisulfite (sulfur dioxide) on DNA synthesis and fidelity by DNA polymerase I.

In an attempt to explain the mechanism of comutagenesis by bisulfite, the extent and accuracy of DNA synthesis by E. coli DNA polymerase I was examined in the presence of sodium bisulfite. Bisulfite concentration of 100 mM caused nearly complete inhibition of dNTP incorporation into activated calf thymus DNA. Other salts (NaCL, Na2SO4) at the same concentration had no effect on enzyme activity. Preincubation of the various DNA synthesis assay components in 100 mM bisulfite showed that only preincubation of DNA polymerase I caused inhibition of DNA synthesis. Exonuclease functions of DNA polymerase I were unaffected by up to 100 mM bisulfite. Accuracy of DNA synthesis in the presence of bisulfite was determined using poly (dA-dT) as a template-primer. Concentrations of bisulfite greater than 50 mM caused a progressive decrease in enzyme accuracy. At 100 mM bisulfite there was an approximate 7.5-fold decrease in the fidelity of DNA synthesis, compared to control values, as measured by the ratio of noncomplementary (dGTP) to complementary (dTTP) nucleotide incorporated. Based on the known chemistry of bisulfite, it is hypothesized that sulfitolysis of the one disulfide group in DNA polymerase I by bisulfite might be responsible for the reduced polymerase activity and accuracy. The exonuclease functions of DNA polymerase I do not seem to require the disulfide linkage. These results suggest that the effects of bisulfite on mutation frequency might be mediated by effects on the fidelity of DNA repair systems.

DNA Polymerase I↗
PubMed1983

The effects of bisulfite on growth and macromolecular synthesis in Escherichia coli.

Bisulfite reversibly inhibits the growth of a variety of microorganisms and has been used as a preservative in foods and beverages for that reason. We have now measured macromolecule synthesis in Escherichia coli K12 after bisulfite treatment. RNA synthesis, the synthesis of total protein, and of an inducible enzyme, beta-galactosidase, stopped almost immediately upon addition of 2 mM (or higher concentrations) of bisulfite. These functions resumed after a lag whose duration depended on the concentration of bisulfite added. The synthesis of DNA was slowed upon bisulfite addition, but did not stop entirely. The inhibition of RNA synthesis by bisulfite took place in both stringent and relaxed strains of E. coli and was not relieved upon addition of chloramphenicol. Stringent control was therefore not involved in this effect. No effect on protein synthesis was observed in the cell-free system of E. coli (using poly(U) or MS2 RNA as messenger) at bisulfite concentrations up to 10 mM. Protein synthesis inhibition in vivo was apparently not due to a reaction of bisulfite with a component of this system. In additional experiments, RNA polymerase was not impaired by bisulfite, and the growth inhibition effect was shown to proceed in the presence of inhibitors of free radical chain reactions.

Escherichia coli↗
PubMed1983

Rat liver S9 preparations contain comutagenic activity.

Rat liver S9 preparations contain material which causes enhancement of UV mutagenesis in Escherichia coli WP2. This comutagenic activity is present in S9 preparations from both uninduced and Aroclor-induced rats. Strains of E. coli which are defective in the uvr-dependent excision repair pathway fail to show comutagenic action by S9. The comutagenic material is heat-labile and non-dialyzable, suggesting that it might be protein. This differs from the small amount of mutagenic material present in rat liver S9, as the latter is dialyzable and can be demonstrated in the repair-deficient strain E. coli WP2s (uvrA).

Animals↗
PubMed1982

Demonstration of recovery from the potentially mutagenic effects of ultraviolet light by replication-inhibited Chinese hamster V79 cells.

The effects of replication-inhibiting conditions on the ability of Chinese hamster (V79) cells to recover from the potentially mutagenic effects of ultraviolet (UV) light were investigated. V79 cells were synchronized by a new technique using a low concentration of hydroxyurea (0.2 mM), which provided a mildly-toxic, nonmutagenic method for producing large quantities of synchronized cells needed for these studies. The protocol developed for this study involved UV-irradiation of synchronized V79 cells which were blocked at the Gl/S boundary. Following UV-irradiation, the cells were either allowed to enter S phase immediately or were blocked for increasing periods of time by the addition of more hydroxyurea. The former cells contained the highest frequencies of ouabain-resistant mutants, while cells whose replication was blocked following UV-irradiation showed decreasing mutation frequencies with respect to time. Therefore, V79 cells are able to demonstrate liquid holding recovery from potentially mutagenic UV-lesions. Since the UV-induced mutation frequency was reduced by almost 50% following 6 h of 'liquid holding', the mutagenic lesions seem to be removed at a faster rate than has previously been reported for the removal of pyrimidine dimers from these cells.

Animals↗
PubMed1981

Effects of inhibitors of de novo protein synthesis on UV-mutagenesis in Chinese hamster cells. Evidence against mutagenesis via inducible, error-prone DNA repair.

Experiments were designed to test whether UV-mutagenesis in animal cells requires an inducible error-phone DNA-repair system similar to the "SOS system" in E. coli. Chinese hamster (V79) cells were exposed to either 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB, a transcription inhibitor), cycloheximide, or puromycin for various times (3-6 h) following UV-irradiation (2-8 J/m2). Post-irradiation treatment with DRB resulted in a reproducible enhancement of UV-induced mutagenesis, whereas post-irradiation treatment with either cycloheximide or puromycin resulted in decreased UV-mutagenesis. Thus, the frequency of UV-mutagenesis does not appear to be dependent on an inducible error-prone DNA-repair pathway, since all 3 agents share the ability to inhibit de novo protein synthesis. In order to understand the effects of these inhibitors on mutation frequency, DNA synthesis in the presence of these agents was examined. DRB stimulated DNA synthesis in both irradiated and unirradiated cells. On the other hand, cycloheximide and puromycin caused an immediate inhibition of DNA synthesis in both irradiated and unirradiated cells. Therefore, it appears that UV-mutagenesis reflects changes in post-irradiation DNA synthesis rather than post-irradiation de novo protein synthesis.

Animals↗
PubMed1981

Bisulfite (sulfur dioxide) is a comutagen in E. coli and in Chinese hamster cells.

The mutagenic and comutagenic effects of bisulfite were investigated. Under moderate exposure conditions (high survival) it was found that bisulfite is not mutagenic to either eukaryotic cells (Chinese hamster V79), or prokaryotic cells (Escherichia coli). However, bisulfite does act as a comutagen with UV irradiation. Bisulfite approximately doubles the mutation frequency in UV-irradiated Chinese hamster V79 cells, and it causes a greater than 8-fold increase in Trp+ revertants in UV-irradiated E. coli. The comutagenic effect occurs whether cells are exposed to bisulfite during or immediately after UV irradiation. Kinetic studies of the comutagenic effect in E. coli shows that it decays in a biphasic manner, with an apparent half-life of 15 min and a persistence of the comutagenic effect for up to 120 min after UV irradiation. Experiments with several strains of E. coli of varying DNA-repair capacities indicate that excision repair is necessary for a comutagenic effect by bisulfite. It is thought that bisulfite acts to inhibit excision repair, perhaps by effects on DNA polymerase I, or DNA ligase.

Animals↗
PubMed1981

Effect of metals on mutagenesis and DNA repair.

Unlike the situation with organic compounds, metals do not show a high correlation between carcinogenicity and mutagenicity. An agent may be mutagenic by causing misreplication of DNA due to alterations of the DNA template, decreased fidelity of DNA polymerase, or inhibition of the proofreading of DNA replication. In addition, bacteria have an inducible, error-prone DNA repair system (SOS repair) whose activity results in mutagenesis. In the best studied example of metal mutagenesis, chromate, there is little evidence for the involvement of the SOS system. Metals may act as comutagens by inhibiting the repair of damage to DNA caused by another agent. This has been demonstrated for arsenite. Comutagens would not be detected by standard screening methods.

Animals↗
PubMed1980

Absence of arsenite mutagenicity in E coli and Chinese hamster cells.

Because exposure to compounds of arsenic has been implicated as a cause of cancer, an investigation of the mutagenicity of sodium aresenite in E coli and in Chinese hamster cells was performed. In the E coli system, Trp + revertants were not induced by arsenite in a variety of experimental protocols including spot tests, treat and plate protocols, and fluctuation tests. The mutator effect of a tif-1 strain was partially blocked by arsenite. Arsenite did not cause the induction of lambda prophage. In Chinese hamster cells, arsenite did not induce ouabain-resistant mutants or thioguanine-resistant mutants. We conclude that arsenite is not mutagenic to E coli or to Chinese hamster cells. When added to the plating medium, arsenite can act as an anti-mutagen by inhibition of induction of the error-prone SOS repair system in E coli.

Animals↗
PubMed1977

Effects of arsenite on DNA repair in Escherichia coli.

Since environmental exposure to arsenicals has been correlated with a high skin cancer risk among populations exposed to sunlight, it is possible that arsenicals might interfere with the repair of damage to DNA (mostly thymine dimers) resulting from the ultraviolet rays in sunlight. To test this hypothesis, strains of E. coli, differing from each other only in one or more repair functions, were exposed to UV light and then plated in the presence or absence of sodium arsenite. Survival after irradiation of wild type E. coli (WP(2)) was significantly decreased by 0.5mM arsenite. This effect was also seen in strains which are unable to carry out excision repair, suggesting that arsenite inhibits one or more steps in the post-replication repair pathways. This is confirmed by the finding that arsenite has no effect on the post-irradiation survival of a recA mutant, which does not carry out post-replication repair. Mutagenesis after ultraviolet irradiation depends on the rec(+) and lex(+) genes. Arsenite decreases mutagenesis in strains containing these genes. In order to determine its mechanism of action, dose-response relationships of arsenite on a number of cellular functions were carried out. The most sensitive cellular functions found were the induction of beta-galactosidase and the synthesis of RNA. Since error-prone repair in E. coli is an inducible process, the inhibition of mutagenesis after UV irradiation may be the result of inhibition of messenger RNA synthesis.

Animals↗
PubMed1977

Culture of cloned cells from the mouse preputial gland tumor ESR 586.

Cells from the mouse preputial gland tumor ESR 586 have been cultured and cloned. Trypsin-ethylenediaminetetraacetate was used to obtain single cells. The cells are grown in a modified CMRL-1415 medium supplemented with 10% fetal calf serum. Clones tend to fall into two classes: Class 1, those that undergo morphological differentiation in liquid medium to form round bodies filled with lipid vaculoes; and Class 2, those that do not differentiate. Preliminary studies on the control of differentiation in Class 1 clones suggest that a minimum cell density is required before differentiation takes place. Analysis of the lipids from differentiating and nondifferentiating clones reveals the presence of sebaceous-type lipids in differentiating clones only. No requirement for testosterone was found for these cells.

Animals↗
PubMed1977

Ultrastructural changes and viral morphogenesis during the growth of the mouse preputial gland tumor ESR 586.

A study was undertaken to observe morphological changes and viral morphogenesis during the growth of the mouse preputial gland tumor ESR 586. The acinar cells of the normal preputial gland have an extensive agranular endoplasmic reticulum and Golgi apparatus and large lipid droplets. No viral particles are present. During tumor growth, and numerous lipid droplets never attain the size of those found in the normal gland. There is a decrease in the Golgi apparatus and agranular endoplasmic reticulum and an increase in the rough endoplasmic reticulum which could reflect a change in composition of tumor secretory product from the normal gland. Indeed, there is a decrease in triglycerides as the tumor ages and an increase in the sterol esters and waxes. In addition, intracisternal A-particles are observed budding from thickened endoplasmic reticulum membranes. Thickening of these membranes occur early in A-particle formation. One side of the membrane is first thickened while the opposing membrane appears is first thickened while the opposing membrane appears morphologically unaffected. The thickening of the affected membrane is initially confined to the outer (cytoplasmic) face of the membrane. In the older tumor, both opposing membranes of the reticulum are thickened and can assume an elongated whorled pattern.

Adenocarcinoma↗