Search PubMed⌕ Search

Biomedical subjects

D G MacPhee

Publications and source records attributed to D G MacPhee.

At least 37 records · Page 2Linked to original sources
PubMed1994

Mutagenesis by 9-aminoacridine in Salmonella typhimurium: inhibition by glucose and other PTS class A carbon sources.

Reversion of the hisC3076 frameshift marker of Salmonella typhimurium has been measured following treatment of cells in growth and non-growth media with 9-aminoacridine (9AA). By varying the carbon source present in a defined medium, it has been shown that mutagenesis is reduced close to the spontaneous level in the presence of glucose whilst significant reductions are also observed with glucosamine, mannose, mannitol, fructose or glucose 6-phosphate. Intermediate mutant yields are observed when lactic acid or glycerol are present, whereas any one of a further group of carbon sources (gluconate, arabinose, ribose, succinate or casein hydrolysate) permit relatively large numbers of mutants to be recovered. Interestingly, when any one of these "high yield" carbon sources is supplemented with glucose the strong inhibitory effect characteristic of glucose is again observed. On the basis of these results, it can be concluded that inhibition of 9AA-induced reversion by a carbon source is not an exclusive property of glucose, although when more than one carbon source is present the inhibitory effect of glucose predominates. Possible explanations for these findings include the active exclusion of 9AA from cells as a direct consequence of glucose transport across the cell membrane. To address this possibility, cells were pre-grown in verapamil, a calcium channel antagonist which is known to increase the mutagenicity of various 9-anilinoacridine derivatives in S. typhimurium. We found that glucose inhibition of 9AA-induced mutagenesis was not relaxed to any significant extent following treatment with verapamil. In a further experiment, two glucose analogues (2'-deoxyglucose and methyl-D-glucoside) known to be actively transported into the cell but not metabolised past the first phosphorylation step were used. These analogues inhibit the transport into the cell of several types of molecules, but since they do not significantly depress 9AA mutagenesis it seems unlikely that blockage of 9AA transport across the cell membrane can be invoked to explain the inhibitory effect of glucose on 9AA mutagenesis. An alternative explanation based on glucose-mediated repression of an error-prone, mutation-generating, DNA-repair process is presented.

Aminacrine↗
PubMed1993

Glucose inhibition of mutagenesis by 9-aminoacridine in Salmonella typhimurium.

Back mutation to prototrophy of the hisC3076 marker of Salmonella typhimurium has been measured following treatment with 9-aminoacridine (9AA) under conditions in which growth is either permitted or not permitted. Cells treated with 9AA in buffer (i.e. under conditions in which little or no replication was possible) were found to respond well to 9AA-induced mutagenesis. This remained true whether or not the plating medium contained trace amounts of histidine to allow residual replication of His- cells (as for example in the Ames test); yields of His+ mutants were also about the same regardless of whether the plating medium contained glucose or glycerol as the sole carbon source. By contrast, 9AA-induced mutagenesis was essentially abolished when cells were treated in buffer containing 1% glucose (i.e. under conditions which strongly favoured replication). Glucose inhibition of 9AA-induced mutagenesis began to be apparent at glucose concentrations of about 0.02%, whilst total abolition was observed at about 0.2%. In addition, glucose inhibition was found to be dependent on the concentration of 9AA, the induced mutation rate increasing at levels of up to 100-150 micrograms/ml of 9AA and then declining steeply at higher levels. Further kinetic studies indicated that glucose could depress the 9AA-induced mutation rate significantly even in cells exposed to the mutagen for up to 18 min prior to the addition of glucose, whilst inhibition of 9AA mutagenesis by glucose was both temporary and reversible.

Aminacrine↗
PubMed1993

Directed mutation: paradigm postponed.

Claims have been made in recent years that spontaneous mutational events are "directed" by (i) the presence in a selective medium of a single carbon source which the cells are unable to utilize (e.g. lactose), or (ii) the absence from a selective medium of an amino acid which the cells are unable to make for themselves (e.g. tryptophan). In resurrecting the previously rejected hypothesis of a "directed" origin for spontaneous mutants, it was noted that selecting for valine-resistant mutants did not allow comparable increases in colony numbers to be observed when overlays containing valine and glucose were added to plates which had accumulated (or were still accumulating) large numbers of "directed" Lac+ or Trp+ mutants. The present paper shows that Valr mutants do occur in much greater numbers if a carbon source other than glucose is added to the plates along with the valine-containing overlays; the evidence is that even small amounts (< 0.02%) of glucose in the overlays prevent the recovery of Valr mutants. From these results, it is argued that the apparent "directed" nature of the spontaneous mutation process is actually a manifestation of the long-known phenomenon of glucose repression.

Animals↗
PubMed1993

Origins of spontaneous mutants: cellular strategies for controlling their formation in response to changing environments.

Spontaneous and induced mutational events may not be as readily distinguishable as has been generally assumed. It is suggested that the later stages of the processes leading to the establishment of fixation of mutations may consist of a 'mutational pathway', with many features in common with the better-known metabolic pathways of organisms like Escherichia coli. If so, the mutational pathway may be controlled by catabolite repression, a cellular mechanism which switches metabolic pathways on and off (or more probably up and down) in response to intracellular levels of a molecular messenger known as cyclic adenosine monophosphate (cAMP). Mutation rates could then be regulated by an intracellular messenger in precisely the way which we would predict they ought to be regulated, if they are to cope with evolutionary pressures for increased variability.

Biological Evolution↗
PubMed1991

Precise excision of Tn10 in Salmonella typhimurium: effects of mutations in the polA, dam, mutH and mutB genes and of methionine or ethionine in the plating medium.

Precise excision of Tn10 occurs at significantly elevated frequencies in cultures of the polA7 mutant strain of Salmonella typhimurium, is further increased in polA7 dam-1 and polA7 mutB strains and decreased in a polA7 mutH background. The numbers of precise excision events occurring in polA7 strains are also significantly increased when methionine (20 micrograms/ml or less) is present in the medium but decreased when ethionine (again, 20 micrograms/ml or less) is present. When both amino present, the outcome is about a 2-fold increase in precise excision events. The involvement of mismatch repair and methylation patterns in precise excision events is discussed.

DNA Repair↗
PubMed1990

Fluctuation tests are more sensitive than plate tests in detection of chemicals which cause enhanced excision of transposon Tn10 in Salmonella typhimurium.

Precise excision of transposon Tn10, as judged by reversion of Salmonella typhimurium strain LT2 trp1014::Tn10 to Trp+, was not detectably enhanced following exposure to 9-aminoacridine, 5-azacytidine or mitomycin C in conventional treat-and-plate assays. By contrast, 7/13 chemicals, including 5-azacytidine and mitomycin C, were found to be capable of enhancing precise excision of Tn10 when tested in modified fluctuation assays. Despite earlier reports, precise excision is one activity of transposons which is not therefore refractory to enhancement by chemical mutagens.

DNA Transposable Elements↗
PubMed1989

Development of bacterial mutagenicity tests: a view from afar.

A brief (and subjective) history of the progressive development of bacterial mutagenicity assays with Salmonella typhimurium and Escherichia coli is presented, with emphasis on the need to view such assays as being capable of detecting genetically active substances rather than being dedicated to the detection of carcinogenic chemicals. The role of mutation-enhancing plasmids in the improvement of Salmonella tester strains and the need for batteries of tests to allow the detection of genetic endpoints that cannot be detected with bacteria (e.g., aneuploidy) are also discussed.

Escherichia coli↗
PubMed1989

Modulation of mutagenesis involving precise excision of transposon Tn10.

Precise excision of transposon Tn10 results in reversion of the Trp- phenotype to Trp+ in a trp-1014::Tn10 strain of Salmonella typhimurium, and also occurs at a markedly higher frequency in a strain carrying the temperature-sensitive polA7 allele. The frequency with which precise excision events occurs can be modified by the plating medium, results indicating that the great majority of mutants which arise on broth-supplemented or tryptophan-supplemented minimal media actually arise on the selective plating medium. Trp+ revertants (1000) arising from excision of Tn10 were purified by re-streaking for single colonies; none were found to retain the Tn10 encoded resistance to tetracycline. Yields of Trp+ revertants of the polA7 strain were consistently higher when glycerol rather than glucose was used as sole carbon source in the selective medium. Clean excision of Tn10 can also be increased by ultraviolet irradiation in (R) plasmid-free strains, and is further increased in strains carrying an N-group plasmid (R205, R46 or pKM101). Ultraviolet-induced precise excision of Tn10 also occurs at a much enhanced frequency in a strain with a deletion through the uvrB gene; in this case, however, the addition of plasmid pKM101 leads to a decrease in yields of ultraviolet-induced precise excision events.

Chromosome Deletion↗
PubMed1989

Mutagenesis by the anti-tumour drug nitracrine in Escherichia coli.

The antitumour drug nitracrine [1-nitro-9-(dimethylaminopropylamino)acridine], known to be a potent frameshift mutagen in strains of Salmonella typhimurium, also strongly reverts the lacZ19124 frameshift marker in Escherichia coli. The results in E. coli indicate that nitracrine causes DNA damage which can be excised by the UvrA,B,C excinuclease, can generate mutations by a recA-dependent mechanism, and gives enhanced yields of mutants when plasmid pKM101 is present. Despite these observations, mutagenesis by nitracrine appears to be independent of the UmuC gene product, and hence nitracrine differs from most (but not all) other chemicals which generate mutations via the SOS response. Given that umuC mutants are about as mutable by nitracine as the wild-type parent strain, it is somewhat surprising that plasmid pKM101 causes an enhancement of nitracrine mutagenesis. Nevertheless, we have found that the observed enhancement of mutagenesis by pKM101 is a function of the mucB gene, normally assumed to be essentially homologous to the umuC gene.

Aminoacridines↗
PubMed1987

RecA-independent mutagenesis in Escherichia coli may be subject to glucose repression.

The frameshift mutagen 9-aminoacridine (9AA) causes DNA damage via a recA+-independent mechanism in Escherichia coli. In this study we have exposed E. coli cells carrying the lacZ19124 frameshift marker to 9AA in defined minimal media, washed them, and plated to score for Lac+ revertants. Our results show that 9AA-induced reversion to Lac+ occurs in the absence of any exogenous carbon source and when cells are plated on media which do not allow much, if any, cell replication prior to expression of the revertant phenotype. When glycerol (1% w/v) was added to the liquid treatment medium, the number of Lac+ E. coli revertants was similar to that obtained when no carbon source was present. By contrast the addition of glucose (1% w/v) during the mutagenesis treatment caused a significant decrease in the number of revertants. Further experiments indicate that the repressing effects of glucose may be due to a reduction in cAMP concentration, since 9AA mutagenesis was abolished in a cya strain in which no adenylate cyclase is produced. These results are consistent with (but do not prove) the notion that at least one part of the process leading to 9AA mutagenesis is subject to catabolite repression.

Adenylyl Cyclases↗
PubMed1987

Frameshift mutagenesis by chloroquine in Escherichia coli and Salmonella typhimurium.

Chloroquine can be detected as a direct-acting mutagen in plate-incorporation assays using the excision-deficient Salmonella typhimurium strain TA97, but very much more effectively using the repair-proficient Escherichia coli strain DG1669 which carries the lacZ19124 marker. When tested at concentrations of 200-1000 micrograms/plate with strain DG1669, the mutagenicity of chloroquine is enhanced by the addition of Aroclor-induced rat-liver S9. Further experiments indicated that chloroquine-induced reversion frequencies were essentially identical in wild-type, recA, umuC and uvrC derivatives of DG1669, as well as in strains carrying the mutation enhancing plasmid pKM101, over a wide range of doses (0-1200 micrograms/plate). These results suggest that neither excision repair nor SOS-type repair are important in chloroquine-induced frameshift mutagenesis.

Animals↗
PubMed1987

Influence of caffeine on mitomycin C induced mutagenesis: a simple explanation.

It has recently been suggested that caffeine probably represses mutations induced by mitomycin C by selectively killing cells with damaged DNA resulting from inhibition of an error-prone repair process which fails to be completed. A more likely explanation is that caffeine exerts its inhibitory effects on mitomycin C mutagenesis because of its well-documented ability to inhibit urvA.B.C-dependent excision repair.

Caffeine↗
PubMed1986

Rapid and complete degradation of thymidine by human peripheral blood platelets: implications for genotoxicity assays.

Small-scale washed cell preparations obtained by Percoll density-gradient fractionation of whole blood were used to study the metabolic fate of [3H]thymidine supplied to isolated human blood mononuclear cells and platelets incubated for up to 24 h in vitro. Two cell fractions were monitored: low molecular weight compounds which were soluble in Triton X-100 and TCA were investigated by thin-layer chromatography, and high molecular weight components, distinguished by their Triton and TCA insolubility, were examined by agarose-gel electrophoresis. Under the conditions used, greater than 99% of added [3H]thymidine was very rapidly degraded. Catabolites were recovered in the Triton-soluble (cytoplasmic) fraction and the extracellular medium. A negligible proportion of added label was associated with Triton- and TCA-insoluble cell fractions. These results confirm and clarify previous data and have important implications for genotoxicity tests which employ in vitro leukocyte cultures.

Blood Platelets↗
PubMed1986

RecA-independent mutagenesis in Escherichia coli: effects of umuC and mucB mutations.

We have studied the effects of a umuC mutation on reversion of the lacZ19124 and lacZ19136 frameshift markers of Escherichia coli. Introduction of a umuC::Tn5 mutation into a strain carrying the lacZ19136 marker resulted in enhanced reversion by 9-aminoacridine and the acridine half-mustard ICR191, whereas reversion of the lacZ19124 marker was decreased (but not abolished) in a umuC strain. The reversion frequency of the lacZ19136 marker was decreased by the presence of plasmid pKM101, and further decreased by a derivative of pKM101 in which the mucB gene was inactivated by a Tn5 insertion. The reversion frequency of the lacZ19124 marker was relatively unchanged by either plasmid. Since both 9-aminoacridine and ICR191 mutagenesis of these strains is independent of the recA+ and lexA+ gene products, these results may suggest a broader role for the UmuC protein in regulating induced mutation frequencies than has previously been suspected. The contrasting effects of the umuC and mucB mutations on reversion of the lacZ19136 marker may suggest a copy number effect, or perhaps more likely that there are inherent (functional) differences between the UmuC and MucB proteins.

Aminacrine↗
PubMed1985

Frameshift mutagenesis by ultraviolet and gamma radiations in Salmonella: SOS inducibility and effects of DNA polymerase I.

Ultraviolet (UV) and gamma-induced mutagenesis have been studied using a doubly auxotrophic strain of Salmonella typhimurium carrying the amber leuA150 mutation (which reverts by base-pair substitution) and the frameshift hisC3076 marker (which reverts by compensating frameshifts). In the initially constructed LT2 background, both markers were poorly revertible by UV and essentially non-revertible by gamma-radiation. A derivative of this strain carrying the mutation-enhancing plasmid pKM101 was however readily reverted by both UV and gamma, with either Leu+ (base substitution) or His+ (frameshift) revertants being observed on appropriate selective media. Photoreactivation experiments suggested that the lesions leading to formation of the two types of mutagenic event were similar if not identical. Support for this suggestion was obtained when it was found that yields of both types of UV-induced revertant were significantly increased in an excision-deficient background, while no revertants of either type were found in a recA background. Yields of gamma-induced revertants were not greatly altered in a uvrB background, but were also reduced to zero (for both markers) in the recA background. These results are consistent with what has previously been well-documented for UV and gamma-induced base-pair substitution mutagenesis, and serve to emphasize the similarities between base-pair substitution mutagenesis and frameshift mutagenesis by these agents. There are differences, however, since although UV-induced reversion of the leuA150 marker was little affected and gamma-induced reversion of leuA150 was somewhat reduced in the presence of a polA mutation (polA3), the yields of His+ frameshift revertants were significantly increased in the polA3 background following treatment with either UV or gamma. Thus while inducible DNA repair (SOS repair) appears to be involved in generating both types of mutational event following either UV- or gamma-irradiation, at some stage in the processing of premutational lesions the level (or type) of DNA polymerase I activity in the cell seems to have an important role in determining whether or not frameshifts or base-pair substitutions will be produced at a particular frequency.

DNA Polymerase I↗