Synergistic effect of an Escherichia coli mutator gene on mutagenesis by ultraviolet radiation and by alkylating agents

E. coli strains differing in a gene responsible for high spontaneous mutability (mut HI) were compared for their mutability by UV radiation and by the alkylating agents ethyl methanesulfonate and methyl methanesulfonate. All three exogenous mutagenic agents induced significantly higher frequencies of mutants with impaired carbohydrate-fermenting ability when the mutator allele rather than the wild-type allele was present. Thus the mut HI gene product possibly increases the probability of replication error due to alterations in the structure of the template strand of DNA. An attempt to detect an synergistic effect for UV-induced suppressor mutations was unsuccessful. The failure may have been due to the particular method used for scoring this type of mutation.     

Assessment of the FAM174A 11G allele as a risk allele for equine metabolic syndrome

An 11G nucleotide repeat in the 3′ UTR of FAM174A was recently postulated as a risk allele with a dominant mode of inheritance for equine metabolic syndrome (EMS) and laminitis status in Arabian horses. The objective of this project was to evaluate this hypothesis in a large and diverse across-breed population. A total of 301 ponies, 292 Morgans, 64 Arabians, 49 Tennessee Walking Horses and 59 Quarter Horses were genotyped for six observed G repeat alleles in the FAM174A 3′ UTR. Phenotype data included laminitis status, baseline insulin, glucose, non-esterified fatty acids, triglycerides, adiponectin, leptin, ACTH, insulin and glucose post oral sugar test, and two proxies for insulin resistance. The 11G allele frequencies were 18.8, 6.9, 1.8, 0.2 and 0.0% in the Arabians, Tennessee Walkers, ponies, Morgans and Quarter Horses respectively. Association analyses between FAM174A genotype and EMS phenotypes, and between allele count and EMS phenotypes, identified no statistically significant associations. When a dominant effect for the 11G allele was evaluated, a statistically significant association with adiponectin levels was identified in the ponies, and pairwise comparisons revealed that the estimated marginal means were higher in ponies with the 11G allele vs. alternative alleles (i.e. the allele had a protective effect). In conclusion, our data do not support the FAM174A 11G allele as a risk allele for EMS in our studied breeds.     

Ethyl methanesulphonate mutagenesis with L5178Y mouse lymphoma cells: a comparison of ouabain, thioguanine and excess thymidine resistance.

Complete inhibition of growth of sensitive L5178Y mouse lymphoma cells in culture was obtained with 10(-3)M ouabain, 1.65 X 10(-3)M thymidine, 1.8 X 10(-4)M thioguanine and 10(-6)M cytosine arabinoside. The toxicity of methotrexate was dependent upon cell density and this compound was excluded from further study. The expression time before addition of the selective agent was important for detecting EMS induced resistant variants. Ouabain-resistant variants appeared immediately after treatment and were present over a broad time span. No excess thymidine- or thioguanine-resistant variants were seen initially; a peak in variant numbers was seen for excess thymidine resistance at 48-96 h and for thioguanine resistance at 144-192 h. Using induced mutation frequencies at optimum expression times, equal EMS treatments yielded substantially more variants resistant to thioguanine than to ouabain. It is suggested that this difference may have origin in possible constraints in the classes of mutants which are permissible in a vital function, maintenance of the Na+/K+ balance, when compared with a non-vital function, salvage purine biosynthesis. Some data are presented on the stability in culture of resistant variants. A limited number of observations were made following treatment in the peritoneal cavity of the mouse which were in broad agreement with the above results.     

The relationship between reaction kinetics and mutagenic action of monofunctional alkylating agents in higher eukaryotic systems. IV. The effects of the excision-defective mei-9L1 and mus(2)201D1 mutants on alkylation-induced genetic damage in Drosophila

Repair-defective mutants of Drosophila melanogaster which identify two major DNA excision repair loci have been examined for their effects on alkylation-induced mutagenesis using the sex-linked recessive lethal assay as a measure of genotoxic endpoint. The alkylating agents (AAs) chosen for comparative analysis were selected on the basis of their reaction kinetics with DNA and included MMS, EMS, MNU, DMN, ENU, DEN and ENNG. Repair-proficient males were treated with the AAs and mated with either excision-defective mei-9L1 or mus(2)201D1 females or appropriate excision-proficient control females. The results of the present work suggest that a qualitative and quantitative relationship exists between the nature and the extent of chemical modification of DNA and the induction of of genetic alterations. The presence of either excision-defective mutant can enhance the frequency of mutation (hypermutability) and this hypermutability can be correlated with the Swain-Scott constant S of specific AAs such that as the SN1 character of the DNA alkylation reaction increases, the difference in response between repair-deficient and repair-proficient females decreases. The order of hypermutability of AAs with mei-9L1 relative to mei-9+ is MMS greater than MNU greater than DMN = EMS greater than iPMS = ENU = DEN = ENNG. When the percentage of lethal mutations induced in mei-9L1 females are plotted against those determined for control females, straight lines of different slopes are obtained. These mei-9L1/mei-9+ indices are: MMS = 7.6, MNU = 5.4, DMN = 2.4, EMS = 2.4 and iPMS = ENU = DEN = ENNG = 1. An identical order of hypermutability with similar indices is obtained for the mus(2)201 mutants: MMS(7.3) greater than MNU (5.4) greater than EMS(2.0) greater than ENU(1.1). Thus, absence of excision repair function has a significant effect on mutation production by AAs efficient in alkylating N-atoms in DNA but no measurable influence on mutation production by AAs most efficient in alkylating O-atoms in DNA. The possible nature of these DNA adducts has been discussed in relation to repair of alkylated DNA. In another series of experiments, the effect on alkylation mutagenesis of mei-9L1 was studied in males, by comparing mutation induction in mei-9L1 males vs. activity in Berlin K (control). Although these experiments suggested the existence of DNA repair in postmeiotic cells during spermatogenesis, no quantitative comparisons could be made.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phenotypic expression time of mutagen-induced 6-thioguanine resistance in Chinese hamster ovary cells (CHO/HGPRT system).

Mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells (referred to as the CHO/HGPRT system) can be quantitated by selection for the phenotype of resistance to 6-thioguanine (TG) under stringently defined conditions. The phenotypic expression time, that is, the time interval after mutagen treatment which is necessary befor all mutant cells are able to express the TG-resistant phenotype, has been found to be 7–9 days in this CHO/HGPRT system when the cells are subcultured every 48 h. Subculture in medium with or without hypoxanthine (HX) utilizing trypsin, ethylenediaminetetraacetic acid (EDTA), or ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) for cell removal yields identical results. When subculture at intervals greater than 48 h is employed, a slight lengthening of the expression time is observed. An alternative method to regular subculture has also been achieved by maintaining the cells in a viable, non-dividing state in serum-free medium. This procedure yields a similar time course of phenotypic expression and thus shows that continued cell division is not essential to this expression process. In addition, this observation offers methodology which can significantly reduce the investment of time and money for mutation induction determinations in this mammalian cell gene mutation assay.     

Effects of chemical and physical mutagens on the frequency of a large genetic duplication in Salmonella typhimurium. I. Induction of duplications.

In Salmonella typhimurium a simple selection has been described to detect bacteria that are merodiploid for almost one-third of the chromosome. The selective procedure is based upon improved utilization of L-malate as the sole carbon source in merodiploid strains. The spontaneous frequency of the duplication in haploid strains is approximately 10(-4) per cell plated. Following the exposure of a haploid strain to mutagenic agents, there is a dose-dependent increase in the duplication frequency above the spontaneous level. In this paper we describe the induction of genetic duplications in Salmonella typhimurium by X-rays, ultraviolet light (UV), ethyl methanesulfonate (EMS), nitrous acid, and the azaacridine half mustard, ICR-372.     

In vitro breakage of plasmid DNA by mutagens and pesticides.

Covalently closed circular molecules of the colicinogenic plasmid E1 can serve as sensitive indicators for detecting in vitro breakage of DNA. After these molecules are radioactively labeled and purified by cesium chloride density-gradient centrifugation, they are incubated with the compounds to be tested. Samples are analyzed on alkaline sucrose gradients to determine the fraction of unbroken molecules and a breakage rate is calculated. Positive results were obtained for all three mutagenic alkylating agents (MMS, EMS, and MNNG) and of the 11 pesticides tested, dexon, dichlorvos, malathion, and methyl parathion induced breaks in molecules at a rate significantly greater than the controls.     

Coupling exo- and endocytosis: An essential role for PIP2 at the synapse

Chemical synapses are specialist points of contact between two neurons, where information transfer takes place. Communication occurs through the release of neurotransmitter substances from small synaptic vesicles in the presynaptic terminal, which fuse with the presynaptic plasma membrane in response to neuronal stimulation. However, as neurons in the central nervous system typically only possess ~ 200 vesicles, high levels of release would quickly lead to a depletion in the number of vesicles, as well as leading to an increase in the area of the presynaptic plasma membrane (and possible misalignment with postsynaptic structures). Hence, synaptic vesicle fusion is tightly coupled to a local recycling of synaptic vesicles. For a long time, however, the exact molecular mechanisms coupling fusion and subsequent recycling remained unclear. Recent work now indicates a unique role for the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP₂), acting together with the vesicular protein synaptotagmin, in coupling these two processes. In this work, we review the evidence for such a mechanism and discuss both the possible advantages and disadvantages for vesicle recycling (and hence signal transduction) in the nervous system. This article is part of a Special Issue entitled Lipids and Vesicular Transport.     

Effects of caffeine-induced defective DNA replication on SCE and chromosome aberrations produced by alkylating agents

The effect of caffeine (CAF) pretreatment (during the first cell cycle) on the frequency of sister-chromatid exchanges (SCE) and chromosome aberrations induced by bifunctional(MC)- and monofunctional(M-MC)-mitomycin C, 4-nitroquinoline N-oxide (4NQO) and ethyl methanesulphonate (EMS) were examined by using a BrdU—Hoechst staining technique. When CAF was added to the cultures during the first cell cycle in the presence of BrdU and then the cultures treated with MC, M-MC, 4NQO or EMS during the second cell cycle, the effect of the CAF was synergistic, i.e., the SCE level achieved was much higher than that expected from a simple additive effect of the agents and CAF. These results do not support the concept that the process of SCE is a manifestation of CAF-sensitive post-replication repair of DNA damage (single-strand exchanges), but, instead, point to exchanges between the double-strands of the DNA duplex present in each chromatid. CAF at certain concentrations is known to significantly slow down the rate of DNA-chain growth, but not appreciably induce strand breaks. Inasmuch as CAF alone induced only a small increase in SCE rates, possible mechanisms which may induce SCE are not only related to the slowing down of the rate of DNA-chain growth, but may also involve breaks in the template strand permitting double-strand exchanges to occur. The mechanisms responsible for chemically induced SCE are also discussed.     

Induction and expression of mutations in mammalian cells in the absence of DNA synthesis and cell division

The induction of mutations by the alkylating agent ethyl methanesulfonate (EMS) was determined with Chinese hamster ovary cells maintained in serum-free medium to arrest DNA synthesis and cell division. The arrested cultures were treated with EMS and maintained in serum-free medium for various time intervals post-treatment before serum containing medium was added to initiate DNA synthesis and cell division. The concentration-dependent increase in 6-thioguanine-resistant mutants in the arrested cultures was similar to that found with exponentially dividing cultures when serum was added to the arrested cultures immediately after the EMS treatment; the time course of phenotypic expression was also similar with both cultures. In addition, maintenance of the arrested cultures in serum-free medium for up to 18 days post-treatment resulted in no change in the mutant frequency. This suggests that the mutagenic damage is not removed in these arrested cultures. Furthermore, maintenance of the arrested state for increasing time intervals before serum addition results in decreases in the time necessary for maximum phenotypic expression. Cultures maintained in serum-free medium for 16 days after mutation treatment show complete expression of the mutations with no need for subculture. This last result suggests that the mutagenic damage induced by EMS in Chinese hamster ovary cells is not removed and that this damage results in both the induction and expression of mutation in the absence of DNA replication.