Isolation of auxotrophic mutants ofAspergillus niger by ethylmethane sulphonate treatment

The alkylating agent EMS can be employed for the isolation of auxotrophic mutants of theAspergillus niger strain K 10. The maximum number of auxotrophic mutants (2.9–5.1%) corresponded approximately to the number of mutants obtained by EMS treatment of yeasts, but it was by order lower than the number of mutants generally obtained by EMS treatment in bacteria. The majority of isolated mutants grew worse than the parent strain in the liquid medium and also formed lower amount of organic acids. The organic acid which was most frequently accumulated by mutants was citric acid.     

Cell killing by various monofunctional alkylating agents in Chinese hamster ovary cells

Cell killing by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU), and methyl methanesulfonate (MMS) was measured in Chinese hamster ovary (CHO) cells using the colony-formation assay. Cell killing by these agents was determined in exponentially growing asynchronous cells, in synchronous cells as a function of cell-cycle position and in nondividing cells. Distinct differences in the cytotoxic effect of the 4 alkylating agents were found in respect to dose-response, cell cycle phase-sensitivity and growth state. MNNG and MNU showed the same biphasic dose-survival relationship in exponentially growing cells, with an initial steep decline followed by a shallow component. The shallow component disappeared in growth-arrested cells. MNNG and MNU differed, however, in the cell-cycle age response. No cell-cycle phase difference was seen with MNNG, whereas cells in G1 seemed more sensitive to MNU than cells in S phase. MMS and ENU both showed shouldered dose-response curves for exponentially growing asynchronous cells, and the same cell-cycle pattern for synchronous cultures with cells in early S phase being the most sensitive. However, survival of nondividing cells versus dividing cells was reduced much more by MMS than by ENU. Caffeine, which interferes with the regulation of DNA synthesis and is known to modify cell killing by DNA-damaging agents, enhanced cell killing by all agents. It is concluded that there must be a number of factors which contribute to cell killing by monofunctional alkylating agents, and that besides alkylation of DNA reaction with other cellular macromolecules should be considered.     

Defective excision repair in a mutant of Micrococcus radiodurans hypermutable by some monofunctional alkylating agents

Summary The lethal and mutagenic effects of methyl methanesulphonate (MMS), ethyl methanesulphonate (EMS), and N-methyl-N-nitro-N-nitrosoguanidine (MNNG) can be dissociated in a mitomycin C (MTC)-sensitive mutant, strain 302, of Micrococcus radiodurans.As regards lethality 302 is extremely sensitive, compared with the wild type, to MTC and decarbamoyl MTC (DCMTC), slightly sensitive to EMS, MNNG, nitrous acid, 7-bromomethylbenz {} anthracene (BrMBA), and N-acetoxy-N-2-acetylaminofluorene (AAAF), and resistant to MMS, hydroxylamine, and ICR 191G. As regards mutability it is, compared to the wild type, very sensitive to MMS, EMS, and MNNG, and slightly sensitive to hydroxylamine and nitrous acid but not to any other agent examined.Alkaline sucrose gradient studies indicate that 302 does not incise DNA containing BrMBA adducts, although it does incise DNA damaged by AAAF but probably not to the same extent as wild type.We put forward the hypothesis that the hypermutability of 302 is due to the non-removal of bases or nucleotides, modified in exocyclic positions, which have altered base-pairing capabilities, while lethality results from the non-removal of bases or nucleotides, also modified in exocyclic positions, which no longer form hydrogen-bonded base pairs.     

Role of the processes of repair of DNA damage in the repair of cytogenetic disorders. Participation of the repair of DNA single-strand breaks in the repair of cytogenetic disorders

A comparison was made between the results of the effect of poly(ADP-ribosylation) inhibitors (e.g. nicotinamide and 3-aminobenzamide) and a chromatin proteinase inhibitor, phenylmethylsulfonylfluoride, on the cytogenetic damages repair, by a micronucleus test and DNA repair in Chinese hamster fibroblasts. The values of the repair half-periods (5-7 min for the cytogenetic damages and 5 min for the rapidly repaired DNA damages) and a similar modifying effect with regard to radiation cytogenetic damages and kinetics of DNA damages repair were found to be close. This confirms the contribution of repair of DNA single-strand breaks in the initiation of structural damages to chromosomes.     

Influence of exogenous DNA on Ypenyl-treated chromosomes ofVicia faba L.

This paper is a study of the effect of exogenous DNA of different genetic origins on the repair of meristematic cells of primary roots ofVicia faba, damaged by 24 hour treatment with 0·01mm solution of Ypenyl. Both kinds of DNA,i.e. isologous and heterologous, stimulated cell proliferation which was decreased by the action of the radiomimetic and influenced both dynamics of production of chromosome aberrations and the interchromosomal distribution of induced damage. While heterologous DNA increased the frequency of aberrations after all recovery periods studied, isologous DNA significantly decreased the number of chromosomal aberrations. Heterologous DNA increased at the same time the relative number of breaks in the group of small chromosomes, while by the action of isologous DNA the number of aberrations related to this group of chromosomes was relatively decreased.     

DNA repair modulates the vulnerability of the developing brain to alkylating agents

Neurons of the developing brain are especially vulnerable to environmental agents that damage DNA (i.e., genotoxicants), but the mechanism is poorly understood. The focus of the present study is to demonstrate that DNA damage plays a key role in disrupting neurodevelopment. To examine this hypothesis, we compared the cytotoxic and DNA damaging properties of the methylating agents methylazoxymethanol (MAM) and dimethyl sulfate (DMS) and the mono- and bifunctional alkylating agents chloroethylamine (CEA) and nitrogen mustard (HN2), in granule cell neurons derived from the cerebellum of neonatal wild type mice and three transgenic DNA repair strains. Wild type cerebellar neurons were significantly more sensitive to the alkylating agents DMS and HN2 than neuronal cultures treated with MAM or the half-mustard CEA. Parallel studies with neuronal cultures from mice deficient in alkylguanine DNA glycosylase (Aag^?/?) or O⁶-methylguanine methyltransferase (Mgmt^?/?), revealed significant differences in the sensitivity of neurons to all four genotoxicants. Mgmt^?/? neurons were more sensitive to MAM and HN2 than the other genotoxicants and wild type neurons treated with either alkylating agent. In contrast, Aag^?/? neurons were for the most part significantly less sensitive than wild type or Mgmt^?/? neurons to MAM and HN2. Aag^?/? neurons were also significantly less sensitive than wild type neurons treated with either DMS or CEA. Granule cell development and motor function were also more severely disturbed by MAM and HN2 in Mgmt^?/? mice than in comparably treated wild type mice. In contrast, cerebellar development and motor function were well preserved in MAM-treated Aag^?/? or MGMT-overexpressing (Mgmt^Tg+) mice, even as compared with wild type mice suggesting that AAG protein increases MAM toxicity, whereas MGMT protein decreases toxicity. Surprisingly, neuronal development and motor function were severely disturbed in Mgmt^Tg+ mice treated with HN2. Collectively, these in vitro and in vivo studies demonstrate that the type of DNA lesion and the efficiency of DNA repair are two important factors that determine the vulnerability of the developing brain to long-term injury by a genotoxicant.     

Thermal enhancement of DNA damage by an alkylating agent in human cells

Human skin cells were incubated at various temperatures during and after treatment with methyl methanesulfonate and the number of single-strand breaks introduced into the cellular DNA then estimated by alkaline sucrose sedimentation. Elevation of temperature above 37° greatly enhanced damage to the DNA caused by methyl methanesulfonate. Inactivation of an essential step in the repair of DNA was indicated by the observation that rejoining of breaks in the DNA was halted above a critical temperature (about 41.5°). Enhancement of damage to DNA increased with temperature, especially above 42°. Similar results were obtained for Chinese hamster cells. A correlation of these results with cell viability is discussed.