Mitotic recombination induced by chemical and physical agents in the yeast Saccharomyces cerevisiae

The treatment of diploid cultures of yeast with ultraviolet light (UV), γ-rays, nitrous acid (NA) and ethyl methane sulphonate (EMS) results in increases in cell death, mitotic gene conversion and crossing-over. Acridine orange (AO) treatment, in contrast, was effective only in increasing the frequency of gene conversion. The individual mutagens were effective in the order UV > NA > γ-rays > AO > EMS. Prior treatment of yeast cultures in starvation medium produced a significant reduction in the yield of induced gene conversion.The results have been interpreted on the basis of a general model of mitotic gene conversion which involves the post-replication repair of induced lesions involving de novo DNA synthesis without genetic exchange. In contrast mitotic crossing-over appears to involve the action of a repair system independent from excision or post-replication repair which involves genetic exchange between homologous chromosomes.     

Effects of sodium acetate and sodium chloride on EMS-induced chlorophyll mutations in barley

Sodium acetate solutions to which sodium chloride was added, and acetate or chloride alone have been used as pre-, simultaneous, and post-treatment of dry and pre-soaked seeds of barley to study their effect on the types and frequencies of ethyl methanesulphonate (EMS)-induced chlorophyll mutations in spring barley, variety Elsa, and winter barley, varieties $\text{436}\text{35}$ and Ager. Application of acetate/chloride on dry seeds before or simultaneously with EMS both resulted in the frequency of chimeral plants with chlorophyll-deficient sectors in M₁ and chlorophyll mutants in M₂ approximately being halved as compared with the controls (EMS treatment alone).An opposite effect was observed after simultaneous treatment with acetate/ chloride and EMS (pH 4.5 and pH 7.0) and application of acetate/chloride after EMS treatment of pre-soaked seeds. In this case the mutagen sensitivity, i.e. the frequency of chimeral plants with induced chlorophyll-deficient sectors in M₁ and of chlorophyll mutants in M₂, was approximately doubled as compared with the control.Separate application of both acetate or chloride as a simultaneous treatment with EMS resulted also in an increase in the chlorophyll mutation frequency as compared with EMS treatment alone.Based on these results some aspects of the acetate/chloride effect are briefly discussed.     

Hycanthone as a specific frameshift mutagen in Saccharomyces cerevisiae

Reversion of mutations of different molecular nature was studied after treatment with hycanthone in mild conditions (0.05–0.4 mM, 4 h in the dark, pH 7.2). The mutagen had a very low reversion activity on 3 missense and 4 nonsense mutations (2 UAA and 2 UAG), although it was very active on 3 frameshift mutations. Our data on intragenic reversion and frameshift suppressors indicate that hycanthone can induce both insertions and deletions.     

Mutagenicity testing in mammalian cells: I. Derivation of a chinese hamster ovary cell line heterozygous for the adenine phosphoribosyltransferase and thymidine kinase loci

As a first step in the development of a multiple-marker, mammalian cell mutagenesis assay system, we have isolated a Chinese hamster ovary (CHO) cell line that is heterozygous for both the adenine phosphoribosyltransferase (aprt) and thymidine kinase (tk) loci. Presumptive aprt^+/? heterozygotes with intermediate levels of APRT activity were selected from unmutagenized CHO cell populations on the basis of resistance to low concentrations of the adenine analog, 8-azaadenine. A functional aprt^+/? heterozygote with ～50% wild-type APRT activity was subsequently used to derive sublines that were also heterozygous for the tk locus. Biochemical and genetic characterization of one such subline, CHO-AT3-2, indicated that it was indeed heterozygous at both the aprt and tk loci. CHO-AT3-2 cells permitted single-step selection of mutants resistant to 8-azaadenine or 5-fluorodeoxyuridine, allowing quantitation and direct comparison of mutation induction at the autosomal aprt or tk loci, as well as in the gene involved in ouabain resistance or at the X-linked, hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus. Significant dose-dependent increases in mutation frequency were observed for all 4 genetic markers after treatment of CHO-AT3-2 cells with ethyl methanesulfonate.     

Differential sensitivity of DNA replication and repair in permeable Escherichia coli exposed to various monofunctional methylating or ethylating agents.

Escherichia coli cells made permeable to deoxynucleoside triphosphates by brief treatment with toluene (permeablized) were used to measure the effect of the following chemical alkylating agents on either DNA replication or DNA repair synthesis: methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and N-ethyl-N′-nitro-N-nitrosoguanidine (ENNG). Replication of DNA in this pseudo-in vivo system was completely inhibited 10–15 min after exposure to MMS at concentrations of 5 mM or higher or to MNU or MNNG at concentrations of 1 mM or higher. The ethyl derivatives of the alkylating agents were less inhibitory than their corresponding methyl derivatives, and inhibition of DNA replication occurred in the following order: EMS < ENNG < ENU. Maximum inhibition of DNA replication by all of the alkylating agents tested except EMS occurred at a concentration of 20 mM or lower. The extent of replication in cells exposed to EMS continued to decrease with concentrations of EMS up to 100 mM (the highest concentration tested).The experiments in which the inhibition of DNA replication by MMS, MNU, or MNNG was measured were repeated under similar assay conditions except that a density label was included and the DNA was banded in CsCl gradients. The bulk of the newly synthesized DNA from the untreated cells was found to be of the replicative (semi-conservative) type. The amount of replicative DNA decreased with increasing concentration of methylating agent in a manner similar to that observed in the incorporation experiments.Polymerase I (Pol I)-directed DNA repair synthesis induced by X-irradiation of permeablized cells was assayed under conditions that blocked the activity of DNA polymerases II and III. Exposure of cells to MNNG or ENNG at a concentration of 20 mM resulted in reductions in Pol I activity of 40 and 30%, respectively, compared with untreated controls. ENU was slightly inhibitory to Pol I activity, while MMS, EMS, and MNU all caused some enhancement of Pol I activity.These data show that DNA replication in a pseudo-in vivo bacterial system is particularly sensitive to the actions of known chemical mutagens, whereas DNA repair carried out by the Pol I repair enzyme is much less sensitive and in some cases apparently unaffected by such treatment. Possible mechanisms for this differential effect on DNA metabolism and its correlation with current theories of chemically induced mutagenesis and carcinogenesis are discussed.     

EMS诱导红阳猕猴桃耐寒突变体的筛选及转录组分析

红阳猕猴桃(Actinidia chinensis var. chinensis ‘Hongyang'')具有较高的经济价值和营养价值,以及较好的市场开发前景。但近年红阳猕猴桃产区如云南、四川等多地多次遭遇倒春寒等极端天气,其抗寒性差的缺点限制了发展空间。该研究通过在组培的过程中使用甲基磺酸乙酯(EMS)诱导红阳猕猴桃突变体,进而筛选出耐寒突变体,并通过转录组分析探究其胁迫响应机制。该研究以红阳猕猴桃叶片为实验材料,在组培时(4.4 g·L^-1 MS+4.5 g·L^-1 琼脂+1.5 mg·L^-1 6-BA+0.1 mg·L^-1 NAA+15 g·L^-1 蔗糖+0.01～0.10 g·L^-1 EMS)利用EMS诱导技术诱导突变体,并在低温环境下筛选出耐寒突变体。选出的耐寒突变体和正常红阳猕猴桃组培苗先进行4 ℃ 12 h寒胁迫处理,再进行转录组测序分析。结果表明:(1)通过初步的表型鉴定,当EMS处理浓度为0.06 g·L^-1时诱导的部分突变体具有一定的耐寒性;(2)在转录组测序数据GO功能富集分析中,富集条目最多的是生物学过程;(3)利用KEGG数据库分析时,共筛选到21个差异表达基因在15条通路中得到注释且均为上调表达,其中内质网中的蛋白质加工通路(ath04141)中富集的差异表达基因最多,并且该通路内的sHSF、Hsp70和NEF可能与耐寒机制调控有关。综上研究结果为红阳猕猴桃耐寒种质资源的研究与利用提供了材料基础及理论依据。     

Screening of ethyl methane sulphonate mutagenized tef [Eragrostis tef (Zucc.) Trotter] population identifies Al-tolerant lines

About 15,000 M₂ seeds of ethyl-methane-sulphonate (EMS)-mutagenized population were screened along with Al-tolerant and sensitive checks and the M₀ variety. Strongly acidic soil with an external application of a toxic Al-solution and exposure to moisture stress was used to maximize selection pressure. Twenty-one M₂ plants with root lengths of greater than the mean of the tolerant check were selected and planted for seed production. Candidate M₃ plants were investigated for Al-tolerance and for morpho-agronomic traits under greenhouse and field conditions, respectively. Highly significant differences were observed for Al-tolerance between the candidate mutant lines and the M₀ (P?<?.001), and between mutant lines and the sensitive check (P?<?.001). Similarly, significant differences were observed between the mutant lines for 16 of the 20 quantitative traits measured. This study is the first to report successful induction of enhanced Al-tolerance in tef by using EMS mutagenized population.     

Isolation of a Drosophila melanogaster desiccation resistant mutant

Mutagenesis provides a powerful way of isolating genetic and physiological processes underlying complex traits, but this approach has rarely been applied to investigating water balance in insects. Here, we describe the isolation of a desiccation-resistant mutant of Drosophila melanogaster. Mutagenesis of a desiccation sensitive line resulted in the isolation of a mutant with two-fold higher resistance. The mutant was partially dominant and mapped to the second chromosome. Mutant flies showed lower rates of water loss, and had a higher water content, but showed no change in body mass, glycogen content, hemolymph volume or water content tolerated at death from desiccation. These physiological differences are contrasted to changes in lines of D. melanogaster mass selected for altered stress resistance. Isolation of this mutant provides an opportunity to identify a gene involved in water balance in insects.