重离子束辐照在优良工业微生物新菌株创建中的应用实践

性能优良的工业微生物菌种是企业保持竞争力的重要保障,因此培育优质、高产的微生物新菌种依旧是生物育种产业创新发展的核心内容之一。重离子束辐照作为一种高效的诱变手段,现已广泛应用于我国工业微生物育种实践中。重点回顾了重离子束辐照诱导生物体DNA突变的分子机制研究以及重离子束微生物育种的研究进展。同时,也展望了我国重离子束育种专用装置的产业化前景。     

重离子诱变微生物育种的研究进展

近年来,重离子束作为一种新的辐射诱变源在微生物育种领域已多有应用.与传统的辐照源相比,重离子束具有更高的传能线密度,可以产生更强的辐射损伤生物效应,因此诱变效率高.本文综述了近年来重离子诱变在微生物育种中取得的进展、诱变后突变体菌株的筛选策略、重离子束引起微生物遗传物质改变的直接和间接机制以及突变后的修复机理,并对其在...     

离子注入微生物诱变育种的研究与应用进展

离子束作为一种新的诱变源虽然在微生物上的应用起步较晚,但成果显著。这项技术适用于多种微生物,也可以和其它方法结合对菌种进行复合诱变,同样离子束介导转基因技术不仅适于植物细胞,对微生物细胞也是可行的。这一技术在对微生物诱变育种的研究中,表现出比传统诱变方法高的诱变效率,利用离子注入进行微生物菌种改良已在生产实践中得到广泛的应用,并取得了显著的经济效益和社会效益。对离子注入微生物诱变育种的理论研究进展和实际应用情况进行了综述。     

工业微生物物理诱变育种技术的新进展

物理诱变技术是当今工业微生物育种中最重要、最有效的技术之一。传统的物理诱变技术主要有紫外线、X射线、γ射线诱变等,它们已在包括青霉素、"-淀粉酶等几乎所有的工业微生物菌种的诱变选育中发挥了巨大的作用。多数菌株在多次重复使用传统诱变源时往往出现抗性饱和的现象。太空环境、离子束、激光等是20世纪70～80年代逐渐兴起的新型诱变技术,因它们具有诱变谱广和在一定程度上能克服菌株对传统诱变源的抗性饱和等优点,而广受工业微生物育种工作者的欢迎。现就空间、离子束、激光等诱变育种技术的作用特点、诱变机理、应用及前景进行阐述。     

离子束生物工程专辑前言

从上个世纪八十年代以来 ,我国科技工作者通过实验发现 ,水稻种子经超低能重离子束辐照后能够诱发变异 ,并在其后代具有遗传性 ,由此开辟了重离子束生物学效应研究与重离子束诱变育种的新领域 ,逐渐形成了一门物理学与生命科学交叉的学科分支。随着时间的推移 ,这项研究也不断地深入和扩展 ,离子种类从较轻的氦离子到较重的锌离子 ,能量从超低能、低能到中能 ,研究对象从植物种子 (包括粮食作物 ,蔬菜 ,瓜果 ,花卉 ,牧草等 )、微生物药物、动物细胞 (例如蚕卵 )到介导外源基因转移等 ,研究内容涉及基础研究、应用研究和技术开发 ,在九十年代…     

离子束诱变育种研究及应用进展

阐述了离子束诱变的生物效应,研究中有待解决的问题,介绍了离子束诱变在农业、工业育种上的研究进展及成果,并展望了今后的研究前景。     

激光诱变微生物技术的研究进展

简要概括了国内外在激光诱变微生物领域内的研究新进展,对激光辐照微生物产生生长刺激作用的实验规律、作用机理进行了总结和讨论,在此基础上探讨了激光辐照微生物进行诱变育种的应用性研究。最后,对本领域的发展进行了展望并提出建议。     

微生物育种物理诱变技术ARTP的应用进展

微生物物理诱变育种技术广泛应用于改善微生物菌种特性、提高微生物产品产量与质量,并且在生物燃料和生物修复方面也具有重要的应用价值。本文重点介绍常压室温等离子体(ARTP)物理诱变技术在微生物诱变育种方面的应用,其对具有重要工农业应用价值的产芽孢菌种具有明显的诱变优越性。进一步分析了微生物物理诱变育种技术未来的发展趋势和重要应用前景,为微生物物理诱变育种工作提供借鉴。     

离子束对生物体的作用原理及应用

离子束是指一束具有一定能量的质量数小于或等于4的带电离子束,离子束注入技术是生物物理技术,具有生理损伤小、突变谱广和突变频率高等特点。离子束与生物体的相互作用是我国具有独立知识产权的生物物理技术,我国科学家在上世纪80年代已经发现了离子束注入的生物效应,并将这一原理应用于植物诱变育种。本文主要概述了低能离子束注入对生物体的作用原理,以及该技术在植物育种、微生物品种改良和遗传改良上的应用,最后还小结了离子束注入技术在研究领域存在的问题并对其未来发展方向提出展望。     

利用紫外诱变选育辅酶q10菌种的研究

微生物育种指的是运用遗传学、诱变、杂交等原理,对负有特定作用的微生物菌种开展筛选、改造,从而得到符合某种需要的技术。本文对微生物菌种选育技术中的紫外诱变来选育辅酶q10高产菌株进行了一系列的试验和研究。。     

Mutagenic effects of allylisothiocyanate inEscherichia coli WP 67

A mutagenic effect of allylisothiocyanate (AITC) onE. coli WP 67 (trp uvrA polA) was only found in a test with metabolic activationin vitro. Mixed function oxidases containing cytochrome P-450 are not responsible for the conversion of AITC to mutagenic products. Liver microsomal fractions of rat, mouse, chinese hamster, goat and monkey were used forin vitro metabolic activation. The mutagenic effect of AITC differed in individual animal species according to the protein content in the microsomel fraction. Allylamine (formed by hydrolysis of AITC) did not exhibit any mutagenic effect.     

Quantitative comparative mutagenesis in bacteria, mammalian cells, and animal-mediated assays A convenient way of estimating genotoxic activity in vivo?

The accumulation of environmental compounds which exhibit genotoxic properties in short-term assays and the increasing lag of time for obtaining confirmation or not in long-term animal mutagenicity and carcinogenicity tests, makes it necessary to develop alternative, rapid methodologies for estimating genotoxic activity in vivo. In the experimental approach used here, it was assumed that the genotoxic activity of foreign compounds in animals, and ultimately humans, is determined among others by exposure level, organ distribution of (DNA) dose, and genotoxic potency per unit of dose, and that knowledge about these 3 parameters may allow to rapidly determine the expected degree of genotoxicity in various organs of exposed animals. In view of the high degree of qualitative correlation between mutagenic activity of chemicals in bacteria and in cultured mammalian cells, and their mutagenic and carcinogenic properties in animals, and in order to be able to distinguish whether mutagenic potency differences were due to differences in (DNA) dose rather than other physiological factors, the results of mutagenicity tests obtained in the present experiments using bacteria and mammalian cells were compared on the basis of DNA dose rather than exposure concentrations, with the following questions in mind: Is there an absolute or a relative correlation between the mutagenic potencies of various ethylating agents in bacteria (E. coli K12) and in mammalian cells (V79 Chinese hamster) after treatment in standardized experiments, and can specific DNA adducts be made responsible for mutagenicity? Is the order of mutagenic potency of various ethylating agents observed in bacteria in vitro representative of the ranking of mutagenic potency found in vivo? Since the answer to this last question was negative, a further question addressed to was whether short-term in vivo assays could be developed for a rapid determination of the presence (and persistence) of genotoxic factors in various organs of mice treated with chemicals. In quantitative comparative mutagenesis experiments using E. coli K12 and Chinese hamster cells treated under standardized conditions in vitro with 5 ethylating agents, there was no indication of an absolute correlation between the number of induced mutants per unit of dose in the bacteria and the mammalian cells. The ranking of mutagenic potency was, however, identical in bacteria and mammalian cells, namely, ENNG greater than ENU greater than or equal to DES greater than DEN congruent to EMS, the mutagenic activity of DEN being dependent on the presence of mammalian liver preparations.(ABSTRACT TRUNCATED AT 400 WORDS)     

低能氮离子注入同源四倍体水稻的生物学效应

以低能氮离子束为诱变源,对同源四倍体水稻进行注入处理后,对其生物学效应进行了研究。研究结果表明,同源四倍体水稻比二倍体水稻对低能氮离子束注入处理更敏感。在注入离子剂量为3×1017N+/cm2时,同源四倍体水稻所受到的损伤比二倍体水稻所受到的损伤更严重。4份同源四倍体水稻(即IR36(4)、IR28(4)、紫血稻(4)、明恢63(4))的成苗率分别是2.0%、3.5%、3.0%和4.0%,致死率非常高。在同样条件下,4份相应的二倍体水稻(即IR36(2)、IR28(2)、紫血稻(2)、明恢63(2))的成苗率分别是33.0%、31.5%、29.0%和24.5%,致死率也比较高。在经过注入处理后的当代群体内,4份同源四倍体水稻的平均变异频率为32.5%,而4份相应的二倍体水稻的平均变异频率为9.5%。在同源四倍体水稻IR36(4)和IR28(4)的变异群体内分别发现22株和14株结实率均达到75.0%以上的单株,其中在IR36(4)群体内有1单株的结实率高达91.89%;在紫血稻(4)的变异群体内发现2株具有双胚苗性状的单株;在二倍体水稻明恢63(2)的变异群体内发现1株具有红心米性状的单株。在第二代群体中,除了叶鞘变异和米质变异这两个变异性状能稳定遗传之外,其它变异性状在群体内都发生了明显的性状分离现象。同源四倍体水稻的高结实率特性和双胚苗特性表现出一定的可遗传性。     

Direct repair of 3,N(4)-ethenocytosine by the human ALKBH2 dioxygenase is blocked by the AAG/MPG glycosylase

Exocyclic ethenobases are highly mutagenic DNA lesions strongly implicated in inflammation and vinyl chloride-induced carcinogenesis. While the alkyladenine DNA glycosylase, AAG (or MPG), binds the etheno lesions 1,N⁶-ethenoadenine (?A) and 3,N⁴-ethenocytosine (?C) with high affinity, only ?A can be excised to initiate base excision repair. Here, we discover that the human AlkB homolog 2 (ALKBH2) dioxygenase enzyme catalyzes direct reversal of ?C lesions in both double- and single-stranded DNA with comparable efficiency to canonical ALKBH2 substrates. Notably, we find that in vitro, the non-enzymatic binding of AAG to ?C specifically blocks ALKBH2-catalyzed repair of ?C but not that of methylated ALKBH2 substrates. These results identify human ALKBH2 as a repair enzyme for mutagenic ?C lesions and highlight potential consequences for substrate-binding overlap between the base excision and direct reversal DNA repair pathways.     

The Salmonella mutagenicity assay in a surface water quality monitoring program based on a 20-year survey

Since 1979, the Environmental Agency of S?o Paulo State in Brazil, CETESB, has been using the Salmonella mutagenicity assay to assess the quality of natural waters. This paper is a compilation of data obtained during the last 20 years from more than a thousand samples. Potencies up to 30,000 revertants/l were observed in 137 positive samples. The Salmonella typhimurium strain TA98 was more sensitive than TA100; 79% of the mutagenicity was detected by this strain, regardless of the presence of S9-mix. A classification of the mutagenic response was proposed to facilitate in the dissemination of the information to the public. The classification was low, moderate, high and extreme for samples with mutagenic potency (revertants/l equivalent) of < 500, 500-2500, 2500-5000 and > 5000, respectively. As a result of this effort to standardize methodologies, compile and classify the mutagenic effect of water pollution, in 1998, the Salmonella mutagenicity assay was officially and systematically included in the S?o Paulo State Water Quality Monitoring Program. This assay has proven to be a useful tool in the identification of important pollution sources. Correction and prevention actions in Water Pollution Control Programs were generated as a result.     

Mutagenicity and DNA adduct formation of PAH, nitro-PAH, and oxy-PAH fractions of atmospheric particulate matter from São Paulo, Brazil

Urban particulate matter (UPM) contributes to lung cancer incidence. Here, we have studied the mutagenic activity and DNA adduct-forming ability of fractionated UPM extractable organic matter (EOM). UPM was collected with a high-volume sampler in June 2004 at two sites, one at street level adjacent to a roadway and the other inside a park within the urban area of the city of S?o Paulo, Brazil. UPM was extracted using dichloromethane, and the resulting EOM was separated by HPLC to obtain PAH, nitro-PAH, and oxy-PAH fractions which were tested for mutagenicity with the Salmonella strains TA98 and YG1041 with and without S9 metabolic activation. The PAH fraction from both sites showed negligible mutagenic activity in both strains. The highest mutagenic activity was found for the nitro-PAH fraction using YG1041 without metabolic activation; however, results were comparable for both sites. The nitro-PAH and oxy-PAH fractions were incubated with calf thymus DNA under reductive conditions appropriate for the activation of nitro aromatic compounds, then DNA adduct patterns and levels were determined with thin-layer chromatography (TLC) 32P-postlabeling method using two enrichment procedures-nuclease P1 digestion and butanol extraction. Reductively activated fractions from both sites produced diagonal radioactive zones (DRZ) of putative aromatic DNA adducts on thin layer plates with both enrichment procedures. No such DRZ were observed in control experiments using fractions from unexposed filters or from incubations without activating system. Total adduct levels produced by the nitro-PAH fractions were similar for both sites ranging from 30 to 45 adducts per 10(8) normal nucleotides. In contrast, the DNA binding of reductively activated oxy-PAH fractions was three times higher and the adduct pattern consisted of multiple discrete spots along the diagonal line on the thin layer plates. However, DNA adduct levels were not significantly different between the sampling sites. Both samples presented the same levels of mutagenic activity. The response in the Salmonella assay was typical of nitroaromatics. Although, more mutagenic activity was related to the nitro-PAH fraction in the Salmonella assay, the oxy-PAH fractions showed the highest DNA adduct levels. More studies are needed to elucidate the nature of the genotoxicants occurring in S?o Paulo atmospheric samples.     

Mutagenicity and genotoxicity of suspended particulate matter in the Seine river estuary

Highly mutagenic compounds such as some PAHs have been identified in surface waters and sediments of the Seine river estuary. Suspended particulate matter (SPM) represents a dynamic medium that may contribute to the exposure of aquatic organisms to toxic compounds in the water column of the estuary. In order to investigate major sources of mutagenic contaminants along the estuary, water samples were taken at 25 m downstream of the outlet of an industrial wastewater-treatment plant (WWTP). SPM samples were analyzed for their genotoxicity with two short-term tests, the Salmonella typhimurium mutagenicity assay (TA98+S9 mix) and the comet assay in the human HepG2 cell line. Sampling sites receiving effluents from a chemical dye industry and WWTP showed the highest mutagenic potencies, followed by petrochemical industries, petroleum refinery and pulp and paper mills. These data indicate that frame-shift mutagens are present in the Seine river estuary. Furthermore, the comet assay revealed the presence of compounds that were genotoxic for human hepatocytes (HepG2 cells). We also observed a high level of mutagenic potency in the sediment of the lower estuary (3 × 10? revertants/g). The source of mutagenic and genotoxic compounds seems to be associated with various types of effluents discharged in the Seine river estuary. Both test systems resulted in the same assessment of the genotoxicity of particulate matter, except for three of the 14 samples, underlying the complementarity of bioassays.     

Key microstructural mechanisms of the 2-aminopurine mutagenicity: Results of extensive quantum-chemical research

As of today, a great amount of experimental and theoretical phenomenological data have been collected in the literature according the mutagenic action of the classical mutagen – 2-aminopurine (2AP). However, so far they have not received proper explanation and substantiation. In this Opinion Piece, we provide an overview of recent progress in computational design and modeling of the physico-chemical mechanisms of the mutagenic action of 2AP. Results of quantum-chemical studies, aimed at the elucidation of the key microstructural mechanisms of the mutagenicity of 2AP, have been summarized here. In this context, for the first time it was outlined the most important surveys: Why 2AP is incorporated into DNA in trace concentrations? Whether classical mechanisms presented in the literature according the formation of the rare tautomers of canonical DNA bases work also for base analogue – 2AP? In what way 2AP induces replication and incorporation errors? Whether the amino-imino tautomerisation of 2AP is related to its mutagenicity, that is whether the 2AP* rare tautomer is mutagenic? It is emphasized that the applied approach has a proper theoretical substantiation, since it is based on our microstructural theory of the spontaneous point mutagenesis in DNA, and at the same time it accumulates scenarios of the origin of the induced point errors – transitions and transversions, which the classical Watson–Crick tautomeric hypothesis permits. Moreover, using author’s methodology, the profiles of the main physico-chemical characteristics for the tautomerisation reactions involving 2AP, which are integral parts of the biologically important tautomerically-conformational transformations, have been presented. Obtained results open new perspectives for prediction and design of the mutagenic derivatives of the nucleotide bases of any structure and origin before their synthesis and also for planning of new experiments and interpretation of the existing data. Abbreviations 2AP 2-Aminopurine

A adenine

C cytosine

DPT double proton transfer

G guanine

IRC intrinsic reaction coordinate

KP key point

T thymine

w wobble

WC Watson–Crick

vdW van der Waals

H-bond hydrogen bond

Communicated by Ramaswamy H. Sarma     

Screening complex hazardous wastes for mutagenic activity using a modified version of the TLC/Salmonella assay

10 complex hazardous wastes were tested for mutagenic activity using a modified version of the TLC/Salmonella assay developed by Bj?rseth et al. (1982). This fractionation/bioassay scheme couples thin-layer chromatography (TLC) with the Salmonella/mammalian-microsome (Ames) assay for the detection of mutagenic constituents in complex mixtures. Crude (unadulterated) hazardous wastes and selected hazardous waste extracts were fractionated on commercially available cellulose TLC plates. Mutagenicity testing was performed in situ by applying a single overlay of minimal growth agar, tester strain TA98 or TA100, and the optional metabolic activation system directly onto the developed chromatogram. A mutagenic effect was indicated either by the appearance of localized clusters of revertant colonies or by an increase in total revertant growth vis-à-vis control plates. 7 of 10 hazardous wastes (including tars, emulsions, sludges, and spent acids and caustics) demonstrated mutagenic activity when tested by this method. To assess the sensitivity of the modified TLC/Salmonella assay, 14 Salmonella mutagens from a wide range of chemical classes and polarities were tested. Selected compounds included heterocyclics, aromatic amines, alkylating agents, antitumor agents, a nitrosamine and a nitroaromatic. 11 of the 14 mutagens were positive in this test system. The 3 compounds refractory to analysis included a polycyclic aromatic hydrocarbon and two volatiles.     

Increase of mutation level in tissues of goby and fry of sturgeon under conditions of block by antibodies against serotonin-modulated anticonsolidation protein

The work present data on studies of a decreased activity of serotoninergic system on the level of mutagenic changes (the micronuclear test) in the goby Neogobius fluviatilis and the fry if sturgeon Acipenser güldenst?dti persicus. It has been shown that the long exposure of the animals to conditions of industrial and oil pollution leads to a significant decrease in their liver of the level of serotonin-modulated anticonsolidation protein (SMAP) correlating directly with the serotonin level as well as to sharp increase of the level of micronuclei in erythrocytes. The intramuscular administration of anti-SMAP polyclonal antibodies to the fry of the sturgeon produces a significant increase of the amount of micronuclei as compared with that in the animals injected with non-immune gamma-globulin. The obtained results allow concluding that the decrease of activity of the serotoninergic system is the mechanism that is triggered with adverse environmental factors and realizes mutagenic damages in the modified genetic apparatus.