鱼微核试验筛检水体诱变物的应用与研究

对五种试验污染物的遗传毒性采用鱼微核试验技术进行了筛检,试验结果表明鱼微核试验用作筛检诱变物的遗传毒性有效的,对监测水体的质量也具有潜在的价值;同时,对国内外学将各种各样的技术应用到鱼微核式试验进行了总结,概述了微核形成的机理;在此基础上,提出应用鱼微核试验筛检水体诱变物时应注意的问题。     

The reaction of the mutagen 1,1′-hexamethylene-bis[(5-p-chlorophenyl)-biguanide] with guanosine and cysteine

The mutagen 1,1′-hexamethylene-bis[(5-p-chlorophenyl)-biguanide] reacts at 37°C with guanosine and guanine to yield xanthosine or xanthine and oxidizes cysteine to cystine. After treatment of a guanosine-labelled DNA sample from Escherichia coli with the mutagen xanthine could be detected as a reaction product. At a slow rate the mutagen is hydrolysed spontaneously yielding urea, 1,6-hexanediol and 4-chloroaniline. The reaction mechanisms both of the hydrolysis and of the reaction with cysteine and guanosine are discussed.     

Genetic characterization of the mei-41 locus in Drosophila melanogaster

Summary The mutagen-sensitive mutant mus(1)104 ^D1 of Drosophila melanogaster maps to a position on the X chromosome very close to the meiotic mutant mei-41 ^D5 . Both mutants have been characterized as mutagen-sensitive and defective in post-replication repair. In the present report we show by complementation studies that mus(1)104 and mus(1)103 are allelic with mei-41. In addition, two reported alleles of mus(1)104 lie between the mei-41 alleles A10 and D5. The size of the mei-41 locus is estimated to be about 0.1 centimorgans (cM). Because several alleles of mei-41 have been shown to reduce recombination and increase meiotic chromosome loss and nondisjunction, mus(1)104 ^D1 females were examined for defects in meiosis. Although there was no evidence for reduced recombination on the second chromosome in homozygous mus(1)104 ^D1 females, heterozygous mus(1)104 ^D1 /mei-41 ^>D5 and mus(1)104 ^D1 /deficiency females showed reduced levels of recombination. However, there was no evidence of an increase in nondijunction in these females.We dedicate this article to the memory of Larry Sandler, who passed away suddenly on February 7, 1987     

Molecular mechanisms of the origin of chromosome aberrations and the structural organisation of eukaryotic DNA

Summary A new hypothesis on the appearance of exchange chromosomal aberrations has been suggested. According to this hypothesis, temporal duplex polynucleotide structure should arise during G₁ and G₂ phases during the correction of DNA. The size of the duplex, as a rule, should be restricted to the size of complementary nucleotide sequences in the regions of repetitions. Any polynucleotide break in a duplex zone would result in chromosome breakage and if complementary broken ends interact with each other, then exchange chromosome aberrations may be formed. This hypothesis would explain such previously obscure phenomena as extremely high frequencies of exchanges after mutagen treatment, the nature of mitotic crossing-over, negative interference, change of aberration types before replication, the low frequency of damaged structural genes during aberration formation, etc.     

Immunization with mutagen-treated (tum−) cells causes rejection of nonimmunogenic rat glioma isografts

The ethyl-N-nitrosourea-induced rat glioma N32 was treated with the mutagenic compoundN-methyl-N-nitro-N-nitrosoguanidine and the surviving cells cloned by limiting dilution. Out of 20 clones tested 8 did not produce tumors subcutaneously even after challenge doses 3 log units above the minimal tumor dose for N32. All of 5 clones grew in a retarded manner intracerebrally but produced tumors in some animals. Preimmunizations with three of the rejected clones (tum^–) gave protection against subcutaneous and intracerebral isografts of the unmutated N32. This effect could be enhanced if the cells used for immunizations were pretreated with interferon (IFN) for 48 h. If immunizations were started subsequent to challenge, only immunization with one of two tested tum^– clones pretreated with IFN induced significant rejection against intracerebral N32 isografts. Both N32 and its tum^– closes were MHC class I positive and MHC class II negative. IFN treatment enhanced the MHC class I expression with 20%–90% on the tum^– clones and with 40% on N32. MHC class II expression could be induced on N32 cells after 7 days of IFN treatment but not on any of the tum^– clones tested. We conclude that the enhancing effect of IFN treatment on tumor isograft rejection may depend on up-regulation of MHC class I but not of MHC class II. This investigation demonstrates that it is possible to induce rejection of weakly immunogenic intracerebral brain tumors by immunization with selected highly immunogenic tumor cell mutants. In conjunction with relevant cytokines, the cross-protective effect of these tum^– variants might be further enhanced and serve as a model for immunotherapy against malignant human brain tumors.     

Induction of streptomycin resistance in the wild tomato Lycopersicon peruvianum

Summary A protoplast mutagenesis and cell selection system was used for the isolation of streptomycin resistant Lycopersicon peruvianum colonies. Protoplasts were treated with the mutagen N-nitroso-methylurea and could be regenerated into fertile plants, carrying the streptomycin resistant character. Several classes of streptomycin resistance could be distinguished. Reciprocal crosses between streptomycin resistant and sensitive plants showed a non-Mendelian transmission of the resistance trait. Streptomycin resistance is the first selectable and maternally inherited cell organelle marker described in tomato.     

几种诱变因子对龟裂链霉菌原生质体的影响

作者应用通电、ＵＶ、ＮＴＧ和亚硫酸氢钠等诱变因子对土霉素产生菌——龟裂链霉菌１３８—ｌ原生质体进行处理。结果表明,各种诱变因子在对原生质体致死率５０％左右时具有较好的诱变效应。经亚硫酸氢钠＋ＬｉＣｌ处理获得的Ｃｌ１８菌株通过１６５００１发酵罐试验,最高发酵水平达３３３３０ｕ／ｍｌ,平均发酵水平为３０５４２．５ｕ／ｍｌ,比出发菌株１３８—１提高２０．６％。     

Evaluation of formation potential of trihalomethanes and mutagens due to chlorination of river water along the Kizu River in the Yodo River system in Japan

The chlorination of river water in purification plants is known to produce carcinogens and genotoxins. The Yodo River system was studied in regard to the trihalomethane formation potential (THMFP) and mutagen formation potential (MFP) by the chlorination of river water and the discharge of sewage effluent. Overall, both THMFP and MFP are higher in the downstream region of the Yodo River system than in the upstream Uji and Katsura River region, whereas the formation potentials are high in the upstream part of the Kizu River. Detailed surveys were carried out on the Nabari River and other tributaries to the Kizu River. It was revealed that the Nabari and the upper Kizu rivers contribute 49.9% and 41.9% of the THMFP and 47.9% and 44.7% of the MFP pollution load to the main stream of the Kizu River, respectively. The contribution rates of the upper Kizu River could be attributed to three rivers, the uppermost Kizu, Hattori, and Kume rivers, and household sewage waterways at 10.6%, 17.3%, 10.9%, and 3.1% for THMFP and at 9.5%, 17.3%, 12.5%, and 5.5% for MFP, respectively. The main cause of water pollution in the upper reaches of the Kizu River may be attributable to both sewage effluent discharged directly into the Kume River and the polluted water of the Tsuge and Yatani rivers, tributaries of the Hattori River. When we examined whether THMFP and MFP depended on the concentrations of organic substances in water, both potentials highly correlated with chemical oxygen demand (COD) in all water samples surveyed. The correlation coefficients between COD and THMFP and between COD and MFP were 0.957 (n = 76, P < 0.01) and 0.804 (n = 76, P < 0.01), respectively. Received: November 29, 2000 / Accepted: July 3, 2001     

A mitochondrial nuclease is modified in Drosophila mutants (mus308) that are hypersensitive to DNA crosslinking agents

Summary The mus308 mutants of Drosophila have previously been demonstrated to be defective in an enzyme that is designated Nuclease 3 (Boyd et al. 1990b). In this study that enzyme is shown to be present in mitochondria of both wild-type flies and embryos. Since the mus308 mutants are hypersensitive to DNA crosslinking agents, Nuclease 3 is potentially required for resistance of the mitochondrial genome to such agents. In support of this hypothesis, electron microscopic studies of mus308 mutant flies that had been exposed to nitrogen mustard revealed an increased frequency of mitochondrial abnormalities. Further investigation of the defect at the enzymological level revealed that the mutants possess a new nuclease activity that is apparently a modified form of the wild-type protein. In the earlier study, enzyme extracts from mus308 mutants were found to lack an enzyme with a pl of approximately 6.2. More precisely defined assay conditions in this study revealed the appearance of a new nuclease activity with a higher pI in extracts from mutants. This observation, together with the finding that only the normal enzyme form is present in heterozygous individuals, supports the hypothesis that the mus308 locus is not the structural gene for the enzyme. Rather, the mus308 gene product is necessary for Nuclease 3 to assume the lower pI. Nuclease 3 has been partially purified and characterized from wild-type embryos. Its activity is stimulated by Mg⁺⁺ and ATP. Optimum activity is found at a pH of 5.5 and a NaCl concentration of 50–100 mM. Nuclease 3 exhibits a temperature optimum of 42°C and is insensitive to N-ethylmaleimide. The enzyme is probably membrane-associated because it exhibits a strong tendency to aggregate and detergent is required for full solubilization.     

ENU诱导带LacZ靶基因的λgt11

采用ENU(乙基亚硝基脲)作用于裸露的 λgt11 DNA,经体外重包装,转染宿主菌　E. coli Y1090,在含底物X-gal,诱导剂IPTG的选择性培养基上铺皿,发现被处理的 λgt11 DNA除了使噬菌体存活率下降外,还出现了靶基因“LacZ”较高频率的突变。其中以二甲基亚砜(DMSO)为溶剂,当存活率分别为3.5×10-3、1.6×10-3和5.5×10-4 时,相应的突变率依次为1.1×10-3、3.2×10-3和5.2×10-3,DMSO溶剂对照突变率则<5.0×10-5 。对ENU诱导的5个阳性突变体进行了扩增,以PCR产物为模板,采用正向引导,对阳性突变体靶基因LacZ进行了部分测序,在被测序的260bp范围内,发现了9个位点的碱基突变。碱基突变的类型有颠换(67%)、转换(11%)和移码突变(22%)。颠换主要以A→T、G→C为主。似乎胞嘧啶(C)更易发生突变(占43%)。 Abstract　To construct molecular mutation detective system of λ DNA with LacZ, naked λ gt11 DNA was treated with mutagen ENU (Ethylnitro sourea). The ENU-damaged DNA was added to Lambda packaging extracts and the resulting phage were grown in host　E.coli　Y1090 on a selective plate containing substract X-gal and inducer IPTG. Under these conditions, the results showed that the higher the viability ratio was, the lower the frequency of clear-plaque mutants occured. In our study, when survival ratios of the host cell survival ratio were 3.5×10-3、1.6×10-3　and 5.5×10-4　respectively, the mutation ratio were 1.1×10-3、3.2×10-3　and 5.2×10-3　accordingly, and the mutation ratio by DMSO (negative control ) was below 5.0×10-5. 260 bases from ENU―induced LacZ　gene were subjected to DNA sequence analysis. There were several mutation sites: transversion (6, 67%), transition(1, 11%), frame shift(2, 22%)(both were insert mutation). Transversions mainly consisted of A→T, G→C. Among the four bases, cytosine seemed to be more sensitive to ENU (43%).