The Contribution of Alu Elements to Mutagenic DNA Double-Strand Break Repair

Alu elements make up the largest family of human mobile elements, numbering 1.1 million copies and comprising 11% of the human genome. As a consequence of evolution and genetic drift, Alu elements of various sequence divergence exist throughout the human genome. Alu/Alu recombination has been shown to cause approximately 0.5% of new human genetic diseases and contribute to extensive genomic structural variation. To begin understanding the molecular mechanisms leading to these rearrangements in mammalian cells, we constructed Alu/Alu recombination reporter cell lines containing Alu elements ranging in sequence divergence from 0%-30% that allow detection of both Alu/Alu recombination and large non-homologous end joining (NHEJ) deletions that range from 1.0 to 1.9 kb in size. Introduction of as little as 0.7% sequence divergence between Alu elements resulted in a significant reduction in recombination, which indicates even small degrees of sequence divergence reduce the efficiency of homology-directed DNA double-strand break (DSB) repair. Further reduction in recombination was observed in a sequence divergence-dependent manner for diverged Alu/Alu recombination constructs with up to 10% sequence divergence. With greater levels of sequence divergence (15%-30%), we observed a significant increase in DSB repair due to a shift from Alu/Alu recombination to variable-length NHEJ which removes sequence between the two Alu elements. This increase in NHEJ deletions depends on the presence of Alu sequence homeology (similar but not identical sequences). Analysis of recombination products revealed that Alu/Alu recombination junctions occur more frequently in the first 100 bp of the Alu element within our reporter assay, just as they do in genomic Alu/Alu recombination events. This is the first extensive study characterizing the influence of Alu element sequence divergence on DNA repair, which will inform predictions regarding the effect of Alu element sequence divergence on both the rate and nature of DNA repair events.     

Studies on the Mutagenic Effect of the Chromosome by 5 MeV Electron

In this experiment, electron and y-ray-radiation were used to irradiate dry seeds of Vicia faba. Experimental results show that electron just as affective as γ-ray has significant mutagenic effect on cells. In the range of the dose, the frequency of chromosomal aberrations on the root tip cells, there are singnificantly dose-effect. But the relationship between the dose and frequency of aberrations is close to straight line and redplication. An analysis from the type of chromosomal aberrations by electron also leads to different results, in which there are more bridges and rings by electron than by γ-ray. Therefore, electron possibly have stronger two hit aberrations, and the frequency of aberration increased in proporti on with the square of dose.     

芦苇细胞培养中的染色体稳定性及诱变效应

培养一年后的芦苇愈伤组织中,仍以80％的八倍体细胞占绝对优势。染色体数目变异范围在105—26之间。而EMS处理的愈伤组织与末经处理的愈伤组织相比较,明显地具有比较高的染色体数目变异和倍性变异。     

Establishment of a Markerless Mutation Delivery System in Bacillus subtilis Stimulated by a Double-Strand Break in the Chromosome

Bacillus subtilis has been a model for gram-positive bacteria and it has long been exploited for industrial and biotechnological applications. However, the availability of facile genetic tools for physiological analysis has generally lagged substantially behind traditional genetic models such as Escherichia coli and Saccharomyces cerevisiae. In this work, we have developed an efficient, precise and scarless method for rapid multiple genetic modifications without altering the chromosome of B. subtilis. This method employs upp gene as a counter-selectable marker, double-strand break (DSB) repair caused by exogenous endonuclease I-SceI and comK overexpression for fast preparation of competent cell. Foreign dsDNA can be simply and efficiently integrated into the chromosome by double-crossover homologous recombination. The DSB repair is a potent inducement for stimulating the second intramolecular homologous recombination, which not only enhances the frequency of resolution by one to two orders of magnitude, but also selects for the resolved product. This method has been successfully and reiteratively used in B. subtilis to deliver point mutations, to generate in-frame deletions, and to construct large-scale deletions. Experimental results proved that it allowed repeated use of the selectable marker gene for multiple modifications and could be a useful technique for B. subtilis.     

SIR Functions Are Required for the Toleration of an Unrepaired Double-Strand Break in a Dispensable Yeast Chromosome

Unrepaired DNA double-strand breaks (DSBs) typically result in G(2) arrest. Cell cycle progression can resume following repair of the DSBs or through adaptation to the checkpoint, even if the damage remains unrepaired. We developed a screen for factors in the yeast Saccharomyces cerevisiae that affect checkpoint control and/or viability in response to a single, unrepairable DSB that is induced by HO endonuclease in a dispensable yeast artificial chromosome containing human DNA. SIR2, -3, or -4 mutants exhibit a prolonged, RAD9-dependent G(2) arrest in response to the unrepairable DSB followed by a slow adaptation to the persistent break, leading to division and rearrest in the next G(2). There are a small number of additional cycles before permanent arrest as microcolonies. Thus, SIR genes, which repress silent mating type gene expression, are required for the adaptation and the prevention of indirect lethality resulting from an unrepairable DSB in nonessential DNA. Rapid adaptation to the G(2) checkpoint and high viability were restored in sir(-) strains containing additional deletions of the silent mating type loci HML and HMR, suggesting that genes under mating type control can reduce the toleration of a single DSB. However, coexpression of MATa1 and MATalpha2 in Sir(+) haploid cells did not lead to lethality from the HO-induced DSB, suggesting that toleration of an unrepaired DSB requires more than one Sir(+) function.     

Structural and Functional Characterization of Human Chromosome 13q14 and Its Potential Tumor Suppressor Genes

Works on chromosome 13 mapping supported by the Russian program Human Genome are reviewed. Emphasis is placed on studies of region 13q14.3, which is often lost in some human tumors and potentially contains tumor suppressor genes (TSG). A strategy of TSG search is described. As the resolution of genome analysis improved, a minimal overlap of genetic loss in B-cell chronic lymphocytic leukemia (B-CLL) was established for chromosome 13. A map of expressed sequences was constructed for the region containing the overlap, and candidate TSG of chromosome 13q14 were identified. The candidate genes were analyzed both structurally and functionally, and their possible role in tumorigenesis was considered. Assuming haploinsufficiency as a genetic mechanism controlling B-CLL, a new strategy was proposed for mutation screening aimed at identifying potential TSG of region 13q14.     

Repeated Intravenous Administration of Liposome-Encapsulated Muramyl Tripeptide-Phosphatidyl-Ethanolamine Diminishes Cytotoxic Potential of Subpopulations of Rat Liver Macrophages

Abstract

In this study we investigated the effects of repeated intravenous (i.v.) administration of liposome-encapsulated muramyl tripeptide-phosphatidylethanolamine (lipMTP-PE) on the antitumor functions of rat liver macrophages. Liver macrophage subtractions, differing in cell-size, were isolated by elutriation 24h after the final injection of liposomes and tested for cytotoxicity against radio-labeled C26 adenocarcinoma cells. Prolonged multiple i.v. injections of lipMTP-PE resulted in decreased tumoricidal activity in all but the small-sized subtraction, compared to a single injection.

Immunohistochemical staining of liver sections revealed that while the number of liver macrophages increased significantly after multiple lipMTP-PE injections, the expression of the la antigens on these cells decreased. Additionally, multiple lipMTP-PE treatment resulted in a reduced nitric oxide release in response to muramyl dipeptide in vitro, while incubation with another immunomodulator, lipopoly saccharide, resulted in substantial levels of NO secretion in all subtractions.

Taken together, these results demonstrate a diminished antitumor response and activated state of a significant part of the liver macrophage population after prolonged repeated administration of lipMTP-PE, and provide further insight in the behavior of the liver macrophage population during in vivo treatment with liposomal immunomodulators.     

Psychological Stress on Female Mice Diminishes the Developmental Potential of Oocytes: A Study Using the Predatory Stress Model

Although the predatory stress experimental protocol is considered more psychological than the restraint protocol, it has rarely been used to study the effect of psychological stress on reproduction. Few studies exist on the direct effect of psychological stress to a female on developmental competence of her oocytes, and the direct effect of predatory maternal stress on oocytes has not been reported. In this study, a predatory stress system was first established for mice with cats as predators. Beginning 24 h after injection of equine chorionic gonadotropin, female mice were subjected to predatory stress for 24 h. Evaluation of mouse responses showed that the predatory stress system that we established increased anxiety-like behaviors and plasma cortisol concentrations significantly and continuously while not affecting food and water intake of the mice. In vitro experiments showed that whereas oocyte maturation and Sr²⁺ activation or fertilization were unaffected by maternal predatory stress, rate of blastocyst formation and number of cells per blastocyst decreased significantly in stressed mice compared to non-stressed controls. In vivo embryo development indicated that both the number of blastocysts recovered per donor mouse and the average number of young per recipient after embryo transfer of blastocysts with similar cell counts were significantly lower in stressed than in unstressed donor mice. It is concluded that the predatory stress system we established was both effective and durative to induce mouse stress responses. Furthermore, predatory stress applied during the oocyte pre-maturation stage significantly impaired oocyte developmental potential while exerting no measurable impact on nuclear maturation, suggesting that cytoplasmic maturation of mouse oocytes was more vulnerable to maternal stress than nuclear maturation.     

Assessing the Mutagenic Potential of Surface Sediments from Beijing Guanting Reservoir to Salmonella typhimurium

Mutagenicity of organic extracts from Beijing Guanting Reservoir sediments was investigated with TA98 and TA100 of Salmonella typhimurium. TA98 and TA100 were employed to detect frameshift mutation and base-pair substitution mutation, respectively. For TA100, no positive result was found, while TA98 was more sensitive and pro-mutagenic frameshift mutagens were mainly detected in sediments. Sediments from northern and southern Guanting Reservoir were at potential mutagenic risk. No mutagenicity was found in the sediments from the entrance of the tributaries, but strong mutagenicity was observed in the sediments from the outlet of the reservoir. Chemical analysis was also performed, and poor correlation was found between mutagenicity and organochlorine pesticides (OCPs). However, significant positive correlation was found for polycyclic aromatic hydrocarbons (PAHs) (r = 0.603–0.946), which showed that PAHs were dominated for the tested mutagenicity in the sediments. Polychlorinated biphenyls (PCBs) might induce mutagenicity or promote the mutagenicity of other substances.     

高效脱硫诱变菌株降硫性能的研究

目的:筛选具有脱硫功能的细菌,为采用生物法脱硫奠定理论基础.方法:从大庆石化废水曝气池中采集5个活性污泥样本,经过富集培养、分离、纯化获得具有典型特征的菌株,采用碘量法对这些菌株进行降硫能力测定,从中选择降硫效率较高的菌株进行诱变.结果:在30℃、转速160r/min的条件下,Z39ay1菌株的最佳生长pH值为7.0,对数生长期为12～32h,当硫离子为102.24mg/L时,该菌株对硫化物的降解率达42.60％,将其置于2000Gry的60Co射线下照射,从存活菌细胞中进行筛选获得1株诱变菌株Z39a,当硫离子浓度为60mg/L时,对硫化物的降解率达98.58％.结论:从大庆石化废水中分离纯化出1株代号为Z39ay1菌株,经鉴定为赖氨酸芽孢杆菌,诱变后获得菌株Z39a,其降硫效果比出发菌株有大幅度的提高.     

Analysis of the Mutagenic Potential of Copper Compounds and Modification of Their Effect by Silver Iodide

Mutagenic potential of copper compounds and its alteration in case of the interaction with silver compounds were analyzed by use of plant test systems. As test systems,Crepis capillarisL., Tradescantia clone 02, and soybean (Glycine max(L.) Merrill) were used. Mutagenic properties of copper iodide and copper sulfate were not detected. CuI, being not a mutagen by itself, remarkably enhanced mutagenic potential of AgI.     

Genetically Modified Vibrio harveyi Strains as Potential Bioindicators of Mutagenic Pollution of Marine Environments

For biodetection of mutagenic pollution of marine environments, an organism naturally occurring in these habitats should be used. We found that marine bacterium Vibrio harveyi may be an appropriate bioindicator of mutagenic pollution. For positive selection of mutants, we developed a simple method for isolation of V. harveyi mutants resistant to neomycin. We constructed genetically modified V. harveyi strains that produce significantly more neomycin-resistant mutants upon treatment with low concentrations of mutagens than the wild-type counterpart. The sensitivity of the mutagenicity test with the V. harveyi strains is at least comparable to (if not higher than) that of the commonly used Ames test, which uses Salmonella enterica serovar Typhimurium strains. Therefore, we consider that the V. harveyi strains described in this report could be used as potential bioindicators of mutagenic pollution of marine environments.     

Low Efficiency of Short-Term Tests in the Assessment of the Potential Mutagenic Hazard of Chemical Compounds to Mammals

A new method of the efficiency assessment of testing mutagenicity chemical pollutants is proposed. The method is based on the selective information criterion and allows one to compare the prognostic significance of results obtained in both individual tests and test batteries. The efficiency of mutagen detection in mammals was estimated in Ames' test, the in vivo test for cytogenetic abnormalities in rodent bone-marrow cells, and the battery combining both these tests. The level of evidence for mutagenicity was determined for chemicals analyzed in these tests. Based on information obtained during the trials, a low efficiency of the analyzed tests and their battery was inferred.     

Mutagenic action of a series of epoxides

The mutagenicity of a series of 13 epoxide compounds was studied using a bacterial plate assay system. The histidine-dependent tester strains TA98 (for frameshift mutagens) and TA100 (for base-pair substitution mutagens) of Salmonella typhimurium were used. Mutagenicity was evaluated both with and without the addition of rat liver microsomal extract. Dieldrin, diglycidyl ether of bis phenol A and 3 of its homologues were not mutagenic. Allyl glycidyl ether, n-butyl glycidyl ether, vinyl cyclohexene diepoxide, glycidol, glycidaldehyde, diglycidyl ether, diepoxybutane and diglycidyl ether of substituted glycerine were mutagenic in the TA100 strain, causing reversion of the bacteria to histidine independence. Dose—response curves of the mutagenicity of the latter 4 compounds were obtained. On a molar basis, glycidaldehyde was about 20–50 times more potent in producing mutation that were the other 3 epoxides in the dose—response test. In general, the mutagenicity of the epoxides was not enhanced or diminished by the addition of microsomal extract.     

冷藏对新鲜冰冻血浆诱导内皮细胞迁移的影响及其分子机制研究

探讨冷藏是否导致新鲜冰冻血浆(fresh frozen plasma,FFP)中的转化生长因子-β(transform growth factor-β,TGF-β)发生变化并降低其对内皮细胞迁移的诱导能力及其分子机制.为证实冷藏可能导致FFP中TGF-β的水平升高进而影响其功能,首先采用ELISA法分析当天解冻FFP(FFP Day 0)和解冻后4℃冷藏1～5天FFP中TGF-β含量,迁移实验及Western blot分析比较FFP(Day 0)和FFP(Day 5)处理人肺微血管内皮细胞(human pulmonary microvascular endothelial cells,HPMECs)后的细胞迁移率及TGF-β信号通路Smad2和Smad3磷酸化,进一步采用ALK5-siRNA和ALK5特异性抑制剂阻断细胞TGF-βⅠ型受体ALK5的活性,分析下调TGF-β/Smad2/3信号传导后的内皮细胞迁移率.结果显示:随着冷藏时间的延长,FFP中TGF-β1水平逐渐增加,其增加率为244.31 ng/(L.d)(P<0.05);与FFP(Day 0)相比,FFP(Day 5)诱导内皮细胞HPMECs的TGF-β信号通路Smad2/3磷酸化显著增加(P<0.05);不管是FFP(Day 0)还是FFP(Day 5)体外均能诱导内皮细胞迁移,与FFP(Day 0)相比,FFP(Day 5)诱导细胞迁移能力显著降低(P<0.05);阻断TGF-βⅠ型受体ALK5的活性、下调Smad 3信号传导可恢复FFP(Day5)诱导细胞迁移能力.上述结果表明,FFP能显著诱导内皮细胞迁移,4℃冷藏增加FFP中TGF-β1水平并增加内皮细胞TGF-β1信号传导,进而降低FFP诱导内皮细胞迁移.提示,FFP复苏疗效的提高可能与增加内皮细胞迁移有关,冷藏导致内皮细胞迁移降低进而降低FFP复苏疗效.     

Differentiation Potential and Chromosome Stability of Pollen Callus of Maize in Subcultures

Pollen calli were subcultured for 20 months. According to the degree of totipotency observed during this period they can be divided into the following four types: 1) The potential of differentiation of the calli was lacking. 2) The calli were able to differentiate into a few root-like structure and green spots. 3). The calli were able to grow into not only a few green spots but also a few shoots. But they could not develop into normal plants. 4). This type of callus possessed a great ability of differentiation and regeneration. They could develop into embryoids as well as shoots in the same medium. After transferring on to the regeneration medium, they readily regenerated into green plantlets with profuse roots. When chromosome counts were made on the ealli, it was found that variations in ploidy were not great. From 1539 randomly chosen cells at metaphase stage, 90% were haploid. As the counts were made on the No. 1 callus type and the root-tip cells of the 35 pants regenerated from this same type 89.7% of the former 427 cells and 87.4% of the latter 645 cells were determined to be haploid. The calli of the Type No. 1 possess unlimited totipotency demonstrated by regeneration. These stability and totipoteney may play an important role in practical application and theoretical research of maize.