The discovery of circadian clock genes and the use of similar strategies to discover unknown genes underlying complex behaviors and brain disorders

Over just the past few years, tremendous progress has been made in unraveling the molecular basis of the circadian clock in mammals. This success has been primarily due to an approach whereby mutations are induced randomly in the germ line and the offspring of the mutagenized animals are tested for abnormal circadian phenotype. Circadian clock genes have been discovered this way in both fruit flies and mice and it is now clear that most, if not all clock genes show homology between flies and mammals, including humans. This ‘forward genetics’ approach is a powerful tool for uncovering genes which underline complex behaviors and brain disorders. Even when a complex neural function involves many, many genes, mutating just one of these genes can have pronounced effects on the expressed behavior and can lead to the discovery of other genes, and their protein products, that interact directly or indirectly with the mutated gene.     

SQA鳌合盐的急性毒性和致突变性研究

目的 检测稀土壳糖胺(SQA)鳌合盐的急性毒性和致突变性。方法 采用霍恩氏法进行急性经口毒性试验，采用小鼠骨髓嗜多染红细胞微核试验、小鼠精子畸形试验和Ames试验对受试物进行致突变性研究。结果 SQA鳌合盐的经口LD50＞10000mg／kg体重；小鼠骨髓嗜多染红细胞微核试验显示无诱发微核作用，小鼠精子畸形试验未见精于畸形率增高，Ames试验在加和不加S9条件下均无致突变性。结论 SQA鳌合盐属实际无毒级物质，在该实验条件下，无致突变作用。     

海洋小单孢菌株产抗肿瘤新化合物FW05-0328-1的诱变#br# 选育及发酵工艺研究

目的 获得抗肿瘤化合物FW05-0328-1高产菌株，提高其发酵产量。方法 采用紫外(UV)及常压室温等离子体技术(ARTP)复合诱变筛选化合物FW05-0328-1高产菌株，结合高产菌株摇瓶发酵特性，优化100L全自动发酵罐发酵工艺。结果 获得1株高产FW05-0328-1菌株Micromonospora sp. FIM0O123，其在100L全自动发酵罐发酵效价达187.3mg/L，较原始菌株提高了17倍。结论 高产菌株的筛选及100L全自动发酵罐发酵工艺优化能有效提高化合物FW05-0328-1产生能力。     

Influence of ethanol induction on the metabolic activation of genotoxic agents by isolated rat hepatocytes

The effects of ethanol-feeding to rats, over a 6-week period, on the activation of genotoxic compounds of different chemical classes, requiring metabolic conversion to exert their mutagenic activity, were studied in isolated rat hepatocytes. The influence of such treatment on cytochrome P-450 content and N-acetylation in isolated hepatocytes was also investigated.Benzidine (BZ), dimethylnitrosamine (DMN), diethylnitrosamine (DEN), isoniazid (INH) and cyclophosphamide (CP) were more effectively activated to products mutagenic towards Salmonella typhimurium by hepatocytes from ethanol-pretreated rats than by hepatocytes from controls. The mutagenic potency of 2-aminofluorene (2-AF) and 2-acetylaminofluorene (2-AAF) was not influenced by ethanol pretreatment. Ethanol consumption was found to be associated with increased cytochrome P-450 content and enhanced N-acetylation in the isolated hepatocytes.Our results support the hypothesis that an alteration of the hepatic drug-metabolizing system may be responsible for the ethanol-induced increase in susceptibility to certain genotoxic compounds.     

突变人CD59分子糖基化前后抗补体活性的变化

目的探讨2种突变CD59分子(HM3、HM4)糖基化前后抗补体活性的变化,为糖尿病血管增殖的发生机制提供理论基础。方法采用重组聚合酶链反应定点诱变技术在CD59易于糖基化的位点“41位赖氨酸—44位组氨酸”将44位的组氨酸基因位置改变,构建2种不同的CD59突变基因,克隆入真核表达质粒pALTER-MAX。利用阳离子脂质体将重组质粒和pcDNA3共转染中国仓鼠卵巢细胞。G418筛选稳定转染细胞克隆,免疫标记技术检测筛选突变CD59基因蛋白高表达株。染料释放实验检测突变CD59分子糖基化前后抗补体活性。结果序列测定及酶切鉴定均证实成功构建了pALTER-突变CD59重组真核表达载体系统。经酶免疫组织化学、荧光免疫组织化学和流式细胞术鉴定出较高表达突变CD59分子的阳性细胞克隆。免疫印迹技术检测出阳性细胞克隆裂解液中有1条相对分子质量约20 000的CD59蛋白表达带。染料释放实验初步证实突变CD59分子具有抗补体活性,糖基化后抗补体活性明显降低。结论2种突变CD59分子均具有抗补体活性,高糖环境下易被糖基化,糖基化后抗补体活性明显降低,为进一步研究糖尿病血管增殖的发生机制提供了线索。     

A knowledge-based system for cassette mutagenesis experimental design

A knowledge-based system for the design and planning of cassette mutagenesis experiments has been developed for scientists working in the field of structural biology and protein engineering. The system applies domain-specific knowledge to manage the menial details and automate most of the decision-making steps involved in the design process. This allows scientists to work at a high abstraction level, and results in significant time savings and increased productivity. The system also includes an automated documentation facility to improve the efficiency and accuracy of record keeping.     

SOS原位法检测蚊香烟气致突变性的研究

采用 SOS/Umu 原位法检测国产蚊香烟气的直接致突变性。结果表明 JC 牌无烟蚊香(不加药)和 E 牌中草药蚊香 SOS/Umu 试验阴性。JL 牌、YC 牌和 TT 牌除虫菊蚊香均为阳性结果,SOS 阳性诱导(R=2)的最小采样体积:JL 蚊香为85.7L,TT 蚊香为108.9L,YZ 蚊香为171.0L。烟气的致突变性主要来源于除虫菊类杀虫剂的燃烧分解产物。SOS 原位法简便、灵敏,适合于检测空气混合性污染物的综合致突变性。     

Loss of heterozygosity: The most frequent cause of recessive phenotype expression at the heterozygous human adenine phosphoribosyltransferase locus

Expression of a recessive phenotype can occur by a number of different mechanisms, such as chromosomal deletion, recombination, and intragenic frameshift mutation or base substitution. To examine the contribution of different mutational events, we isolated and characterized a human fibroblast cell line heterozygous at the adenine phosphoribosyltransferase (APRT) locus. Cells that subsequently lost APRT activity were selected, cloned, and analyzed for the mechanisms contributing to the loss of APRT activity. Loss of APRT activity occurred at a rate of 7.8 x 10^-5 per allele per cell generation. Molecular analysis of DNA from 21 independent APRT^- clones demonstrated that 62% of mutants had lost the functional allele and that the rest had incurred intragenic mutations. Loss of the functional allele was frequently accompanied by loss of the proximal marker D16S77 but not the more distant proximal marker D16S4, indicating that a high frequency of mitotic recombination or deletion occurred at the region between D16S77 and D16S4 on chromosome 16. Loss of APRT activity in the remaining 38% of the clones was predominantly due to point mutations. These data demonstrate that the mechanisms for loss of heterozygosity at the APRT locus are similar to those found in retinoblastoma and other tumors. The autosomal location of the APRT gene and the ease with which its phenotype can be selected make this gene useful for modeling mutational events at loci important to carcinogenesis.     

双链DNA直接测序法在环境诱变剂致突变分子机制研究中的应用Ⅱ.甲基丙烯酸环氧丙酯诱导质粒抗性基因突变的测定

运用我所建立的双链DNA直接测序法测定了GMA诱导的质粒抗性基因片段(Tc~R)EcnRI/SaII片段(～0.3Kb)的序列,并与相应正常基因片段序列作一比较,所得结果具有一定意义。本实验表明:双链DNA直接测序法可应用于环境诱变剂研究。     

Double and quadruple deletion mutant of EHV-1 is highly attenuated and induces optimal immune response

Equid alphaherpesvirus 1 (EHV-1) infection causes significant health problems in equines. The EHV-1 infection leads to abortion storm in mares, respiratory disease and myeloencephalopathy. Despite the wide use of vaccines, the outbreaks of EHV-1 infections keep occurring globally, suggesting the need for the development of improved vaccines. Gene deletion attenuated mutant viruses could be a good candidate for the development of modified live vaccines. Here, we report the generation of mutant EHV-1 by deleting virulence (glycoprotein E & internal repeat 6; IR6) and immune evasive (pUL43 & pUL56) associated genes either individually or in combinations; and comprehensive evaluation of mutants through in vitro characterization followed by in vivo study in murine model to adjudge the attenuation of the virus and immune responses generated by mutants vis-à-vis wild type (wt) virus. The EHV-1 mutants with deletion of IR6 and gE genes (vToH-DMV) and four genes (i.e., gE, IR6, pUL43 and pUL56) (vToH-QMV) revealed a significant reduction in plaque size with minimal loss in replication efficiency in comparison to the wt virus. Further, in vivo studies showed virus attenuation adjudged through significant reduction in clinical signs, weight loss, gross and histopathological lesions in comparison to wt virus also revealed improved immune responses estimated through serum neutralization and flow cytometric analysis of CD4 + and CD8 + cell populations. Thus it can be concluded that EHV-1 mutants viz. vToH-DMV and vToH-QMV (novel combination) are promising vaccine candidates and qualify to be studied for adjudging the protective efficacy with wt virus challenge.