Synthesis of dinucleoside tetraphosphates in transfected cells by a firefly luciferase reporter gene

The firefly luciferase gene is widely used as a reporter gene and its expression is generally considered to be non-toxic. In addition to its light-producing reaction, luciferase can synthesise dinucleoside polyphosphates, intracellular signalling molecules, in vitro. Here we show that COS-7 cells transfected with a luciferase expression vector accumulate up to 0.5 mM adenine-containing dinucleoside tetraphosphates (Ap₄N) during the 24 h following luciferin addition. The optimal external concentration of luciferin was 0.4–0.6 mM. In agreement with its poor ability to synthesise adenine-containing dinucleoside triphosphates in vitro, the level of these compounds did not increase after transfection. Consequently, the results of experiments involving luciferase-mediated light production by live cells should now be viewed in the light of the possible effects of an increased intracellular Ap₄N concentration on the properties of the system under investigation. This observation also points to a useful non-invasive procedure for the specific enhancement of intracellular Ap₄N for studies directed at understanding the functions of these compounds.Received 12 November 2003; received after revision 10 December 2003; accepted 12 December 2003     

Adenovirus-mediated<Emphasis Type="Italic">CTLA4-FasL</Emphasis> gene transfer prevents autoimmune diabetes in mice induced by multiple low doses of streptozotocin

Type 1 diabetes is the result of a selective destruction of insulin-producing β cells in pancreatic islets by autoreactive T cells. Depletion of autoreactive T cell through apoptosis may be a potential strategy for the prevention of autoimmune diabetes. Simultaneous stimulation of Fas-mediated pathway and blockade of costimulation by a CTLA4-FasL fusion protein has been reported to lead to substantial inhibition of mixed lymphocyte reaction and enhanced in vitro apoptosis of peripheral lymphocytes. To test the feasibility of CTLA4-FasL-based gene therapy to prevent autoimmune diabetes, we developed recombinant adenovirus containing human CTLA4-FasL gene (AdCTLA4-FasL). A single injection of 2×10~8 plaque forming units (PFU) of AdCTLA4-FasL via tail vein dramatically reduced the incidence of autoimmune diabetes in mice induced by multiple low doses of streptozotocin. AdCTLA4-FasL administration maintained islet insulin content, significantly increased apoptosis of pancreatic lymphocytes, quantitatively     

哈拉维及其"赛博格"神话

堂娜·哈拉维是当代西方著名的跨学科学者.她提出了著名的赛博格女性主义理论和女性主义后现代主义的科学观,还对基因食品进行了文化解读.介绍了哈拉维的生平和著作,概括和分析了哈拉维的主要思想.     

分析伯氏疏螺旋体密码子使用的倾向性

本文采用非线性映射的方法分析伯氏疏螺旋体前导链和后随链上的基因结构，发现基因分布存在明显的差异，同义密码子的使用亦具有明显的倾向性．此外，高表达基因分布具有不对称性，其同义密码子使用与其它基因亦有不同，这表明原核生物基因组复制起始点两侧的碱基分布及翻译机制均影响基因的密码子使用．     

人tumstatin基因的克隆、表达和抗体制备

应用PCR技术从人胎肺cDNA文库中扩增tumstatin基因编码序列，克隆至pET-3c载体构建非融合表达的重组质粒pET-3c-tum，并在大肠杆菌E．coli B121（DE3）中获得高效表达。表达蛋白经SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离、切胶回收后免疫新西兰大白兔，获得ELISA效价高达1：1000的特异性兔抗人tumstatin抗血清。     

利用回交群体对小麦雌性不育基因的SSR标记

对普通小麦新601、雌性不育材料XND126及其BC1群体的育性进行了观察.分析结果表明,此组合中雌性不育表现为1对隐性基因的遗传.结合集群分析(Bulked Segregant Anal-ysis,BSA)法在亲本间筛选了1 080对微卫星引物,并利用回交群体对小麦雌性不育基因进行了SSR分子标记,确定微卫星引物cfd36标记与雌性不育基因连锁,其遗传距离为13.0 cM.     

植物基因分离的方法

总结了目前植物基因分离的方法．概括出四大类植物基因分离的方法，即序列克隆法，功能克隆法。作图克隆法。表型差异克隆法，并对其特点和适用范围进行了评述。     

突变型大肠杆菌二氢叶酸还原酶基因的合成

利用PCR定点突变技术，以野生型大肠杆菌二氢叶酸还原酶基因为模板，获取3种突变型(Cys85Ser．Cysl51Ser，Cys85／151Ser)二氢叶酸还原酶(DHFR)基因．用限制性内切酶BamH Ⅰ与Pst Ⅰ将3种突变基因片段插入到克隆载体pUC18上，进行蓝白筛选，将筛选的克隆进行DNA序列测定．