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Analysis of the relationships between the DNase I or II enzymes and apoptosis

DNase I或II酶与细胞凋亡的关系分析

基本信息

批准号：
12307011
负责人：
KISHI Koichiro
金额：
$ 25.08万
依托单位：
Gunma University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2003
项目状态：
已结题

项目摘要

1.We have previously found human deoxyribonuclease (DNase) II to be a genetic marker : DNase II levels in human vary depending on whether the individual has the DNASE2^*H or ^*L allele. It was clarified that the A-75G transition in the proximal promoter region causes the allelic difference in the promoter activity of the gene, underlying its genetic polymorphism.2.We devised a simple DNA extraction procedure suitable for STR typing of urine samples using a commercially available DNA/RNA extraction kit. This method allows urine samples to be kept in both a frozen or aqueous state for forensic use. Furthermore, we devised a procedure that combines a simple extraction method, isoelectric focusing and activity-staining for DNase I typing from aged urine stains. DNase I polymorphism could be considered the first biochemical marker found to be well suited for individualization from small aged urine stains.3.We confirmed the population genetic basis for DNase I polymorphism in a Japanese population for forensic application. Our examination of DNase I types revealed a decreasing north-to-south gradient in the DNASE1 allele.4.We discovered a novel variable number of tandem repeat of 56bp unit in intron 4 of human DNase I gene, resulting in elevation of individualization efficiency of DNase.5.In order to devise the highly sensitive-and specific-immunological typing for DNase I and II polymorphisms, we succeeded in production of each murine monoclonal anti-human DNase I and DNase II with high specificity using newly developed screening procedure for antibodies. Immunoaffinity procedure using these monoclonal antibodies made the purification of human DNases I and II easier, faster and more effective than the conventional procedure. Furthermore, in order to obtain human DNase I as an immunogen, we developed a procedure consisting of transient expression of the enzyme in transfected mammalian cells with DNase I cDNA and purification of the enzyme from the culture medium.

1.我们先前发现人类脱氧核糖核酸酶（DNase）II是一种遗传标记：人类的DNase II水平取决于个体是否具有DNASE 2 ^*H或^*L等位基因。说明该基因启动子近端A-75 G的突变导致了基因启动子活性的等位基因差异，是其遗传多态性的基础。2.我们设计了一种简单的DNA提取方法，适用于使用市售的DNA/RNA提取试剂盒对尿样进行STR分型。这种方法允许尿液样本保持在冷冻或含水状态下供法医使用。此外，我们设计了一个程序，结合一个简单的提取方法，等电聚焦和活性染色的DNA酶I分型从老年尿污渍。DNA酶I多态性可以被认为是第一个被发现的生化标记，很好地适用于个体化从小年龄尿染色。3.我们证实了群体遗传学基础的DNA酶I多态性在日本的法医应用。4.在人DNase Ⅰ基因内含子4中发现了一个新的可变数目的56 bp串联重复序列，它提高了DNase的个体化效率。5.为了设计高灵敏度、高特异性的DNase Ⅰ和DNase Ⅱ多态性免疫分型方法，我们使用新开发的抗体筛选方法成功地生产了具有高特异性的每种鼠单克隆抗人DNA酶I和DNA酶II。使用这些单克隆抗体的免疫亲和方法使人DNA酶I和II的纯化比常规方法更容易、更快和更有效。此外，为了获得人DNA酶I作为免疫原，我们开发了一种程序，包括瞬时表达的酶在转染的哺乳动物细胞与DNA酶I cDNA和纯化的酶从培养基中。