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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.我们以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DNASE2^*H或^*L等位基因。结果表明，A-75G在近端启动子区域的转变导致了该基因启动子活性的等位基因差异，从而导致了其遗传多态性。我们设计了一种简单的DNA提取程序，适用于使用市售DNA/RNA提取试剂盒对尿液样本进行STR分型。这种方法允许尿液样本以冷冻或水的状态保存，以供法医使用。此外，我们设计了一种结合了简单提取方法、等电聚焦和活性染色的程序，用于从老年人尿液染色中分型DNase I。dna酶I多态性可以被认为是第一个被发现的生化标记物，非常适合于从小的老年尿渍个体化。我们在日本人群中确认了dna酶I多态性的群体遗传基础，以供法医应用。我们对DNASE1型的检查显示，DNASE1等位基因的南北梯度递减。我们在人类dna酶I基因的内含子4中发现了一个新的56bp单位的可变数目串联重复序列，从而提高了dna酶I的个体化效率。为了对DNase I和DNase II的多态性设计高敏感性和特异性的免疫分型，我们成功地使用新开发的抗体筛选程序生产了具有高特异性的小鼠单克隆抗人DNase I和DNase II。使用这些单克隆抗体的免疫亲和程序使人dna酶I和II的纯化比传统程序更容易、更快和更有效。此外，为了获得人类DNase I作为免疫原，我们开发了一种程序，包括在转染了DNase I cDNA的哺乳动物细胞中瞬时表达酶，并从培养基中纯化酶。