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.我们以前已经发现人脱氧核糖核酸酶（DNase）II是遗传标记：人类中的DNase II水平因个人是否具有dnase2 ^*h还是 ^*l等位基因而变化。澄清说，近端启动子区域中的A-75G转变会导致基因启动子活性的等位基因差异，这是其遗传多态性的基础。2。我们设计了一个简单的DNA提取程序，适用于使用市售的DNA/RNA提取盒的尿液样品进行STR键入。这种方法允许将尿液样品保存在冷冻或水性状态下，以供取证使用。此外，我们设计了一种结合了一种简单的提取方法，从老年尿液中键入的DNase I的等电聚焦和活动染色的过程。 DNase I多态性可以被认为是第一个生化标记物非常适合来自小老年尿液染色的个性化。3。我们确认了日本人群中DNase I多态性的种群遗传基础用于法医。我们对DNASE I类型的检查显示，在DNase1等位基因中，北部梯度下降了。4。我们发现了人类DNase I基因内含子4中56bp单位的新颖数量的串联重复，导致DNase的个性化效率提高DNase.5。使用新开发的抗体筛选程序具有高特异性的鼠单克隆抗人DNase I和DNase II。使用这些单克隆抗体的免疫亲和力程序使人类DNase I和II的纯化比常规程序更容易，更快，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，该程序由用DNase I cDNA在转染的哺乳动物细胞中的瞬时表达以及从培养基中纯化酶的纯化。