Molecular-biological analysis of age-related markers present in body fluids and its forensic applications

体液中年龄相关标记物的分子生物学分析及其法医学应用

• 批准号: 09357004
• 负责人: KISHI Koichiro
• 金额: $ 18.11万
• 依托单位: Gunma University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A).
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09357004/
• 关键词: estimation of age; age-related markers; gene expression; cDNA cloning; Ugl-Y; Molecular-biological analysis; forensic application; body fluid; PCR; differential display; 分子生物学; 年齢; cDNA; 尿; アミノ酸配列; 鑑識科学

Although individualization in legal medicine is one of very important research field, a number of age-related markers suitable for estimation of age is extremely limited. In this study, we have examined biochemical markers specific to each step of aging and develpment and tried to establish the method to estimate the age from forensic samples by biochemical and molecular-biological detection of these markers1. Ugl-Y is a glycoprotein which has been discovered as an age-related marker detected in urine of only young subjects. In this study, we has determined its primary structure. Ugl-Y consisted of 180 amino acid residues, and from the database search, the amino acid sequence of Ugl-Y was found to be entirely identical to that of a part of fibronectin. From the result, it was assumed that Ugl-Y might be derived from a limited digested product or alternative spliced form of primary gene transcript of fibronectin.2. We have surveyed gene expressed in the age-related manner in the mouse k … More idney to find novel age-related markers. Consequently, seven kinds of age-dependent expressed cDNA could be identified using a fluorescence differential display-PCR (FDD-PCR) technique and comparative RT-PCR technique. Therefore, these methods were found to be very useful for detecting new age-related genes expressed differentially.3. We have elucidated molecular and biological properties of one of age-related genes described above. Its cDNA was 3,058 bp long and consisted of 194 amino acid residues encoding the 2.2 kb protein. From BLAST searching, it was clarified that this protein had high homology to peroxisomal protein, Mpv 17 and PMP 22. Therefore, we named this novel protein as M-LP belonging to Mpv 17 domain family. Moreover, its gene was 5 kb long with three exons, and age-related expression of the gene was confirmed. From these findings made in this research project, we could establish the basis for the molecular and biological approach which is indispensable for analysis and forensic application of age-related markers. Less

尽管个体化在法律医学中是非常重要的研究领域之一，但适合年龄估计的年龄相关标记物却极其有限。在本研究中，我们检查了衰老和发育各个阶段特有的生化标记，并试图建立通过对这些标记进行生化和分子生物学检测来估计法医样本年龄的方法1。 Ugl-Y 是一种糖蛋白，被发现是一种与年龄相关的标记物，仅在年轻受试者的尿液中检测到。在这项研究中，我们确定了其主要结构。 Ugl-Y由180个氨基酸残基组成，通过数据库检索，发现Ugl-Y的氨基酸序列与纤连蛋白的部分氨基酸序列完全相同。由此推测Ugl-Y可能来源于纤连蛋白初级基因转录物的有限消化产物或可变剪接形式。 2．我们调查了小鼠中以与年龄相关的方式表达的基因，以寻找新的与年龄相关的标记。因此，使用荧光差异显示PCR（FDD-PCR）技术和比较RT-PCR技术可以鉴定七种年龄依赖性表达的cDNA。因此，这些方法对于检测差异表达的新的年龄相关基因非常有用。3．我们已经阐明了上述年龄相关基因之一的分子和生物学特性。其 cDNA 长 3,058 bp，由 194 个氨基酸残基组成，编码 2.2 kb 的蛋白质。 BLAST检索表明该蛋白与过氧化物酶体蛋白Mpv 17和PMP 22具有高度同源性。因此，我们将这种新蛋白命名为M-LP，属于Mpv 17结构域家族。此外，其基因长5 kb，具有三个外显子，并且证实了该基因的年龄相关表达。根据本研究项目的这些发现，我们可以为分子和生物学方法奠定基础，这对于年龄相关标记的分析和法医应用是必不可少的。较少的

项目成果

Tamiko Nakajima: "Rapid purification of human Dnase I using mouse monoclonal anti-Dnase I antibodies and characterzation of the antibodies"Experimental and Clinical Immunogenetics. (in press).

Tamiko Nakajima：“使用小鼠单克隆抗 Dnase I 抗体快速纯化人 Dnase I 并表征抗体”实验和临床免疫遗传学。

Osamu Hosomi: "Molecular cloning of cDNA encoding Xenopus Laevis deoxyribonuclease I"DNA Sequence. (in press).

Osamu Hosomi：“编码非洲爪蟾脱氧核糖核酸酶 I 的 cDNA 的分子克隆”DNA 序列。

Yasuda,T.: "Chromosomal localization of a human deoxyribonuclease II gene (DNASE2) to 19p13.2-p13.1 using both the polymerase chain reaction and fluorescence in situ hybridization analysis" Biochemical and Biophysical Research communications.

Yasuda,T.：“使用聚合酶链式反应和荧光原位杂交分析将人脱氧核糖核酸酶 II 基因 (DNASE2) 染色体定位到 19p13.2-p13.1”《生物化学和生物物理研究通讯》。

Haruo Takeshita: "Mammalian deoxyribonuclease I are classified into three types"Biochemical and Biophysical Research Communications. 269(2). 481-484 (2000)

Haruo Takeshita：“哺乳动物脱氧核糖核酸酶 I 分为三种类型”《生物化学和生物物理研究通讯》。

Koichiro Kishi: "DNase I : structure, function, and use in medicine and forensic science"Legal Medicine. (in press). (2001)

岸晃一郎：《DNase I：结构、功能以及在医学和法医学中的应用》法律医学。