权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Studies on the genes of the regulatory proteins for blood coagulation and fibrinolysis.

凝血和纤溶调节蛋白基因的研究。

基本信息

批准号：
62480260
负责人：
AOKI Nobuo
金额：
$ 3.78万
依托单位：
Tokyo Medical and Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

项目摘要

1. We have isolated overlapping phage genomic clones covering an area of 26 kilobases that encodes the human alpha_2-plasmin inhibitor (alpha_2PI). The alpha_2PI gene contains 10 exons and 9 introns distributed over 16 kilobases of DNA. To our knowledge, the number of introns is the highest yet reported for a member of the serine protease inhibitor (serpin) superfamily. All introns are located in the 5'-half of the corresponding mRNA. The 5'-untranslated region and the leader sequence are interrupted by 3 introns totaling 6 kilobases. A "TATA box" sequence is located 17 nucleotides upstream from the proposed transcription initiation site. Multiple "GC box" sequences, G+C-rich sequences, and "CCAAT box"-like sequence, the hepatitis B virus enhancer element-like sequence and the human immunodeficiency virus enhancer-like sequence appear in the 5'-flanking region.2. The human alpha_2PI gene (PLI) was mapped by in situ hybridization using a genomic DNA probe which contained exons coding for the signal peptide and a portion of the mature protein. The results allowed the chromosome localization of the gene to 18q11.1 q11.2.3. The molecular genetic basis of a familial deficiency of alpha_2PI was studied. Southern blot hybridization analysis with human alpha_2PI cDNA and genomic DNA probes demonstrated no gross deletion or rearrangement of the gene. BY sequencing all the coding exons and exonintron boundaries of the gene of a homozygote, we identified a single cytidine nucleotide insertion in the exon coding for the carboxyl-terminal reqion. This frameshift mutation leads to an alteration and elongation of the carboxyl-terminal portion of the deduced amino acid sequence. In a transient expression assay, the alpha_2PI level in the culture medium of the cells transfected with the mutated alpha_2PI expression vector was very low and only 4% of that of the cells transfected with the normal vector.

1.我们已经分离出覆盖26个编码人α_2-纤溶酶抑制剂（α_2PI）的酶的重叠噬菌体基因组克隆。α_2PI基因由10个外显子和9个内含子组成，分布在16个碱基上。据我们所知，内含子的数量是最高的丝氨酸蛋白酶抑制剂（丝氨酸蛋白酶抑制剂）超家族的成员尚未报道。所有的内含子都位于相应mRNA的5 '-一半。5 '-非翻译区和前导序列被3个内含子中断，共6个内含子。“TATA盒”序列位于所提出的转录起始位点上游17个核苷酸处。在5 '侧翼区出现多个“GC box”序列、富含G+ C序列、“CCAAT box”样序列、B型肝炎病毒增强子元件样序列和人类免疫缺陷病毒增强子样序列.用含有编码信号肽和部分成熟蛋白的外显子的基因组DNA探针，通过原位杂交对人α_2PI基因（PLI）进行了定位。结果允许染色体定位的基因18q11.1 q11.2.3。本文对一个α_2 PI家族性缺陷症的分子遗传学基础进行了研究。用人α_2PI cDNA和基因组DNA探针进行Southern杂交分析，结果表明该基因无明显缺失或重排。通过对一个纯合子基因的所有编码外显子和外显子内含子边界进行测序，我们发现在编码羧基末端区域的外显子中插入了一个胞苷核苷酸。这种移码突变导致推导的氨基酸序列的羧基末端部分的改变和延长。在瞬时表达试验中，用突变的α_2PI表达载体转染的细胞的培养基中的α_2PI水平非常低，仅为用正常载体转染的细胞的4%。