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Analysis of physiological role of the plasma histidine-rich glycoprotein(HRG)

血浆富含组氨酸糖蛋白（HRG）的生理作用分析

基本信息

批准号：
05680561
负责人：
WAKABAYASHI Sadao
金额：
$ 1.34万
依托单位：
HIMEJI INSTITUTE OF TECHNOLOGY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05680561/
关键词：
Plasma protein Gene structure Histidine-rich glycoprotein control of gene expression

项目摘要

Human genomic library was screened for plasma histidine-rich glycoprotein (HRG) gene using a cDNA for HRG as a screening probe. The nucleotide sequence of the gene for human plasma histidine-rich glycoprotein (15,499 bp) was determined. It composed of 7 exons and 6 introns, in contrast to 9 exons previously reported. The 5'end of each intron has GT sequence and 3'AG and structure around the intron-exon boundaries were all well conserved. As about 100 bases at the 5'end of the reported cDNA for HRG was found to be identical to the part of yeast mitochondrial DNA,human liver cDNA library was rescreened for the real cDNA for HRG in order to determine the transcriptional initiation site. But the obtained clones had various DNA fragments coding for other proteins at their 5'end and, therefore, the initiation point was not firmly established. Various length fragments located just upstream of putative transcriptional intiation site were inserted into CAT expression vector and transfected into the cultured cells originated human hepatocytes using electoporator. After 48 h culture, the cell extracts were prepared and assayd for CAT,but almost no activity was found. Then these fragments were inserted into CAT expression vector which contains SV40 enhancer sequence. This time the CAT activity was detected. The 145 bp fragment could induce the expression of CAT while 57bp could not, suggesting that the essential elements for HRG expression are present between -57 and -145 bp from the putative initiation point. There are recognition sequences for HNF-4 and HNF-1 transcription factors at around -100 and -140 bp, respectively. The other experiment is chemical cross-linking under the physiological conditions to identify the real partner of HRG in the plasma. Three different crosslinkers, EDC,DMA and DSP,were used for this purpose, but at present no closs-linked product was identified.

以人血浆富组氨酸糖蛋白(HRG)基因为筛选探针，对人基因组文库进行筛选。测定了人血浆富组氨酸糖蛋白基因的核苷酸序列(15,499个碱基)。它由7个外显子和6个内含子组成，而不是以前报道的9个外显子。每个内含子的5‘端都有GT序列和3’AG，内含子-外显子边界附近的结构都很保守。由于已报道的HRG基因的5‘端约100个碱基与酵母线粒体DNA的部分序列完全相同，为了确定转录起始点，我们重新筛选了人肝脏cDNA文库，以寻找HRG的真实基因。但获得的克隆在其5‘端有多种编码其他蛋白质的DNA片段，因此起始点并不确定。将位于转录起始点上游的不同长度片段插入到CAT表达载体中，利用电位仪将其导入培养的人肝细胞中。培养48h后，提取的细胞提取液进行过氧化氢酶活性测定，但几乎没有活性。然后将这些片段插入含有SV40增强子序列的CAT表达载体中。这一次检测到了CAT活性。145bp的片段可以诱导CAT的表达，而57bp的片段不能诱导CAT的表达，这表明HRG的表达所必需的元件存在于推测的起始点的-57~-145bp之间。HNF-4和HNF-1转录因子的识别序列分别位于-100和-140bp左右。另一项实验是在生理条件下进行化学交联，以确定血浆中HRG的真正伴侣。三种不同的交联剂，EDC，DMA和DSP被用于这一目的，但目前还没有鉴定出Coss连接的产物。