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

以血浆富组氨酸糖蛋白（HRG）cDNA为筛选探针，从人基因组文库中筛选HRG基因。测定了人血浆富组氨酸糖蛋白基因的核苷酸序列（15，499 bp）。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 '端有GT序列，3'端有AG序列，内含子-外显子边界附近的结构都很保守。由于HRG cDNA的5 '端约100个碱基与酵母线粒体DNA的部分序列完全相同，故对人肝cDNA文库进行了重新筛选，以确定HRG的真实的cDNA的转录起始位点。但是，所获得的克隆在其5 '末端具有编码其它蛋白质的各种DNA片段，因此，起始点没有被牢固地确立。将位于转录起始位点上游的不同长度的片段插入CAT表达载体中，用电穿孔仪转染培养的人肝细胞。培养48 h后，制备细胞提取液，检测CAT活性，但几乎没有检测到CAT活性，然后将这些片段插入含有SV 40增强子序列的CAT表达载体中。这次检测到CAT活性。145 bp的片段能诱导CAT的表达，而57 bp的片段不能诱导CAT的表达，这表明HRG表达的必需元件存在于距假定起始点的-57 ~-145 bp之间。HNF-4和HNF-1转录因子的识别序列分别在约-100和-140 bp处。另一个实验是在生理条件下进行化学交联，以鉴定血浆中HRG的真实的配偶体。三种不同的交联剂，EDC，DMA和DSP，用于此目的，但目前没有确定的紧密连接的产品。

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.