| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1994: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1993: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
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.
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