| Project/Area Number |
09671181
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Kidney internal medicine
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| Research Institution | The Jikei University School of Medicine |
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Principal Investigator |
YOSHIDA Hiroaki The Jikei University School of Medicine, Instructor, 内科学講座・第2, 助手 (10256464)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 1998: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1997: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Keywords | ANGIOTENSIN CONVERTING ENZYME / GENE POLYMORPHISM / TRANSCRIPTION / NEGATIVE REGULATORY ELEMENT / QUANTITATIVE TRAIT LOCUS / アンジオテンシン変換酵素 / 慢性腎不全関連遺伝子 / reporter gene assay / gel-shift assay |
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| Research Abstract |
Recent studies by us and others have demonstrated that the homozygote of the D allele (DD) of the ACE insertion/deletion (l/D) polymorphism is a potential risk factor for poor prognosis in slowly progressive renal diseases. The ACE I/D polymorphism has been reported to have a significant association with the systemic and local levels of ACE.Recent segregation-linkage analyses suggest that one of quantitative trait loci might be the I/D locus itself or a locus in close proximity to the ACE I/D locus. The I allele of the ACE gene has a 287 bp fragment (insert) within intron l6 of the gene, which is lacking in the D allele. While our sequence analysis has revealed that the insert has a motif highly homologous to negative regulatory element (NRE) of a renin gene (NRE like sequence), the functional significance of the l/D locus remains to be determined. In this study, we tested the functionality of the insert using a reporter gene analysis.
A reporter gene firefly luciferase (LUC), which was
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expressed from a truncated SV4O promoter (pLuc) fused to a DNA fragment of the intron 16 with the insert (pLuc-I), was transected into human JEG-3 cells. As a control, the reporter gene fused to a DNA fragment of the intron 16 without the insert (pLuc-D) was used. Cells were co-transected with co-reporter vector having renilla LUC gene with HSV-tk promoter. Cells transected transiently with these vectors were harvest for LUC assay 48 hr after transfection. Measured firefly LUC activity was normalized by renilla LUC activity. Our results demonstrated that luciferase gene expression in cells which transected pLuc-I was significantly lower than that in pLuc-D, indicating that the insert supressed the expression of luciferase reporter gene. Following analyses using gel-shift assay found protein complexes that bind to a oligo nucleotide with the NRE like sequence. We made mutant oligo nucleotides of the NRE like sequence and found a mutant oligo does not bind a protein complex. Then, pLuc-I with the mutation was made by site directed mutagenesis. The effects of the insert on the luciferase reporter gene expression was decreased in the pluc-I with the mutation on the NRE like sequence in the insert.
These results therefore indicate that the insert located in the intron 16 of the ACE gene significantly suppresses the expression of the reporter gene. These results are consistent with the possibility that the ACE I/D locus per se has a functional significance for controlling the ACE level. Less
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