Genetic Trait for Progression of Chronic Renal Disease

慢性肾病进展的遗传特征

• 批准号: 09671181
• 负责人: YOSHIDA Hiroaki
• 金额: $ 1.79万
• 依托单位: The Jikei University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09671181/
• 关键词: 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

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 … More 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

我们和其他人最近的研究表明，ACE插入/缺失（L/D）多态性的D等位基因（DD）的纯合子是较差的进行性肾脏疾病的潜在危险因素。据报道，ACE I/D多态性与ACE的全身和局部水平有着显着关联。截至隔离 - 链接分析表明，定量性状局部可能是I/D基因座本身，或与ACE I/D轨迹接近接近的基因座。 ACE基因的I等位基因在基因的内含子L6中具有287 bp的片段（插入），而D等位基因缺乏。尽管我们的序列分析表明，该插入物具有肾素基因的负调节元件（NRE）高度同源的基序（NRE类似序列），但L/D基因座的功能意义仍有待确定。在这项研究中，我们使用报告基因分析测试了插入物的功能。一个报告基因萤火虫荧光素酶（LUC），该酶是……从截短的SV4O启动子（PLUC）融合到内含子16的DNA片段16的DNA片段（pluc-i）的DNA片段（PLUC-I），被转化为人类Jeg-3细胞。作为对照，使用了融合到内含子16的DNA片段的基因，而没有插入物（PLUC-D）。与具有HSV-TK启动子的Renilla luc基因的共生载体共转移。翻译后48小时的LUC测定法收集了与这些载体瞬时转移的细胞。测得的萤火虫LUC活性通过Renilla Luc活性标准化。我们的结果表明，翻译PLUC-I的细胞中的荧光素酶基因表达明显低于PLUC-D中的荧光素酶基因，这表明该插入物将荧光素酶报告基因的表达浸透。在使用凝胶换档测定的分析后，发现与NRE类似序列结合的寡核苷酸结合的蛋白质复合物。我们制作了NRE序列的突变寡核苷酸，发现突变寡核苷不结合蛋白质复合物。然后，通过位点定向诱变进行突变的PLUC-I。插入物对荧光素酶报告基因表达的影响在插入物中的NRE序列上的突变中得到了改善。因此，这些结果表明，ACE基因内含子16中的插入物可显着抑制报告基因的表达。这些结果与ACE I/D基因座本身具有控制ACE水平具有功能意义的可能性一致。较少的

项目成果

吉田裕明: "糖尿病性腎症の発症・進展に関わる遺伝因子" 糖尿病. 41. 3-5 (1998)

Hiroaki Yoshida：“糖尿病肾病发生和进展的遗传因素”糖尿病。41. 3-5 (1998)。

Yoshida H: "Genetic factor in Diabetic nephropathy. "Pathogenesis in incidence and progression of diabetic nephropathy" (IN JAPANESE)" J Japan Diabetic Soc. 41. 3-5 (1998)

吉田 H：“糖尿病肾病的遗传因素。“糖尿病肾病发病和进展的发病机制”（日语）”日本糖尿病协会杂志。

Yoshida H: "Functional significance of the ACE I/D locus for controlling the ACE gene expression (abstract)" J Am Soc Nephrol. 8. 633A (1997)

Yoshida H：“ACE I/D 位点对于控制 ACE 基因表达的功能意义（摘要）”J Am Soc Nephrol。

Yoshida H: "ACE gene Polymorphism and chronic renal diseases." Experimental Nephrology. in press (1998)

Yoshida H：“ACE基因多态性与慢性肾脏疾病。”

Yoshida H: "ACE gene Polymorphism and chronic renal diseases." Experimental Nephrology. (In press).

Yoshida H：“ACE基因多态性与慢性肾脏疾病。”