Genetic Trait for Progression of Chronic Renal Disease

近年来我们和其他学者的研究表明，ACE插入/缺失（I/D）多态性的D等位基因（DD）纯合子是慢性进展性肾脏疾病预后不良的潜在危险因素。ACE I/D多态性与ACE的系统和局部水平有显著的相关性，最近的分离-连锁分析表明，数量性状位点之一可能是I/D位点本身或与I/D位点邻近的位点。ACE基因的I等位基因在基因的内含子16内具有287 bp的片段（插入片段），其在D等位基因中缺失。虽然我们的序列分析已经揭示了插入片段具有与肾素基因的负调控元件（NRE）高度同源的基序（NRE样序列），但I/D位点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性。 关于我们 将从与具有插入片段的内含子16的DNA片段（pLuc-I）融合的截短的SV 4 O启动子（pLuc）表达的pLuc转染到人JEG-3细胞中。作为对照，使用与没有插入片段的内含子16的DNA片段融合的报道基因（pLuc-D）。用具有海肾LUC基因和HSV-tk启动子的共报道载体共横切细胞。转染后48小时收获用这些载体瞬时横切的细胞用于LUC测定。测量的萤火虫LUC活性通过海肾LUC活性标准化。我们的结果表明，荧光素酶基因的表达在细胞中的横切pLuc-I显着低于pLuc-D，表明插入抑制荧光素酶报告基因的表达。使用凝胶迁移测定进行分析后，发现与具有NRE样序列的寡核苷酸结合的蛋白质复合物。我们制备了NRE样序列的突变寡核苷酸，发现突变寡核苷酸不结合蛋白质复合物。然后，通过定点诱变制备具有突变的pLuc-I。插入片段对荧光素酶报告基因表达的影响在插入片段中NRE样序列突变的pluc-I中降低，因此，位于ACE基因内含子16的插入片段对报告基因的表达有明显的抑制作用。这些结果与ACE I/D位点本身对控制ACE水平具有功能意义的可能性一致。少

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