Genetic Trait for Progression of Chronic Renal Disease

我们等人最近的研究表明，ACE插入/缺失（l/D）多态性的D等位基因（DD）的纯合子是缓慢进展性肾脏疾病预后不良的潜在危险因素。据报道，ACE I/D多态性与全身和局部ACE水平有显著关联。最近的分离连锁分析表明，数量性状的一个位点可能是I/D位点本身，也可能是ACE I/D位点附近的一个位点。ACE基因的I等位基因在其16内含子中有287 bp的片段（插入），而D等位基因则缺乏这一片段。虽然我们的序列分析显示，插入物具有与肾素基因负调控元件（NRE样序列）高度同源的基序，但l/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来测试插入物的功能。短句来源将截断的sv40o启动子（pLuc）与内含子16的DNA片段（plug - i）融合后表达的报告基因萤火虫荧光素酶（LUC）插入人JEG-3细胞中。作为对照，报告基因融合到内含子16的DNA片段中，没有插入物（pla - d）。用带HSV-tk启动子的肾细胞LUC基因共报告载体共横切细胞。用这些载体短暂横切的细胞在转染48小时后收获用于LUC检测。测量到的萤火虫LUC活性被归一化为肾细胞LUC活性。我们的研究结果表明，荧光素酶基因的表达在剪切了pluck - i的细胞中显著低于剪切了pluck - d的细胞，这表明插入物抑制了荧光素酶报告基因的表达。接下来的分析使用凝胶移位法发现蛋白质复合物结合寡核苷酸与NRE样序列。我们制作了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，通过定点诱变法制备了具有该突变的plap - 1。插入物对荧光素酶报告基因表达的影响随着插入物中NRE样序列的突变而降低。因此，这些结果表明，位于ACE基因的内含子16中的插入物显著抑制了报告基因的表达。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是一致的。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