BIOLOGIC EFFECTS OF DNA BINDING BY 52 KD RO/SS-A

52 KD RO/SS-A 结合 DNA 的生物学效应

基本信息

批准号：
2457979
负责人：
DANIEL P MCCAULIFFE
金额：
$ 12.04万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-08-01 至 1999-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2457979
关键词：
DNA binding protein autoantigens chemical binding computer assisted sequence analysis gel mobility shift assay gene expression genetic library genetic regulatory element human genetic material tag nucleic acid sequence oligonucleotides polymerase chain reaction protein structure protein structure function tissue /cell culture transfection

项目摘要

One of the recently characterized Ro/SS-A (Ro) genes encodes a 52 kD autoantigen that contains a zinc finger motif. The 52 kD Ro protein's novel zinc finger motif has significant homologies with sequences found in a number of DNA binding proteins. Zinc finger motifs have been described in numerous DNA binding proteins, most of which have gene regulatory functions. To test the hypothesis that 52 kD Ro is a DNA binding protein, DNA cellulose binding experiments were conducted. The results reveal that the native and recombinant 52 kD Ro proteins bind DNA with high affinity and in a zinc-dependent fashion. Whole genome polymerase chain reaction methods were utilized to isolate human genomic DNA fragments that are specifically bound by the 52 kD Ro protein. Naturally occurring Ro autoantibodies prevent 52 kD Ro protein from binding DNA. Studies are now proposed to characterize the DNA sequences that are bound by the 52 kD Ro protein, and to authenticate the DNA binding sites by using synthetic oligonucleotides in competitive binding experiments and gel shift assays. The target sequence(s) will be compared to previously characterized regulatory elements. Genes that contain these target sequences will be identified by searching the DNA databases or if necessary by screening a human genomic DNA library. Gene fragments that contain the target sequence will be subjected to competitive binding assays, gel shift assays and (or) DNase protection assays to confirm that the 52 kD Ro protein binds to them. Transfection studies will investigate the cellular effects caused by over-expressing the 52 kD Ro protein in human cells. These studies will: 1) determine DNA sequence(s) bound by the 52 kD Ro antigen; 2) identify genes whose expression is regulated by the 52 kD Ro antigen; and 3) elucidate the cellular function of the 52 kD protein. The outcome of these studies should have a significant impact on our understanding of the role that this autoantigen plays in the Ro autoantibody-associated immune disorders and thus may expedite the development of better strategies for treating those afflicted.

最近特征的RO/SS-A（RO）基因编码52 kD 含有锌指图案的自动抗原。 52 kd RO蛋白新颖的锌指基序具有重要的同源性，并在许多DNA结合蛋白。已经描述了锌指图案在许多DNA结合蛋白中，其中大多数具有基因调节性功能。为了测试52 kD RO是DNA结合蛋白DNA的假设进行了纤维素结合实验。结果表明天然和重组52 kD RO蛋白与高亲和力结合DNA，以锌的方式。整个基因组聚合酶链反应利用方法来隔离人类基因组DNA片段特别由52 kD RO蛋白结合。天然发生的RO 自身抗体预防52 kD RO蛋白结合DNA。现在提出了研究以表征结合的DNA序列通过52 kD RO蛋白，并通过在竞争结合实验中使用合成寡核苷酸和凝胶转移测定。目标序列将与先前的特征是监管元素。包含这些目标的基因序列将通过搜索DNA数据库或if 通过筛选人类基因组DNA文库所必需的。基因碎片包含目标序列将受到竞争性结合测定，凝胶轮班测定法和（或）DNase保护分析，以确认 52 kD RO蛋白与它们结合。转染研究将调查通过过表达52 kD RO蛋白的细胞效应人类细胞。这些研究将：1）确定由52 kD RO结合的DNA序列抗原; 2）识别其表达受52 kD RO调节的基因抗原; 3）阐明52 kD蛋白的细胞功能。这这些研究的结果应该对我们的了解这种自动抗原在RO中的作用自身抗体相关的免疫疾病，因此可能会加快制定更好的策略来治疗受苦者。