PATHOGENESIS OF MEMBRANOUS GLOMERULONEPHROPATHY

描述（改编自申请人的摘要）：长期目标是 了解膜性肾小球肾病（MGN）的机制。 他们 将与公认的实验模型MGN，海曼肾炎， (HN)的大鼠，并集中他们的研究对推定的抗原（gp 330）， 机制的主要参与者。 具体目的是：1）通过筛选获得gp 330的全长cDNA 用已获得的gp 330部分cDNA克隆构建大鼠肾cDNA文库， 用限制性内切酶检查其它克隆的重叠区域 和Southern印迹分析，并确认每个克隆与gp 330结合 通过北方分析和S1核酸酶保护测定的mRNA。 2)隔离 gp 330的基因，通过用各种 gp 330 cDNA克隆。 3)分析gp 330基因的结构， 转录起始位点，转录因子和调节序列， 以及内含子/外显子组织。 这将通过以下方式实现： 对基因的5 ′启动子区以及内含子和外显子进行测序 S1核酸酶和引物延伸分析，以确定 转录起始位点;限制性作图和Southern分析 具有全长cDNA的各种片段的基因组克隆，以及 作为S1核酸酶分析以确定内含子/外显子大小和位置。 四、 gp 330基因启动子区的分析 肾小球上皮细胞 gp 330 5'启动子的各种构建体 将该区域连接到CAT表达载体上，并且得到的 磷酸钙沉淀法转染GEC的质粒 法 对这些细胞中CAT表达的分析将确定 gp 330基因启动子区的序列控制其表达。 5)HN肾小球gp 330基因表达水平的研究 在HN中的MGN病变的进展和解决通过原位 杂交方法 6)gp 330基因在胎儿期表达的研究 和出生后发育的肾脏原位杂交和 核糖核酸酶保护测定。 7)研究gp 330 mRNA是否存在于其他 使用核糖核酸酶保护通过定量gp 330 mRNA 比色法 从基因水平研究gp 330产生的数据将提供 它的结构，与其他蛋白质的关系， 表达、肾发育和HN期间的表达水平以及 存在于其他组织中。 这些新知识将直接影响到 了解这种蛋白质的正常功能以及长期的 项目的长期目标。

DESCRIPTION (Adapted from Applicant's Abstract): The long term goal is to understand the mechanisms of membranous glomerulonephropathy (MGN). They will work with the accepted experimental model of MGN, Heymann Nephritis (HN) of rat, and concentrate their studies on the putative antigen (gp330), a key participant in the mechanisms. The specific aims are: 1) Obtain a full length cDNA of gp330 by screening rat kidney cDNA library with already obtained partial cDNA clones of gp330, examining additional clones for overlapping regions by restriction enzyme and Southern blot analysis, and confirming that each clone binds to gp330 mRNA by Northern analysis and S1 nuclease protection assays. 2) Isolate the gene for gp330 by screening a rat genomic library with the various gp330 cDNA clones. 3) Analyze the structure of the gp330 gene for transcription start sites, transcription factor and regulatory sequences, as well as intron/exon organization. This will be accomplished by: sequencing the 5' promotor region of the gene as well as intron and exon junctions; S1 nuclease and primer extension analysis to determine transcription start sites; and restriction mapping and Southern analysis of the genomic clones with various fragments of the full length cDNA, as well as S1 nuclease analysis to determine intron/exon size and locations. 4) Analyze the promotor region controlling gp330 gene expression in cultured glomerular epithelial cells. Various constructs of the gp330 5' promotor region will be ligated to a CAT expression vector and the resulting plasmids used to transfect GEC by the calcium phosphate precipitation method. The analysis of CAT expression in these cells will determine what sequences in the promotor region of the gp330 gene control its expression. 5) Study the level of gp330 gene expression in HN glomeruli during the progression and resolution of the MGN lesion in HN by in situ hybridization. 6) Study the expression of the gp330 gene during prenatal and postnatal development of the kidney by in situ hybridization and the ribonuclease protection assay. 7) Study if gp330 mRNA is present in other tissues by quantitation of gp330 mRNA using the ribonuclease protection assay. The data generated from studying gp330 at the gene level will provide information on its structure, relationship to other proteins, regulation of expression, level of expression during renal development and HN as well as existence in other tissues. This new knowledge will directly bear on the understanding of the normal function of this protein as well as the long term goal of the project.