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.
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