The application's long-term objectives are to provide a comprehensive understanding of the structure, biosynthesis and regulation of expression for the third component of human complement (C3).
Specific aims to be addressed during the proposed funding period include (1) determination of the complete human C3 mRNA structure, (2) delineation of the structure of the human C3 gene, (3) assessment of differences in C3 tissue-specific biosynthesis and the relationship to genomic transcriptional control structures and (4) determination of the molecular basis for human C3 deficiency. Primer-extension studies utilizing human liver mRNA as template will be employed to ascertain sequence for the 5' end of C3 mRNA. Sequence analysis of appropriate restriction fragments derived from C3 cDNA isolated in this laboratory will delineate structure for the 3' end of C3 mRNA. Cloned genomic DNA spanning the 5' end of the C3 gene and beta- chain coding region will be identified and isolated from cosmid libraries using a restriction fragment from the 5' end of an existing C3 cDNA as probe. Sequence analysis by the Sanger dideoxy procedure of pre-selected, exon-containing phage templates subcloned from sheared DNA will allow a rapid determination of the remaining exon/intron boundaries for the C3 gene. The kinetics of C3 synthesis by established cell lines and primary cell cultures will be assessed by biosynthetic labeling studies. These studies will be extended to the RNA level. The putative low molecular weight Raja cell C3 transcript will be cloned and sequenced. The transcription start site(s) and the transcriptional control region will be mapped employing cloned genomic DNA representative of the 5' end of the C3 gene. Protein factors involved hepatic transcription of the C3 gene will be identified and regions that serve as protein binding sites mapped by performing DNA """"""""footprint"""""""" studies with nuclear protein extracts from hepatic (HepG2) and non- hepatic cells. The basis for C3 deficiency (C3D) in a well characterized C3D kindred will be assessed by Southern analysis of DNA prepared form EBV-transformed B cells of deficient and normal individuals. The C3 mRNA in deficient and normal family members will be examined by Northern analysis and the defect at the protein level assessed by sequences analysis of potential transcripts of the defective gene.
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