The fifth component of human complement (C5) is an important participant in inflammatory and cell killing processes. The activation peptide (C5a) possesses potent spasmogenic and chemotactic activity. The macromolecular cleavage product (C5b) expresses a transient binding site for C6. The C5b, 6 complex is the foundation upon which the membrane attack complex (C5b-9) is assembled. Elucidation of the molecular features germane to these biological properties will require knowledge of the complete primary structure of C5. Available protein sequence data for C5a will be used to synthesize a mixed sequence oligodeoxyribonucleotide hybridization probe complementary to C5 mRNA which will be used to screen a human liver cDNA clone bank. Confirmed cDNA clones greater than 1kb will be subjected to sonication, thereby producing random overlapping fragments of 400 to 500 bp, shotgun cloned in the bacteriophage M13 vector and sequenced using the Sanger dideoxy termination method. The complete nucleotide coding sequence for C5 will be derived in this way. Concurrently, random tryptic peptides generated from purified C5 will be sequenced to assist in generating an unambiguous complete primary sequence. Poly (A)+ mRNA specifying C5 will be prepared from multiple tissues and cell lines and analyzed using C5 cDNA probes by Northern transfer analysis. The molecular basis for C5 deficiency states will be studied. The chromosomal locus for the C5 gene will be deduced by Southern analysis of DNA samples prepared from interspecies somatic cell hybrids. C5 cDNA clones will also be used as hybridization probes to screen two human genomic libraries. The gene for C5 will be isolated and characterized. Correlation of the intron/exon arrangement of the C5 gene with structural and/or functional domains of the protein molecule will be made. Recent data has indicated that the complement proteins C3, C4, and C5 and the protease inhibitor Alpha 2M are structurally homologous. Available Alpha 2M protein sequences will be used to synthesize oligonucleotide probes for identification and isolation of Alpha 2M cDNA clones. Cloned Alpha 2M cDNA fragments will then be used to identify genomic clones. Information derived from these studies of C5 and Alpha 2M gene structures will be used to analyze the pattern of protein structural domain segregation.
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