The project proposes to study (a) the structure of the gene encoding complement protein C3, (b) the controls of transcription of this gene into RNA, (c) the molecular defects that lead to inherited C3 deficiencies in man and (d) the isolation of mouse cDNA. The study will take advantage of mouse C3 cDNA clones and C3 genomic DNA clones that have been prepared in the applicant's laboratory and will extend currently ongoing work. First, the tissue specific expression of C3 will be investigated. C3 mRNA concentrations will be titrated in hepatocytes, macrophages and in a variety of C3 producing and non-producing tissue culture cell lines and correlated with the state of methylation of the gene. The 5'-ends of liver and macrophage C3 mRNAs will be compared in order to determine whether the gene is transcribed in both tissues from the same promoter. It is intended to establish the total nucleotide sequence of C3 mRNA as a basis for future studies of particular regions of the C3 polypeptide, such as the binding sites for cellular C3d receptors. The second part proposes to isolate one intact copy of the human C3 gene from a gene library to be constructed in cosmid vectors. The human gene will be characterized and assayed for function by transfection into tissue culture cells. A defective allele carried by homozygous C3 deficient members of a Dutch family will be analyzed. In the third part an approach is described for the production of cloned mouse C4 cDNA clones. The last section outlines how C4 cDNA clones will be characterized and distinguished from cDNA clones for the C4 variant of mice, the Slp-protein. These clones will be used to screen mouse gene libraries for C4 genomic clones. Work on the human C4 gene will follow according to the initial results obtained with the mouse C4 gene. The proposed studies on the C3 and C4 genes will provide basic information on the structure and the regulation of these genes and also on inherited human disorders associated with these genes.
