4TH COMPONENT OF XENOPUS-LAEVIS COMPLEMENT - CDNA CLONING AND LINKAGEANALYSIS OF THE FROG MHC

Complement C4 shows extensive structural and functional similarity to complement C3, hence these components are believed to have originated by gene duplication from a common ancestor. Although to date C3 cDNA c lones have been isolated from all major classes of extant vertebrates including Xenopus, C4 cDNA clones have been isolated from mammalian sp ecies only. We describe here the molecular cloning and structural anal ysis of Xenopus C4 cDNA. The cDNA sequence encoding the thioester regi on of Xenopus C4 was amplified by reverse transcriptase-polymerase cha in reaction using Xenopus liver mRNA as a template, and then used to s creen a liver cDNA Library. The amino acid sequence of Xenopus C4 dedu ced from a clone containing the entire protein-coding sequence showed 39%, 30%, 25%, and 20% overall identity with those of human C4, C3, C5 , and alpha 2-macroglobulin, respectively. The predicted amino acid se quence consisted of a 22-residue putative signal peptide, a 634-residu e beta chain, a 732-residue alpha chain, and a 287-residue gamma chain . Of 30 cysteine residues, 27 were found in exactly the same positions as in human C4. Genomic Southern blotting analysis indicated that C4 is a single copy gene in Xenopus and is part of the frog MHC cluster. These results clearly demonstrate that C3/C4 gene duplication and link age between the C4 gene and the major histocompatibility complex preda te mammalian/amphibian divergence.