Isolation of cDNA clones for the human complement protein factor B, a class III major histocompatibility complex gene product.

Abstract

cDNA clones corresponding to a major histocompatibility class mI antigen, the complement protein factor B, have been isolated from a human adult liver cDNA library. The clones, ranging in size from 1.0 to 2.3 kilobases, were identified by direct hybridization with two synthetic oligonucleotide mixtures. Two regions of the factor B amino acid sequence, each with minimal ambiguity in codon assignment, were chosen for synthesis of the oligonucleotides. The sequences of two clones have been partially determined. They contain coding information for the amino acid sequence of the Bb fragment of factor B and the entire 3'-untranslated region.The genes for three ofthe serum complement proteins, the second (C2) and fourth (C4) components and factor B, have been localized to the major histocompatibility complex (MHC) in humans, mice, and guinea pigs (for review, see ref. 1) and have been designated class III MHC antigens. The availability of cDNA probes for genes within the MHC will permit detailed analysis of the structure of this region and an understanding of the molecular basis of polymorphic variants important in regulation of the immune response. Recently, cDNA clones corresponding to human and mouse class I and II histocompatibility antigens were described (2-7).Factor B, a single-chain serum glycoprotein (Mr, -95,000), is a component of the alternative pathway of the complement system. Activation of factor B by factor D generates two fragments, Ba (Mr, 30,000), the amino-terminal segment, and Bb (Mr =60,000). The latter fragment is associated with a cleavage product ofthe third complement (C3) component, C3b, to generate an unstable C3-cleaving enzyme (C3bBb, the C3 convertase). Factor B is an unusual serine protease, the active site of which is contained in the Bb fragment.Factor B accounts for approximately 0.1% of total protein synthesized in human liver (8). Several techniques have recently been applied to the identification of cDNA clones derived from mRNA species present in low abundance. Among these is the approach in which synthetic oligonucleotides are used indirectly as primers for cDNA synthesis and directly as probes for the detection of unique genes in Southern blot filter hybridization and colony or bacteriophage library screening (3, 9-11). By using this method, we have isolated cDNA clones for a human class III MHC antigen, factor B.METHODS mRNA Isolation and Translation and Immunoprecipitation. Fresh human adult liver was obtained from a cadaver donor (a white male, HLA A1,2; Cw3,w6; B15,w39; complotype Bf FS; C2C; C4A4,4; C4B2,QO) in accord with the Anatomical Gift Act and with the cooperation of the New England Organ Bank. Within 10 min after the tissue was obtained, it was finely minced and homogenized in 0.25 M sucrose/0.14 M NaCl/0.01 M Tris'HCI, pH 7.5/1.5 mM Mg C12 (TNMS buffer) at 00C in a glass Dounce homogenizer. The homogenate was centrifuged at 16,000 x g for 5 min at 4°C and the postmitochondrial supernatant was made 1% in NaDodSO4. Total cytoplasmic RNA was isolated by using...

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