Somatomedins (SM) or insulin-like growth factors (IGF) constitute a heterogeneous group of peptides with important growth-promoting effects in vitro as well as in vivo. Amino acid sequences have been determined for only two of them, IGF-I and IGF-II, which are highly homologous. IGF-I, which is identical with SM-C, is composed of 70 amino acid residues and IGF-II contains 73 amino acids and may be identical with SM-A. Other peptides with different charge properties but with similar SM-like or insulin-like behaviour in biological and receptor assays, have been described but have not yet been fully characterized. The liver is known to be a major site of production of these peptides, but many other tissues--especially in the fetus--may synthesize them as well. We report here the nucleotide sequence of a human liver cDNA encoding the complete amino acid sequence of IGF-I. The IGF-I coding region is flanked by sequences encoding an amino-terminal peptide of at least 25 amino acid residues and a carboxyl-terminal peptide of 35 amino acids. This provides evidence that IGF-I is synthesized as a precursor protein and that formation of IGF-I from this precursor requires proteolytic processing at both ends.

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...

Eighteen overlapping cosmid clones spanning 240 kilobases and encoding the gene for factor B and two genes related to the fourth component of complement (C4) were isolated from a murine H-2d genomic library. Cosmid clones were identified by hybridization to human cDNA probes for factor B and C4 and were linked by chromosomal walking procedures. The cluster of clones contains two regions with sequences homologous to the C4 cDNA probe, both in the same orientation, representing a direct duplication of at least 55 kilobases of chromosomal DNA, separated by a shorter (<25 kilobases) segment of nonduplicated DNA. Restriction fragment-length polymorphism seen by using C4 probes maps these sequences to the S region of the major histocompatibility complex. 5' to the two C4-like sequences is an :40-kilobase-long region of -chromosomal DNA remarkable for its lack of restriction fragment-length polymorphism, containing sequences homologous to the human factor B cDNA probe. These experiments demonstrate that the structural gene for factor B is located in the S region of the murine major histocompatibility complex and that this region contains an extensive direct duplication that contains the structural gene for mouse C4 and, we presume, for the sex-limited protein variant, SIp. RNA transfer blot analysis of total liver RNA from high C4-and low C4-producing strains showed that steady-state levels of C4-hybridizing RNA were much greater in high C4-producing strains. Regulation of circulating C4 levels in high C4 and low C4 strains is at least partly at the level of mRNA transcription, processing, or degradation.The S region of the murine major histocompatibility complex (MHC), located between the I and D regions, contains the structural genes encoding the fourth component of murine complement (C4) and the sex-limited protein variant, Slp (reviewed in refs. 1 and 2). Slp shares extensive structural homology with C4 but has no demonstrable hemolytic activity and no known function (3). There are two major C4 alleles, C4-high (C4h) and C4-low (C41), which control ==20-fold differences in serum C4 levels. Slp is produced in only some C4h strains and is found only in males (1). In man, the structural genes for the second component of complement (C2) and for factor B of the alternate pathway also map to the M HC (2). Recent studies suggest that the structural gene for mouse factor B maps to the MHC as well and that control of mouse C2 hemolytic levels maps to the S region (4, 5). Together, these proteins make up the class III molecules of the MHC.The class I and class II molecules of the MHC are cell-surface glycoproteins that are involved in many immune reactions (reviewed in refs. 6 and 7). They are notable for the extensive allelic polymorphism that they demonstrate. A great deal has already been learned about the structure and evolution of these genes. Recent experiments have used cosmid cloning to isolate >13 clusters of clones containing sequences encoding class I genes and including >800 kilobases (kb) of genomic DNA...

A cDNA clone for the fourth complement component (C4), pC4AL1, has been isolated from a human adult liver cDNA library by using a synthetic oligonucleotide mixture containing all 384 possible sequences coding for residues 14-21 of the C4 y-chain amino acid sequence. This clone spans the entire.C4 y-chain coding sequence and includes a short 3' untranslated region, a poly(A) recognition site, and 16 nucleotides of the poly(A) tail. The 5' end of-the clone-begins 18 nucleotides upstream from the amino terminus of the C4 y chain and codes for Arg-Asn-ArgArg-Arg-Arg, a highly charged proteolytic cleavage site involved in the processing of pro-C4 to native C4. Liver mRNA preparations from C4-deficient guinea pigs were incapable of directing synthesis of pro-C4 or C4 peptides in cell-free translation experiments. Southern blot analysis using pC4AL1 as a hybridization probe of C4-deficient guinea pig DNA established that the deficiency is not the result of deletion of the entire C4 gene. RNA blot analysis using pC4AL1 as a hybridization probe of normal -guinea pig liver mRNA revealed a C4 mRNA of 5.0 kilobases (kb). No such mRNA species was observed in C4-deficient guinea pig liver mRNA; however, a 7.0-kb RNA was detected, indicating the presence of a C4 precursorRNA. These results suggest that the basis of C4 deficiency in the guinea pig is a post-transcriptional defect in the processing of C4 precursor RNA to mature C4 mRNA.The complement system consists of more than 20 plasma proteins that interact in a highly specific manner to mediate several functions associated with host defense against infection (1). The structural genes for.three of the complement proteins (the second component, C2; the fourth component, C4; and factor B) have been localized to the major histocompatibility complex (MHC) in man, mouse, and guinea pig (2) and have been designated class III MHC genes.C4 is a serum glycoprotein of approximately 200 kilodaltons (kDa) that is comprised of.disulfide-linked polypeptide chains of 93 kDa (a chain), 73 kDa (P chain), and 33 kDa (y chain) (3). C4 in man, mouse, and guinea pig is synthesized as a singlechain precursor molecule, pro-C4 (4-6), in the order NH2-,3-