The expanding wealth of human, model and other organism's genomic data has allowed the identification of a distinct gene family of apoptotic related genes. Most of these genes are currently unannotated or have been subsumed under two questionably related gene families in the past. For example the transmembrane Bax inhibitor 1 (BI1) motif family has been reported to play a role in apoptosis and to consist of at least seven mammalian protein genes, GRINA, BI1, Lfg/FAIM2, Ghitm, RESC1/Tmbim1, GAAP/Tmbim4, and Tmbm1b. However, a detailed sequence and phylogenetic analysis shows that only five of these form a clear and unique protein family. This now provides information for understanding and investigating the biological roles of these proteins across a wide range of tissues in model organisms. The evolutionary relationships among these genes provide a powerful prospective for extrapolating to human conditions.

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...

We have recently proposed that the major secreted isoprotein form of human apolipoprotein A-I (designated apo A-I2) is modified extracellularly to become the predominant apo A-I form seen in plasma (designated apo A-I4). In the current report we demonstrate that the primary translation product of human apo A-I (designated apo A-I2p) has a 24-amino-acid NH2-terminal extension with a sequence of Met-Lys-Ala-Ala-Val-LeuThr-Leu-Ala-Val-Leu-Phe-Leu-Thr-Gly-Ser-Gln-Ala-Arg-His-PheTrp-Gln-Gln. The first 18 amino acids of this NH2-terminal extension are cleaved intracellularly by the signal peptidase, resulting in the formation of apo A-I2, which is the secreted form of apo A-I. Sequence analysis of apo A-I2 confirmed that it contains a hexapeptide extension at its NH2 terminus compared to apo A-14. This observation demonstrates that apo A-I2 is a propeptide and that the apo A-I2 to apo A-I4 conversion involves the removal of the NH2-terminal hexapeptide of apo A-I2 by a protease in plasma, lymph, or both. Our findings indicate that apo A-I is synthesized as a prepropeptide, which undergoes intracellular and extracellular proteolysis to attain the major plasma apo A-I4 isoprotein form.

To study structural variants of human serum amyloid A (SAA), an apoprotein of high-density lipoprotein, complementary DNA clones were isolated from a human liver library with the use of two synthetic oligonucleotide mixtures containing sequences that could code for residues 33-38 and 90-95 of the protein sequence. The SAA-specific cDNA clone (pA1) contains the nucleotide sequence coding for the mature SAA and 10 amino acids of the 18-residue signal peptide. It also includes a 70 nucleotide long 3'-untranslated region and approximately 120 bases of the poly(A) tail. The derived amino acid sequence of pA1 is identical with the alpha form of apoSAA1. A fragment of pA1 containing the conserved (residues 33-38) region of SAA also hybridized with RNA from human acute phase liver and acute phase stimulated, but not unstimulated, mouse and rabbit liver. In contrast, a fragment corresponding to the variable region hybridized to a much greater extent with human than with rabbit or murine RNA. Human acute phase liver SAA mRNA (approximately 600 nucleotides in length) directs synthesis of preSAA (Mr 14 000) in a cell-free translating system. In a Xenopus oocyte translation system preSAA is synthesized and processed to the mature Mr 12 000 product. The complete 18 amino acid signal peptide sequence of preSAA was derived from sequencing cDNA synthesized by "primer extension" from the region of SAA mRNA corresponding to the amino terminus of the mature product. Two other SAA-specific cDNA clones (pA6 and pA10) differed from pA1 in that they lack the internal PstI restriction enzyme site spanning residues 54-56 of pA1.(ABSTRACT TRUNCATED AT 250 WORDS)