Structure of the murine complement factor H gene

Factor H is a regulatory protein of the alternative pathway of complement activation comprised of 20 tandem repeating units of 60 amino acids each. A factor H cDNA clone was used to identify 17 genomic clones from a cosmid library. Four clones were selected for analysis of intron/exon junctions and 5' and 3' regions of the gene and for mapping of the exons. The factor H gene was found to be comprised of 22 exons. Each repeating unit is encoded by one exon, except the second repeat, which is coded by two exons; the leader sequence is encoded by a separate exon. The exons range in size from 77 to 210 base pairs (bp) and average 178 bp. They span a region of approximately 100 kilobases (kb) on chromosome 1. The leader sequence exon is 26 kb upstream of the first repeat exon, representing the largest intron. The other introns range in size from 86 bp to 12.9 kb, and the average intron size is 4.7 kb. Analysis of the genomic organization of the factor H gene has provided insight into the protein structure and will enable the construction of deletion mutants for functional studies.     

Genomic organization and polymorphisms of the human C3d/Epstein-Barr virus receptor

The human C3d/Epstein-Barr virus receptor (CR2/CD21) is a 145-kDa protein primarily expressed on mature B lymphocytes. CR2 is a member of the regulators of complement activation (RCA) gene family found on band q32 of chromosome 1. The RCA proteins are characterized by the presence of 60-70 amino acid short consensus repeats (SCR). A full length CR2 cDNA was cloned and used to identify overlapping cosmid genomic clones. Analysis of CR2 exon-intron junctions revealed the presence of three types of exons in the short consensus repeat region of CR2. First, four exons each of which encodes two SCR are present. Five exons encode a single SCR. Six exons encode SCRs which are split in identical positions. The order of these types of exons is in a repeated array of four SCRs, indicating that the contemporary CR2 gene likely evolved from a more primitive gene containing four SCRs. The CR2 full length cDNA clone was used to find restriction fragment length polymorphisms (RFLPs). Restriction enzyme TaqI generated 2.55- and 2.10-kilobase (kb) polymorphic bands. This RFLP was mapped near the exon containing the first two SCRs. HaeIII digestion generated polymorphic bands of 1.45, 1.55, and 1.75 kb. The HaeIII 1.45-kb RFLP band maps near the exon containing the 15th SCR. The TaqI and HaeIII RFLPs will provide tools for the genetic analysis of CR2. The organization of the CR2 gene provides insights into the evolution of human CR2 and the RCA gene family.     

Structure of the gene for human complement protein decay accelerating factor.

Decay accelerating factor (DAF) is a glycophospholipid-anchored membrane protein that is part of the regulators of complement activation (RCA) gene family located on human chromosome 1, band q32. These proteins, beginning at their amino terminus, consist largely of multiple copies of an approximately 60 amino acid short consensus repeat (SCR). A DAF cDNA clone was used to identify overlapping bacteriophage genomic clones. The human DAF gene spans approximately 40 kb and consists of 11 exons. The length of these exons and introns varies considerably, with the exons ranging from 21 to 956 bp and the introns ranging from approximately 0.5 to 19.8 kb. SCR I, II, and IV are all encoded by single exons; however, SCR III is encoded by two separate exons, with the splice junction occurring after the second nucleotide of the codon for the glycine residue at position 34 of the consensus sequence. This feature has also been found in CR1, CR2, membrane cofactor protein, and murine factor H. Following the SCR in DAF is a 76 amino acid serine/threonine-rich domain encoded on three separate exons. Exon 10 encodes the Alu family sequence that has been found as an insert in a minor class of DAF cDNA, thus indicating that this mRNA arises by standard alternative splicing. The last DAF exon, which comes after the largest intron of 19.8 kb, encodes the hydrophobic carboxy terminus and the 3'UT region. The nature of the signal that directs posttranslational attachment of a glycophospholipid anchor to DAF is not known, but that signal is apparently spread over three exons and greater than 20 kb. An analysis of the DAF gene provides additional evidence for the common evolutionary heritage of the RCA gene family. The exon/intron structure of this gene will facilitate experiments aimed at understanding the functions of the various domains of DAF.     

Complete exon-intron organization of the human leukocyte common antigen (CD45) gene

Ten genomic DNA clones encoding the human leukocyte common Ag (LCA, CD45) gene were isolated by screening human genomic DNA libraries with LCA cDNA probes. One genomic DNA clone contains the promoter region and the first two exons, as determined by primer extension analyses and S1 nuclease protection studies as well as nucleotide sequence determination. The first exon does not encode a peptide, while the second exon contains the initiation ATG codon and encodes the signal peptide. The other nine genomic DNA clones, which are separated from the first genomic clone by an unknown distance, are connected and span a total of 73 kb. The nine connected genomic clones encode a total of 31 exons. The 33 exons encoded by these 10 genomic clones account for the entire cDNA sequences including the 5' and 3' untranslated sequences. Exon 3 and exons 7 through 15 encode the extracellular domain sequences that are common to all LCA isoforms. Differential usage of exons 4, 5, and 6, generates at least five distinct LCA isoforms. Exon 16 encodes the transmembrane peptide. The cytoplasmic region of the leukocyte common antigens is composed of two homologous domains. Exons 17 through 24 encode the first domain, and exons 25 through 32 encode the second domain. The comparison of these exons indicated that the homologous domains were generated by duplication of several exons. The most 3' exon (exon 33) encodes the carboxy terminus of the LCA molecules and includes the entire 3' untranslated sequence.     

Conservation of the RD-BF-C2 organization in the pig MHC class-III region: mapping and cloning of the pig RD gene

The RD gene, named after the arginine (R) and aspartic acid (D) repeat in the central part of its protein, was initially mapped in the mouse H-2S subregion between C4 and BF. It was later mapped in the same position in the human MHC and here we show it is also conserved in the pig MHC class III region, close to the complement BF gene. A pig RD genomic clone was isolated from a γ-phage library. Hybridizations on genomic DNA separated with pulsed field gel electrophoresis identified common 220kb Nrul, 130 kb EagI and 200 kb Mlul bands for RD, BF and C2. The RD gene has also a 17 kb Kpnl and 11 kb Sad fragment in common with BFbut not with C2. The close linkage of the RD and BF genes was further established by hybridization of BF to a genomic γ-phage clone also containing the RD gene. This genomic RD clone overlaps with a γ -phage clone previously isolated and containing the complete BF gene and the 3' part of C2. The distance between RD and BF is about 6 kb. The junction between the two complement genes BF and C2 was sequenced and the BF 5' promoter region, overlapping the 3' noncoding region of C2, was compared with that of the human BF promoter. The overall homology was about 80% and all but one identified promoter elements were found in the same position in both genes. The results obtained demonstrate the RD-BF-C2 organization is strongly conserved between human, mouse and pig. No polymorphisms were detected in either the RD gene or in the BF promoter region using polymerase chain reaction and restriction fragment polymorphism analysis.     

Structure of the gene encoding potato cytosolic pyruvate kinase.

The polymerase chain reaction (PCR) has been used to generate a series of overlapping genomic clones representing 43 bp of 5' untranslated sequence, 63 bp of 3' untranslated sequence and the entire coding sequence of the gene encoding potato cytosolic pyruvate kinase (PKc). This portion of the gene is approximately 4.5 kb in length and is interrupted by three introns, one of which is present in the 5' untranslated region. Southern blot analysis indicates that PKc is encoded by a small gene family, and sequence data from a number of PCR-derived genomic clones indicate that there are as many as six PKc genes. Sequence differences between the PCR-generated genomic clones and a PKc cDNA clone are discussed with respect to the fidelity of Taq polymerase. An alignment of intron placement in the potato PKc gene with intron placement in PK genes from other sources indicates that two of the potato introns correspond to intron positions in other species.     

Duplication and divergence of the amino-terminal coding region of the complement receptor 1 (CR1) gene. An example of concerted (horizontal) evolution within a gene

Human C3b/C4b receptor or complement receptor type one (CR1) is one of a family of receptor and regulatory glycoproteins that are encoded at a single genetic region (1q32) and are composed largely of a tandemly repeated motif (short consensus repeat or SCR) of approximately 60 amino acids. In addition, CR1 features an internal homology of seven SCRs in length, known as a long homologous repeat, that is reiterated four times, in the major polymorphic size variant, from SCR-1 to SCR-28, and may be reiterated three, five, and six times in other polymorphic forms. In the course of studying CR1, we detected sequences closely related to CR1 on several overlapping genomic clones. We have characterized a 40-kilobase CR1-like genomic region containing 10 potential exons that are 95% homologous to the amino-terminal coding portion of CR1. This region appears to be a partial duplication of CR1 and may encode a related gene. A comparison of CR1 and CR1-like sequences suggests that unequal crossing-over and concerted evolution have occurred within the most precisely reiterated subregion of CR1. Similar mechanisms have been important in the evolution of tandemly repeated genes and could provide the means for generation of the CR1 polymorphic size variants.     

The murine Slp gene. Additional evidence that sex-limited protein has no biologic function

Sex-limited protein (Slp) is a mouse serum protein of unknown function that has approximately 95% amino acid sequence identity with murine complement component C4 but is inactive in the complement pathway. The gene for Slp lies in the S region of the murine H-2 complex adjacent to the gene Cyp21 that encodes the Cytochrome P-450 enzyme steroid 21-hydroxylase. We report the sequence of a 26,307 bp long segment of the mouse genome that includes both the Slp and Cyp21 genes. The sequence reported was assembled from the sequences of three overlapping lambda phage genomic clones from mouse strain B10.WR, which carries four tandem pairs of Slp and Cyp21 genes. We also report the sequence of a fourth lambda clone, 12,539 bp in length, carrying parts of a distinct pair of Slp and Cyp21 genes from B10.WR mice. The Slp gene at 14.3 kb in length is about 1 kb shorter than the C4 gene; this difference is due primarily to absences of a simple repetitive sequence and a middle repetitive MT element in the corresponding introns 14 and 15, respectively. The gene sequence reveals an intron/exon organization identical to that of the murine C4 gene, and also that the 9 nucleotide deletion in exon 18, which appears to be directly responsible for the absence of complement activity, is unrelated to differences in intron sequences. Detailed comparisons of C4 and Slp gene sequences indicate that nucleotide substitutions in the Slp gene are occurring at approximately the same rate in both exons and introns. This implies that the murine Slp gene resembles a pseudogene and supports previously reported evidence that the Slp protein has no biologic function.     

Molecular cloning of a human serum protein structurally related to complement factor H.

Two cDNA clones termed H36-1 and H36-2 were isolated from a human liver cDNA library. Clone H36-1 appears to represent the recently isolated human serum proteins h37 and h42, which are two differently glycosylated forms of a protein antigenically related to human complement factor H. The H36-1 deduced protein sequence is 327 amino acid long and possesses a leader sequence. The secreted part of the protein is comprised of five tandem repeating units, termed short consensus repeats (SCRs). SCR 1 and 2 display high homology to the corresponding region of the recently isolated murine factor H-related cDNA clone 13G1. In contrast, the 3'-end of the H36-1 clone shows sequence homology to the 3'-end of human complement factor H. The second clone, H36-2, is nearly identical to H36-1. Within 1148 base pairs, where the two clones overlap, their nucleotide sequences differed at nine positions. One nucleotide exchange in the sequence of H36-2 which was located within SCR 1 creats a stop codon (TAA). Consequently, the corresponding mRNA cannot code for a functional protein, suggesting that this clone is a transcribed pseudogene. These two clones represent new human members of the family of proteins structurally related to complement factor H.     

Characterization of the murine Icam-1 gene.

Intercellular adhesion molecule-1 (ICAM-1, CD54) is a cell adhesion molecule that interacts with the leukocyte beta 2 integrins, LFA-1 and Mac-1. Murine inflammatory models are being utilized increasingly to define the role that ICAM-1 induction plays in the initiation of inflammation. We have isolated murine genomic clones that contain the Icam-1 gene including over 2 kb of 5' flanking sequence. The gene for murine Icam-1 spans over 13 kb and is composed of seven exons and six introns. Each of the extracellular immunoglobulin-like domains of ICAM-1 is encoded by a single exon that ends with the first base of the next codon. Examination of ICAM-1 expression in vivo shows that mRNA levels of ICAM-1 are low in all organs except for the lung but increase markedly in multiple organs at 3 h after administration of endotoxin. The 5' flanking region of the murine gene contains a putative TATA box and potential SP-1, AP-1, and AP-3 sites in positions nearly identical to those in the human gene.     

Structure of the 3' portion of the bovine elastin gene

A bovine genomic library constructed by partial Sau3A digestion and contained in lambda Charon 30 was screened by in situ hybridization with a 1.3-kilobase (kb) sheep elastin cDNA clone [Yoon, K., May, M., Goldstein, N., Indik, Z., Oliver, L., Boyd, C., & Rosenbloom, J. (1984) Biochem. Biophys. Res. Commun. 118, 261-269]. Three clones encompassing 10 kb of the bovine elastin gene were identified and characterized by restriction mapping and DNA sequencing of the 6.2 kb of the most 3' region of the gene. These analyses have permitted localization of eight exons in the 6.2 kb in which the translated exons vary in size from 27 to 69 base pairs, and there is an approximately 1-kb untranslated region at the 3' end. In addition to identification of sequences homologous to those found in porcine tropoelastin, the analyses defined a 58 amino acid sequence that forms the carboxy-terminal region of tropoelastin, and this sequence, which contains two cysteine residues, was previously not observed in the protein sequence data. The analyses also suggest that functionally distinct cross-link and hydrophobic domains of the protein are encoded in separate exons.     

Genomic cloning of the gene for an IgE-binding lectin reveals unusual utilization of 5' untranslated regions.

epsilon BP (for epsilon binding protein) is a M(r) 31,000 S-type animal lectin that binds to IgE and has been identified as the homologue of Mac-2, a macrophage cell-surface marker, as well as the lectins RL-29, CBP35, and L-34. The protein is composed of two domains with the amino-terminal portion containing tandem repeats of nine amino acids and the carboxyl-terminal half containing consensus sequences shared by S-type animal lectins. We determined the genomic map in both rat and mouse and isolated overlapping genomic clones that contain the 5' two-thirds of the murine gene. The remaining portion of the gene was obtained by polymerase chain reaction (PCR) amplification of genomic murine DNA followed by subcloning into plasmid vectors. The epsilon BP gene is composed of six exons separated by five introns. The entire amino-terminal repetitive sequence is contained in exon III, and the carboxyl-terminal domain is encoded by the three succeeding exons (IV, V, VI). The latter three exons correspond well in size and share sequence homology with three exons coding for 14-kDa S-type lectins. The sequence in exon I offers an explanation for the generation of two mRNAs differing only in their 5' untranslated sequences, previously reported in Mac-2 cDNA clones. Using cDNA synthesis and PCR amplification, we determined that two alternative splice sites are used in many different types of cells. This alternative splicing results in different 5' untranslated regions of the murine epsilon BP mRNA.     

The Bos taurus casein genes. Isolation and characterization of the kappa-casein gene

A region spanning 25 kb of genomic DNA containing the kappa-casein gene, has been isolated from two genomic libraries in EMBL3 and EMBL4 phage vectors. Five phage clones containing kappa-casein gene have been found. Gene organisation has been determined using restriction mapping and a partial sequencing the 5' and 3' flanking regions. The kappa-casein gene includes 5 exons, the first of them coding for 64 nucleotides from the 5' untranslated mRNA zone. The gene is 12.5 kb long, which is almost 16 times longer than the corresponding mRNA. The first intron spans 2.5 kb, the second is the largest one and spans 5.5 kb. The 5' flanking region sequence has been analysed; it contains a TATA box from -30 to -25 bp, somewhat different from the canonic sequence, and a CAAT box at -80 bp.