Complete exon-intron organization of the human gene for the alpha1 chain of type XV collagen (COL15A1) and comparison with the homologous COL18A1 gene

The human gene for the alpha1 chain of type XV collagen (COL15A1) is about 145 kilobases in size and contains 42 exons. The promoter is characterized by the lack of a TATAA motif and the presence of several Sp1 binding sites, some of which appeared to be functional in transfected HeLa cells. Comparison with Col18a1, which encodes the alpha1(XVIII) collagen chain homologous with alpha1(XV), indicates marked structural homology spread throughout the two genes. The mouse Col18a1 contains one exon more than COL15A1, due to the fact that COL15A1 lacks sequences corresponding to exon 3 of Col18a1, which encodes a cysteine-rich sequence motif. Twenty-five of the exons of the two genes are almost identical in size, six of them contain conserved split codons, and the locations of the respective exon-intron junctions are identical or almost identical in the two genes. The homologous exons include the closely adjacent first pair of exons and the exons encoding a thrombospondin-1 homology found in the N-terminal noncollagenous domain 1, which are followed by the most variable part of the two genes, covering the C-terminal half of their noncollagenous domain 1 and the beginning of the collagenous portion, after which most of the exons are homologous. The lengths of the introns are not similar in these genes, with two exceptions, namely the first intron, which is very short, less than 100 base pairs, and the second intron, which is very large, about 50 kilobases, in both genes. It can be concluded that COL15A1 and Col18a1 are derived from a common ancestor.     

Physical and linkage mapping of the gene for the alpha3 chain of type IX collagen, COL9A3, to human chromosome 20q13.3

Type IX collagen is a minor cartilage component which associates with mixed fibrils of types II/XI collagen. We have determined the precise physical and genetic locations for the gene encoding the alpha3 chain of type IX collagen, COL9A3. Utilizing fluorescence in situ hybridization, radiation hybrid mapping, and multipoint linkage analysis, we have mapped COL9A3 to human chromosome 20q13.3, 13 cM telomeric to D20S173.     

Oxidant-antioxidant imbalance in the experimental interstitial lung disease induced in sheep by visna-maedi virus

X-linked Alport syndrome (AS) is a heritable disorder which is associated with mutations in the type IV collagen alpha 5 (IV) chain gene (COL4A5) located on chromosome X. Following renal transplantation, an average of 6% of male AS patients develop anti-GBM nephritis. We studied the specificity of the antibodies against type IV collagen in the serum of a patient with COL4A5 partial deletion. The specificity of these alloantibodies was determined against collagenase-digested GBM, as well as against recombinant non-collagenous (NC1) domains of the type IV collagen alpha 1(IV)-alpha 6(IV) chains expressed in escherichia coli. Immunoblotting and ELISA demonstrated that these antibodies bound specifically to the NC1 domain of alpha 5(IV) collagen. There was no binding to the NC1 domain of the other chains, including the Goodpasture antigen. Competitive ELISA confirmed the results obtained by ELISA and immunoblotting. This patient developed alloantibodies directed against antigens present in the grafted kidney, but absent from his Alport kidney. The pathogenesis of post-transplantation glomerulonephritis in the Alport patient studied is thus similar to that of Goodpasture syndrome, with the exception that the pathogenic antibodies are targeted to another alpha chain of type IV collagen.     

Targets of alloantibodies in Alport anti-glomerular basement membrane disease after renal transplantation

A minority of patients with Alport syndrome develop anti-GBM disease in their allografts after renal transplantation. Clinically, the renal disease appears indistinguishable from Goodpasture's disease of native kidneys, in which the target of autoantibodies had been identified as the NC1 domain of the alpha 3 chain of type IV collagen, alpha 3(IV)NC1. However, in the majority of cases, Alport syndrome is due to mutations in the gene encoding the alpha 5 chain of type IV collagen, located on the X chromosome. Neither chain is detectable in the glomerular basement membrane (GBM) of most patients with Alport syndrome. We investigated the targets of the alloantibodies of 12 Alport patients who developed post-transplant anti-GBM disease by Western blotting onto recombinant NC1 domains made in insect cells. Binding to these antigens, for both typical Goodpasture and Alport anti-GBM antibodies, was strong and conformation-sensitive. Nine antibodies showed selective binding to alpha 5(IV)NC1. This specificity was confirmed by the demonstration of binding to a 26 kDa band of collagenase-solubilized human GBM, and/or binding to normal epidermal as well as renal basement membranes by indirect immunofluorescence. One antibody showed binding to alpha 5 and alpha 3(IV)NC1, while two showed predominant binding to alpha 3(IV)NC1. All seven patients whose pedigree or mutation analysis showed X-linked inheritance had predominant anti-alpha 5 reactivity. One with predominant anti-alpha 3 reactivity had a COL4A3 mutation. These findings show that human anti-GBM disease can be associated with antibodies directed towards different molecular targets. Alpha 5(IV)NC1 is the primary target in most patients with X-linked Alport syndrome who develop post-transplant anti-GBM disease.     

Structure of the murine CD156 gene, characterization of its promoter, and chromosomal location

The murine cell surface antigen mCD156 is a glycoprotein that is expressed in monocytic cell lines and consists of a metalloprotease domain, a disintegrin domain, a cysteine-rich domain, and an epidermal growth factor-like domain in the extracellular region. The mCD156 gene is composed of 24 exons and 23 introns and spans approximately 14 kilobases. The first exon encodes most of the signal peptide sequence, and the transmembrane region is encoded by a single exon (19). In contrast, the other regions are composed of multiple exons. Of these, exons 7-12 and 12-15 encode a metalloprotease domain and a disintegrin domain, respectively. Sequence analysis of the 5'-flanking DNA revealed many potential regulatory motifs. Chloramphenicol acetyltransferase analysis demonstrated that nucleotides at positions -183, -334, and -623 contained cis-acting enhancing elements in a mouse monocytic cell line, aHINS-B3. Nucleotides at positions -183 and -390 contained elements responsible for lipopolysaccharide (LPS) inducibility, although several other 5'-flanking regions were also involved in LPS responsiveness. Regions -202, -507, and -659 play a role in interferon-gamma inducibility. Some of the potential regulatory motifs and other unknown cis elements may be involved in the constitutive expression, and LPS and interferon-gamma inducibilities. The mCD156 gene was mapped to chromosome 7, region F3-F4.     

Structural organization of a gene encoding a novel calmodulin-binding kinesin-like protein from Arabidopsis

Kinesin-like calmodulin-binding protein (KCBP) is a recently identified microtubule motor protein that appears to be unique to plants. Here we report isolation and sequence analysis of a gene encoding Arabidopsis KCBP. KCBP gene contains 21 exons and 20 introns. All exons except exon 3 are short (94-272 nt). Exons 1-9 code for the globular tail region whereas the coiled-coil region is coded by exons 10-15. The conserved motor domain is coded by exons 16-20. Calmodulin-binding domain that is present in the C-terminal region of the protein and unique to KCBP is coded by the last exon. The size of introns ranged from 71 (intron 17) to 320 (intron 19) nucleotides. As in most plant introns, the content of AT is very high in all introns (up to 76%). Phylogenetic analysis of KCBP using motor domain sequence grouped KCBP with other known C-terminal microtubule motor proteins. However, Arabidopsis KCBP together with its homologs from potato and tobacco constitute a distinct group within the C-terminal subfamily of motors which is consistent with structural and functional features of KCBP.     

Genomic organization of IFI16, an interferon-inducible gene whose expression is associated with human myeloid cell differentiation: correlation of predicted protein domains with exon organization

The human IFI16 gene is a member of an interferon-inducible family of mouse and human genes closely linked on syntenic regions of chromosome 1. Expression of these genes is largely restricted to hemopoietic cells, and is associated with the differentiation of cells of the myeloid lineages. As a prelude to defining the mechanisms governing IFI16 expression, we have deduced its genomic organization using a combination of genomic cloning and polymerase chain reaction amplification of genomic DNA. IFI16 consists of ten exons and nine intervening introns spanning at least 28 kilobases (kb) of DNA. The reiterated domain structure of IFI16 protein is closely reflected in its intron/exon boundaries, and may represent the evolutionary fusion of several independent functional domains. Thus, exon 1 consists of 5' untranslated (UT) sequences and contains sequence motifs that may confer interferon-inducibility, and exon 2 encodes the lysine-rich amino-terminal ("K") region, which possesses DNA-binding activity. Exon 3 codes for a domain which is poorly conserved between family members, except for a strongly retained basic motif likely to provide localization. The first of two 200 amino acid repeat domains that are the hallmark of this family (domain A) is represented jointly on exons 4 and 5, which are reiterated as exons 8 and 9, respectively, to encode the second 200 amino acid domain (B). Two intervening serine-threonine-rich domains (C and C'), unique to IFI16, are each encoded by single exons of identical length (exons 5 and 6). These domains are predicted to encode semi-rigid "spacer" domains between the 200 amino acid repeats. The reiterated nature of exons 4 to 6 and the insertion of introns into a single reading frame strongly suggest that IFI16 and related genes arose by a series of exon duplications, some of which antedated speciation into mouse and humans. Several alternative mRNA cap sites downstream of a TATA consensus sequence were defined, using primer extension analysis of mRNA. Sequencing of approximately 1.7 kb of DNA upstream of this region revealed no recognizable consensus elements for induction by interferon-alpha (interferon-alpha/beta-stimulated response elements), but two motifs resembling interferon-gamma activation sites were located. IFNs alpha and gamma both induce IFI16 mRNA expression in myeloid cells. Interferon-alpha inducibility of IFI16 may be regulated by an interferon-alpha/beta-stimulated response consensus element in the 5' UT exon, as a similar motif is conserved in the corresponding position in the related myeloid cell nuclear differentiation antigen gene.(ABSTRACT TRUNCATED AT 400 WORDS)     

Determination of the genomic structure of the COL4A4 gene and of novel mutations causing autosomal recessive Alport syndrome

Autosomal recessive Alport syndrome is a progressive hematuric glomerulonephritis characterized by glomerular basement membrane abnormalities and associated with mutations in either the COL4A3 or the COL4A4 gene, which encode the alpha3 and alpha4 type IV collagen chains, respectively. To date, mutation screening in the two genes has been hampered by the lack of genomic structure information. We report here the complete characterization of the 48 exons of the COL4A4 gene, a comprehensive gene screen, and the subsequent detection of 10 novel mutations in eight patients diagnosed with autosomal recessive Alport syndrome. Furthermore, we identified a glycine to alanine substitution in the collagenous domain that is apparently silent in the heterozygous carriers, in 11.5% of all control individuals, and in one control individual homozygous for this glycine substitution. There has been no previous finding of a glycine substitution that is not associated with any obvious phenotype in homozygous individuals.     

Modulation of disease severity of dystrophic epidermolysis bullosa by a splice site mutation in combination with a missense mutation in the COL7A1 gene

Dystrophic epidermolysis bullosa (EBD) is a clinically heterogeneous skin disorder, characterized by abnormal anchoring fibrils (AF) and loss of dermal-epidermal adherence. EBD has been linked to the COL7A1 gene at chromosome 3p21 which encodes collagen VII, the major component of the AF. Here we investigated two unrelated EBD families with different clinical phenotypes and novel combinations of recessive and dominant COL7A1 mutations. Both families shared the same recessive heterozygous 14 bp deletion at the exon-intron 115 boundary of the COL7A1 gene. The deletion caused in-frame skipping of exon 115 and the elimination of 29 amino acid residues from the pro-alpha1(VII) polypeptide chain. As a result, procollagen VII was not converted to collagen VII and the C-terminal NC-2 propeptide which is normally removed from the procollagen VII prior to formation of the anchoring fibrils was retained in the skin. All affected individuals also carried missense mutations in exon 73 of COL7A1 which lead to different glycine-to-arginine substitutions in the triple-helical domain of collagen VII. Combination of the deletion mutation with a G2009R substitution resulted in a mild phenotype. In contrast, combination of the deletion with a G2043R substitution led to a severe phenotype. The G2043R substitution was a de novo mutation which alone caused a mild phenotype. Thus, different combinations of dominant and recessive COL7A1 mutations can modulate disease activity of EBD and alter the clinical presentation of the patients.     

The exon/intron organization and chromosomal mapping of the mouse ADAMTS-1 gene encoding an ADAM family protein with TSP motifs

ADAM is a recently discovered gene family that encodes proteins with a disintegrin and metalloproteinase. ADAMTS-1 is a gene encoding a new member protein of the ADAM family with the thrombospondin (TSP) type I motif, the expression of which is associated with inflammatory processes. In the present study, we have characterized the exon/intron organization of the mouse ADAMTS-1 gene. The ADAMTS-1 gene is composed of nine exons, all of which are present within the 9.2-kb genomic region. Among the nine exons, exons 1, 5, and 6 encode a proprotein domain, a disintegrin-like domain, and a TSP type I motif, respectively, of the ADAMTS-1 protein, suggesting that there is a correlation between exon/intron organization and functional domains. In addition, the exon/ intron organization of the ADAMTS-1 gene is very different from that of the metalloproteinase-like/disintegrin-like/ cysteine-rich protein gene (MDC) (ADAM11), suggesting that the genomic structure of ADAM family genes is not necessarily conserved. Furthermore, fluorescence in situ hybridization revealed that the ADAMTS-1 gene is located in region C3-C5 of chromosome 16, to which none of the previously identified ADAM genes have been mapped.     

Identification of domains responsible for von Willebrand factor type VI collagen interaction mediating platelet adhesion under high flow

We have identified type VI collagen (Col VI) as a primary subendothelial extracellular matrix component responsible for von Willebrand factor (vWF)-dependent platelet adhesion and aggregation under high tensile strength. Intact tetrameric Col VI was the form of the collagen found to be capable of promoting vWF-mediated platelet adhesion/aggregation under this shear condition, whereas removal of the predominant portion of the terminal globules by pepsin treatment abrogated its activity. The inability of the pepsin-digested Col VI to support any platelet interaction at high flow was because of the failure of the A3(vWF) domain to bind to this form of collagen, suggesting a stringent requirement of a tridimensional conformation or of intactness of its macromolecular structure. In contrast, the A1(vWF) domain bound to both intact and pepsin-digested Col VI tetramers but, in accordance with the cooperating function of the two vWF domains, failed to support platelet adhesion/aggregation under high shear onto Col VI by itself. The putative A1(vWF) binding site resided within the A7(VI) module (residues 413-613) of the globular amino-terminal portion of the alpha3(VI) chain. Soluble recombinant A7(VI) polypeptide strongly perturbed the vWF-mediated platelet adhesion to Col VI under high shear rates, without affecting the binding of the vWF platelet receptor glycoprotein Ibalpha to its cognate ligand A1(vWF). The findings provide evidence for a concerted action of the A1(vWF) and A3(vWF) domains in inducing platelet arrest on Col VI. This is accomplished via an interaction of the A1(vWF) domain with a site contained in the alpha3 chain A7(VI) domain and via a conformation-dependent interaction of the A3(vWF) domain with the intact tetrameric collagen. The data further emphasize that Col VI microfilaments linking the subendothelial basement membrane to the interstitial collagenous network may play a pivotal role in the hemostatic process triggered upon damage of the blood vessel wall.     

Structure and stability of the triple-helical domains of human collagen XIV

Two triple-helical domains, Col 1 and Col 2, were obtained from a pepsin digest of human placental collagen XIV and separated from each other under nondenaturing conditions. Edman degradation demonstrated 106 amino acids residues in the Col 1 and 149 residues in the Col 2 domain. All except one of the 37 prolines in the Yaa position of the Gly-Xaa-Yaa triplets were completely hydroxylated to 4-hydroxyproline, and there were three imperfections in the triplet repeat. Partial or complete hydroxylation and glycosylation were found for all seven lysines in the Yaa position. Domain Col 1 was joined by disulfide bonds into a trimer, while Col 2 appeared as a mixture of monomers and disulfide-linked dimers. Circular dichroic spectra were typical for the collagen triple helix and revealed relatively high melting temperatures for Col 1 (38 degrees C) and Col 2 (43 degrees C). An almost perfect refolding of the triple helix was observed for Col 1 but not for Col 2, emphasizing the importance of disulfide bonds for the folding kinetics and in part the stability of the triple helix. Circular dichroic spectra of the large nontriple helical domain, NC3, of collagen XIV indicated 11% alpha helix and 63% beta structure. Comparative melting profiles of NC3 and intact collagen XIV indicated that the triple helices in intact collagen XIV have a melting temperature of 44 degrees C.