Human collagen gene COL5A1 maps to the q34.2----q34.3 region of chromosome 9, near the locus for nail-patella syndrome.

Type V collagen is a fibrillar collagen that is widely distributed in tissues as a minor component of extracellular matrix and is usually composed of one pro alpha 2 (V) and two pro alpha 1 (V) chains. In this report, recently isolated cDNA and genomic clones, which encode the pro alpha 1 (V) chain, are used as probes for hybridization to filter-bound DNA from a panel of human-mouse hybrid cell lines and for in situ hybridization to metaphase chromosomes. These studies establish the chromosomal location of the COL5A1 gene, which encodes the pro alpha 1 (V) chain, within segment 9q34.2----q34.3. These findings add to the previously characterized dispersion of collagen genes in the human genome, as this is the first example of a collagen locus on chromosome 9. In addition, these studies place COL5A1 near the locus for the genetic disorder, nail-patella syndrome (hereditary osteo-onychodysplasia), which also maps to 9q34.     

The human alpha 2(XI) collagen gene (COL11A2) maps to the centromeric border of the major histocompatibility complex on chromosome 6

Type XI collagen, a minor structural component of cartilage fibrils, is composed of three chains, alpha 1(XI), alpha 2(XI), and alpha 3(XI). Using a cloned fragment of the human alpha 2(XI) collagen gene (COL11A2) as a molecular probe for in situ hybridization and somatic cell hybrid mapping, we have localized the gene to the short arm of chromosome 6, region 21.3. By exploiting the rich source of probes provided by the major histocompatibility complex (MHC) genes, which also map to this chromosomal band, we have constructed macrorestriction maps of the region by pulsed-field gel electrophoresis and have localized the alpha 2(XI) collagen gene to the centromeric extreme of the MHC. Finally, we have demonstrated, by the isolation of overlapping cosmid clones, that the gene is 45 kb centromeric to the HLA-DPB2 locus and oriented with the 3' end toward the MHC. The COL11A2 locus thus demarcates the proximal boundary of the MHC. This finding may have implications for the understanding of certain MHC-linked diseases.     

Assignment of the human collagen alpha 1 (XIII) chain gene (COL13A1) to the q22 region of chromosome 10

Type XIII collagen is a recently described collagen that resembles in structure the short-chain collagens of types IX, X, and XII. Unlike any other collagen, the type XIII is found in several different forms generated through alternative splicing. A 2.0-kb genomic fragment from the human alpha 1 (XIII) collagen gene was isolated and shown by DNA sequencing to contain exon 12 as counted from the 3' end. This fragment was used as a probe to localize the gene. The gene (COL13A1) was assigned to chromosome 10 by hybridization of the probe to DNA isolated from a panel of human-mouse somatic cell hybrids containing different human chromosomes. Furthermore, the gene was mapped to the q22 region by in situ hybridization to metaphase chromosomes.     

Further evidence for the dispersion of the human fibrillar collagen genes.

Recombinant DNA probes specific for the human pro alpha 1(II) and pro alpha 1(III) collagen chains have been used for the chromosomal localization of the two genes. Restriction endonuclease analysis of DNA from human-rodent hybrid cell lines in conjunction with in situ hybridization of human metaphasic chromosomes have shown that the gene coding for the pro alpha 1 chain of type II collagen (COL2A1) is located on chromosome 12 in the segment 12q131----12q132. Likewise, the gene coding for the pro alpha 1 chain of type III collagen (COL3A1) was assigned to the segment 2q31----2q323 of chromosome 2.     

The genes coding for human pro alpha 1(IV) collagen and pro alpha 2(IV) collagen are both located at the end of the long arm of chromosome 13.

We have isolated and characterized a cDNA clone containing DNA sequences coding for the noncollagenous carboxy-terminal domain of human pro alpha 2(IV) collagen. Using this cDNA clone in both Southern blot analysis of DNA isolated from human-mouse somatic-cell hybrids and in situ hybridization of normal human metaphase chromosomes, we have demonstrated that the gene coding for human pro alpha 2(IV) collagen is located at 13q33----34, in the same position on chromosome 13 as the pro alpha 1(IV) collagen gene.     

The human alpha 2(XI) collagen (COL11A2) chain. Molecular cloning of cDNA and genomic DNA reveals characteristics of a fibrillar collagen with differences in genomic organization

We have isolated three overlapping cDNA clones encoding the pro alpha 2(XI) collagen chain from a human chondrocyte cDNA library. Together, the cDNAs code for 257 uninterrupted Gly-X-Y triplets (almost 80% of the triple helical domain) and about 200 amino acid residues of the carboxyl telopeptide and carboxyl propeptide. The identification of the clones as pro alpha 2(XI) cDNAs was based on the complete identity between the amino acid sequences of three tryptic peptides derived from human alpha 2(XI) collagen and the cDNA-derived sequence. We have also sequenced six exons within a human genomic alpha 2(XI) cosmid clone. This sequence shows that although type XI collagen belongs to the fibril-forming class of collagens, there are substantial differences in exon sizes at the 3' end of the gene when comparing the alpha 2(XI) gene with those of human types I, II, and III collagens. Finally, pro alpha 2(XI) cDNA has been used as a probe to determine the location of the gene by in situ hybridization of chromosome spreads. The results demonstrate that the gene is located close to the region p212 on chromosome 6. Northern blot analysis shows that the gene is expressed in cartilage but not in adult liver, skin, and tendon.     

Physical mapping by PFGE localizes the COL3A1 and COL5A2 genes to a 35-kb region on human chromosome 2

The genes encoding the alpha 1 chain of Type III collagen (COL3A1) and the alpha 2 chain of Type V (COL5A2) collagen have been mapped to the long arm of human chromosome 2. Linkage analysis in CEPH families indicated that these two genes are close to each other, with no recombination in 37 informative meioses. In the present study, DNA probes from the 3' ends of each gene have been physically mapped by pulsed-field gel electrophoresis. The probes recognized 11 macrorestriction fragments in common, ranging from greater than 1000 kb MluI and NotI fragments to a 35-kb SfiI fragment. Therefore, the COL3A1 and COL5A2 genes appear to exist as a gene cluster on chromosome 2. This is the third example of a collagen gene cluster. Other examples include the COL4A1-COL4A2 genes on chromosome 13q and the COL6A1-COL6A2 genes on chromosome 21q. The physical proximity of these genes may indicate common evolution and/or regulation.     

Assignment of the gene coding for the alpha 1 chain of collagen type XIII (COL13A1) to human chromosome region 10q11----qter

The gene coding for the alpha 1 chain of human type XIII collagen. COL13A1, is assigned to chromosome region 10q11----qter by Southern blot hybridization of DNA from 24 human x rodent somatic cell hybrids using a cloned cDNA as probe. A number of previous reports indicate that 10 of the collagen genes are located on six autosomes, but no other collagen genes have been found on chromosome 10. The data therefore provide further evidence for the dispersion of members of the collagen gene family throughout the genome.     

Assignment of the human pro alpha 2(I) collagen structural gene (COLIA2) to chromosome 7 by molecular hybridization.

A cDNA for the pro alpha 2 chain of human type I collagen has been recently cloned and amplified. We have used this specific probe to identify the human chromosome carrying the pro alpha 2(I) collagen gene. The DNA from 17 independent human/hamster and human/mouse somatic cell hybrids was digested by Eco RI and the restriction pattern analyzed in Southern blot experiments, using the 32P-labeled cDNA as a hybridization probe. The gene coding for the pro alpha 2 collagen subunit could be unambiguously assigned to human chromosome 7. All the other chromosomes, including chromosome 17, were excluded.     

Cloning and chromosomal localization of human genes encoding the three chains of type VI collagen.

Type VI collagen is a heterotrimer composed of three polypeptide chains, alpha 1(VI), alpha 2(VI), and alpha 3(VI). By immunological screening of an expression cDNA library, human cDNAs specific for each chain were isolated and characterized. Major mRNA species encoding these chains have a size of 4.2 kb (alpha 1), 3.5 kb (alpha 2), and 8.5 kb (alpha 3). The cDNA clones were also used to map the genes on human chromosomes by somatic cell hybrid analysis and in situ hybridization. The alpha 1 (VI) and alpha 2(VI) collagen genes were both located on chromosome 21, in band q223. This represents a third example of a possible physical proximity of two collagen loci. The alpha 3(VI) collagen gene was localized to chromosome 2, in the region 2q37. The alpha 3(VI) collagen gene is the fifth extracellular matrix gene to be localized to 2q, as four other extracellular matrix genes--i.e., the alpha 1(III) and alpha 2(V) collagen genes, the elastin gene, and the fibronectin gene--have been previously mapped to the distal region of the long arm of chromosome 2.     

Genomic organization and characterization of the human type XXI collagen (COL21A1) gene

We cloned a 4.1-kb full-length cDNA based on a reported human genomic clone containing a partial open reading frame (ORF) coding for a novel collagen-like protein. Sequence analysis indicated that the ORF codes for the alpha(1)-chain of type XXI collagen. Assembly of the genomic data reveals a complete sequence of the human gene COL21A1. COL21A1 is localized to chromosome 6p11.2-12.3, spanning 337 kb in size. The gene contains 31 exons, in which the 5'-untranslated exons 1 and 1a are alternatively spliced. The exon/domain organization of COL21A1 resembles that of the reported FACIT collagen genes, including COL9A1, COL9A2, COL9A3, and COL19A1, suggesting that these genes may have derived from the same ancestor FACIT gene by duplication. The expression of COL21A1 in human tissues is developmentally regulated, with a higher level at fetal stages. Type XXI collagen is an extracellular matrix component of the blood vessel walls, secreted by smooth-muscle cells. Platelet-derived growth factor (PDGF) has a pronounced effect on the stimulation of COL21A1 expression in cultured aortic smooth-muscle cells, suggesting that alpha1(XXI) collagen may contribute to the extracellular matrix assembly of the vascular network during blood vessel formation.     

Chromosomal assignment of a gene encoding a new collagen type (COL15A1) to 9q21 --> q22.

The collagens constitute a large family of extracellular matrix components primarily responsible for maintaining the structure and biological integrity of connective tissue. These proteins exhibit considerable diversity size, sequence, tissue distribution, and molecular composition. Fourteen types of homo- and/or heterotrimeric molecules, thus far reported, are encoded by a minimum of 27 genes. Nineteen of these genes, including several that are closely linked, have been assigned to 10 separate autosomes, and one collagen gene has been mapped to the X chromosome. We have isolated a 2.1-kb human cDNA clone coding for a collagen molecule different in sequence and structure from types I-XIV collagens. This polypeptide has been designated the alpha 1 chain of type XV collagen. To determine the location of the corresponding gene, the cDNA clone was hybridized to rodent-human hybrid DNAs and to human metaphase chromosomes. The results obtained using the hybrid cell lines showed that this newly identified collagen gene, COL15A1, is present in the pter --> q34 region of chromosome 9. In situ hybridization allowed sublocalization to 9q21 --> q22, a region to which no other collagen genes had previously been assigned. Our data further demonstrate the complex arrangement of the many collagen genes in the human genome.     

The chicken alpha 1 (XI) collagen gene is widely expressed in embryonic tissues.

Complementary DNA and genomic DNA clones corresponding to the chicken alpha 1 (XI) collagen gene were isolated and characterized. These recombinant DNA clones covered 2667 base pairs of the mRNA and encode 624 amino acids of the triple helical region plus the entire carboxyl-terminal propeptide. Northern blot analysis showed a major band of approximately 6.5 kilobases and a minor band of approximately 7.5 kilobases. A combination of Northern blot and in situ hybridization analyses showed that, in addition to its presence in cartilage, this mRNA also is present in a wide variety of chicken noncartilaginous embryonic tissues including brain, heart, skeletal muscle, calvaria, and skin, but was not detected in liver. Type II collagen mRNA has also been detected at low levels in these same tissues. Also, similar to the mRNA for the alpha 1 chain for type II collagen, the alpha 1 (XI) collagen mRNA is detected in limb mesenchyme prior to condensation and differentiation of the core mesenchyme into cartilage.     

Differential expression of two exons of the alpha1(XI) collagen gene (Col11a1) in the mouse embryo.

The amino terminal domain of collagen XI has a unique structure, which is believed to participate in the regulation of matrix assembly. Interestingly, several distinct isoforms of the amino terminal domain of alpha1(XI) and alpha2(XI) collagen chains exist as a result of alternative splicing. Here we report the analysis of the alternative splicing pattern of the mouse alpha1(XI) collagen gene (Col11a1). Like other vertebrate species, the mutually exclusive expression of exons 6A and 6B of Col11a1 results in the inclusion in the alpha1 chain of either an acidic peptide (pI 3.14) or a basic peptide (pI 11.66). Expression of these two exons was monitored in several tissues of the 16.5-day mouse embryo by in situ hybridization and immunohistochemistry, with exon-specific cDNA probes and peptide-specific antibodies, respectively. The results documented that isoforms containing the exon 6B-encoded peptide accumulate predominantly in the vertebrae, skeletal muscles and intestinal epithelium. By contrast, exon 6A products were found to be most abundant in the smooth muscle cells of the intestine, aorta and lung. The results using in situ hybridization confirmed those using immunohistochemistry. Albeit correlative, the evidence suggests distinct contributions of the two peptides to the differential assembly of tissue-specific matrices.     

Cloning of the human and mouse type X collagen genes and mapping of the mouse type X collagen gene to chromosome 10.

Type X collagen, a homotrimer of alpha 1 (X) polypeptide chains, is specifically expressed by hypertrophic chondrocytes in regions of cartilage undergoing endochondral ossification. We have previously described the isolation of a small fragment of the human type X collagen gene (COL10A1) and its localization to the q21-q22 region of human chromosome 6 [Apte, S., Mattei, M.-G. & Olsen, B. R. (1991) FEBS Lett. 282, 393-396]. Using this fragment as a probe to screen genomic libraries, we report here the isolation of human and mouse genomic clones which contain the major part of the human and mouse type X collagen genes. In both species, the 14-kb genomic clones which were isolated contain a long open reading frame (greater than 2000 bp in length) which codes for the entire C-terminal non-collagenous (NC1) domain, the entire collagenous (COL) domain and part of the N-terminal non-collagenous (NC2) domain of the alpha 1(X) collagen chain. The human genomic clone contains the major part of the COL10A1 gene, in addition to the region we have previously cloned, and is highly similar to the corresponding portions of the mouse genomic clone (84.5% similarity at the nucleotide level, and 86.1% at the level of the conceptual translation product). The identification of the mouse genomic clone as the alpha 1(X) collagen gene (Col10a1) was confirmed by in situ hybridization of a fragment of the mouse genomic clone to sections from newborn mice. Hybridization was restricted to the hypertrophic chondrocytes of developing chondroepiphyses, being absent in small chondrocytes and in other tissues. Using interspecific backcross analysis, the locus for the mouse alpha 1 (X) collagen gene was assigned to chromosome 10. The cloning and chromosomal mapping of the human and mouse alpha 1 (X) collagen genes now permit the investigation of the possible role of type X collagen gene defects in the genesis of chondrodysplasias in both species and provide data essential for the generation of transgenic mice deficient in type X collagen.     

The mouse alpha 1(XII) and human alpha 1(XII)-like collagen genes are localized on mouse chromosome 9 and human chromosome 6.

Type XII collagen is a member of the FACIT (fibril-associated collagens with interrupted triple helices) group of extracellular matrix proteins. Like the other members of this group, collagen types IX and XIV, type XII has alternating triple-helical and non-triple-helical domains. Because of its structure, its association with collagen fibrils, and its distribution in dense connective tissues, type XII is thought possibly to act as a cross-bridge between fibrils and resist shear forces caused by tension. A portion of the ffuse gene was isolated by screening a genomic library with a chicken alpha 1 (XII) cDNA probe, followed by subcloning and sequence analysis. Comparison of exon sequences with the sequence of a mouse cDNA clone allowed the mouse gene to be identified as the alpha 1 (XII) collagen gene. In the mouse, Col12a1 is located on chromosome 9, as determined by linkage analysis using DNA from interspecific backcrosses with Mus spretus. Screening of a human genomic library also allowed the isolation of a human alpha 1(XII)-like gene (CoL12A1). This gene was mapped to chromosome 6 by blot hybridization to DNA from human/hamster hybrid cell lines. This information should prove useful in determining the role of type XII collagen genes as candidate genes in inheritable connective tissue diseases.     

Molecular cloning of rat and human type IX collagen cDNA and localization of the alpha 1(IX) gene on the human chromosome 6

Type IX collagen is found in hyaline cartilage, where it is associated with type II collagen in quarter-staggered collagen fibrils. Chicken type IX collagen has been extensively characterized and shown to contain molecules with three triple-helical domains, interspersed with non-triple-helical sequences. The molecule contains three, genetically distinct, subunits and one of these subunits carries a covalently bound glycosaminoglycan side chain. In the present report, we describe for the first time the primary structure of mammalian type IX collagen chains, based on cloning and sequencing of cDNA from rat and human cDNA libraries. The results suggest that mammalian alpha 1(IX) chains have the same multi-domain structure as the avian protein. We also demonstrate, by in situ hybridization of chromosome spreads, that the human alpha 1(IX) collagen gene is located on the long arm of chromosome 6. The cloning of human type IX collagen cDNA provides a probe for molecular studies of human chondrodysplasias that may involve abnormalities in this extracellular collagen-proteoglycan.