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.     

Mapping of a human fibrillar collagen gene, pro alpha 1 (XI) (COL11A1), to the p21 region of chromosome 1

Type XI collagen is a minor and poorly characterized structural component of cartilage. Recently, cDNA and genomic clones coding for the pro alpha 1 chain of human Type XI collagen, formerly 1 alpha collagen, have been isolated and fully characterized. Here we have used one such probe to establish the chromosomal localization of the pro alpha 1 (XI) collagen gene (COL11A1) by hybridization to filter-bound DNA isolated from flow-sorted chromosomes and by in situ hybridization on metaphase chromosomes. This combination of approaches has enabled us to locate COL1A11 in the p21 region of chromosome 1. This represents the first mapping of a Type XI collagen gene and the first assignment of a collagen locus to chromosome 1. These studies also provide additional evidence for the nearly uniform dispersion of the human fibrillar collagen genes in the human genome.     

Nucleotide sequence of a cDNA clone for mouse pro alpha 1(I) collagen protein

A cDNA clone for mouse pro alpha 1(I) collagen has been isolated and sequenced. A 1.8-kb PstI fragment spans nucleotides -1991 to -159 of the alpha-domain of mouse pro alpha 1(I) collagen.     

The complete primary structure of a nematode alpha 2(IV) collagen and the partial structural organization of its gene.

We have isolated and characterized cDNA and genomic DNA clones which encode an alpha 2(IV) collagen chain from the parasitic nematode Ascaris suum. In addition we have determined, by nucleic acid sequence analysis, the structural organization of approximately two-thirds of the gene. This analysis has shown that the gene contains at least 15 introns, and those that have been characterized range in size from 141 to 854 base pairs. The derived protein sequence contains 1763 amino acids and includes a putative 26-amino acid signal sequence. The collagenous triple-helical region contains 17 interruptions, many of which occur in the same positions as those in the human alpha 1(IV) and alpha 2(IV) chains. Comparison of the genomic DNA sequence with the cDNA sequence has revealed the presence of a sequence within the gene which appears to be an intact and normal exon that is not represented in our cDNA sequence. The presence of this putative exon raises the possibility that the A. suum alpha 2(IV) collagen gene may undergo alternative splicing.     

Molecular cloning of alpha 5(IV) collagen and assignment of the gene to the region of the X chromosome containing the Alport syndrome locus.

Type IV collagen is a major structural component of basement membranes. Four constituent polypeptides have been described and characterized to different degrees. Whereas the primary structure of the alpha 1(IV) and alpha 2(IV) chains has been completely established, only short protein sequences have been reported for the recently recognized alpha 3(IV) and alpha 4(IV) subunits. We have isolated overlapping human cDNA clones whose derived amino acid sequence is highly homologous to the alpha 1(IV) and alpha 2(IV) chains. However, these clones code for neither alpha 3(IV) nor alpha 4(IV), and thus this new polypeptide has been designated the alpha 5 chain of type IV collagen. To determine whether the gene encoding the alpha 5(IV) chain is syntenic with the contiguously arranged alpha 1(IV) and alpha 2(IV) genes at 13q34, the alpha 5(IV) cloned DNA was hybridized to genomic DNA from somatic cell hybrids and to metaphase chromosomes. The results demonstrated that the alpha 5(IV) collagen gene is located on the long arm of the X chromosome. Since 14 collagen genes have previously been assigned to nine autosomes, these data represent the first mapping of a collagen gene to the X chromosome. Most important, the alpha 5(IV) gene has been sublocalized to bands Xq22----q23, which are in the same region known to contain the locus for the X-linked form of Alport syndrome. It is therefore possible that this severe dominantly inherited nephritis, manifested by splitting of the glomerular basement membrane, could be caused by mutations in the alpha 5(IV) collagen gene.     

Major rearrangements in the alpha 5(IV) collagen gene in three patients with Alport syndrome.

The gene coding for the alpha 5 chian of type IV collagen (alpha 5(IV) collagen), which maps to Xq22, is a candidate gene for the X-linked dominant disease Alport syndrome (AS). Using three cDNA clones, covering the 3' end of the alpha 5(IV) collagen gene, 3 of 38 patients have been identified with mutations in this gene. Each of these patients shows a gross rearrangement of DNA: a deletion of at least 35 kb, an insertion/deletion event involving approximately 25 kb, and a duplication of at least 35 kb of DNA.     

Isolation and partial characterization of the entire human pro alpha 1(II) collagen gene.

Using a cDNA probe specific for the bovine Type II procollagen, a series of overlapping genomic clones containing 45 kb of contiguous human DNA have been isolated. Sequencing of a 54 bp exon, number 29, provided direct evidence that the recombinant clones bear human Type II collagen sequences. Localization of the 5' and 3' ends of the gene indicated that the human Type II collagen gene is 30 kb in size. This value is significantly higher than that of the homologous avian gene. The segregation of a polymorphic restriction site in informative families conclusively demonstrated that the Type II gene is found in a single copy in the human haploid genome. Finally, sequencing of a triple helical domain exon has confirmed that a rearrangement leading to the fusion of two exons occurred in the pro alpha 1(I) gene, following the divergence of the fibrillar collagens.     

Analysis of cDNA and genomic clones coding for the pro alpha 1 chain of calf type II collagen.

A bovine cDNA library constructed from fetal cartilage RNA was screened with a pro alpha 1(II) collagen specific chicken cDNA. A recombinant clone (Bc 7), with an insert of 1 kb, was identified and shown to contain sequences exhibiting 85% homology with the chicken pro alpha 1(II) collagen C-propeptide. Interspecies comparison strongly suggested that one potential glycosylation site present in the avian C-propeptide is not utilized, since this site is absent in the bovine chain. In addition, two overlapping genomic clones (Pal 3 and Pal 4) were isolated and partially characterized. These clones span 23 kb of DNA and contain approximately 17 kb of the pro alpha 1(II) calf gene. Sequencing of exon 1 has determined the length of the 3' untranslated region and the exact location of the polyadenylation attachment site.     

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.     

The pro alpha 2(V) collagen gene is evolutionarily related to the major fibrillar-forming collagens.

A number of overlapping cDNA clones, covering 5.2 kb of sequences which code for the human pro alpha 2(V) collagen chain, have been isolated. Analysis of the structural data have indicated a close evolutionary kinship between the pro alpha 2(V) chain and the major fibrillar collagen types. Isolation and analysis of an 8 kb genomic fragment has further supported this notion by revealing a homologous arrangement of nine triple-helical domain exons. These studies have therefore provided conclusive evidence which categorizes the Type V collagen as a member of the Group 1 molecules, or fibrillar-forming collagens.     

Proposed alignment of helical interruptions in the two subunits of the basement membrane (type IV) collagen

We have isolated a cDNA clone for part of the alpha 2 type IV collagen (pCIV-2-176). Deoxynucleotide sequence analysis shows that this clone codes for 439 amino acids from the helical domain adjacent to the C-terminal globular domain of the alpha 2 (IV) chain. By aligning the deduced amino acid sequence of the alpha 2 (IV) chain with the published sequence for the alpha 1 (IV) chain, we find that all interruptions in the alpha 1 (IV) chain coincide with an interruption in the alpha 2 (IV) chain. Additional interruptions in the alpha 2 (IV) chain exist, however, three out of the four analysed only slightly disturb the collagen triple helix.     

Isolation and characterization of genomic DNA coding for alpha 2 type I collagen.

We have isolated and characterized a segment of the chick alpha 2 collagen gene by screening a library of chick genomic fragments using as hybridization probe an alpha 2 collagen cDNA clone. Several clones were isolated and one of them, lambda gCOL 204, was used for further studies. The DNA of lambda gCOL 204 hybridizes to a unique species of mRNA the size of alpha 2 collagen mRNA. This mRNA can be translated into a unique polypeptide which comigrates in SDS-gel electrophoresis with pro-alpha 2 collagen. Electron microscopic analysis by R-loop technique indicates that lambda gCOL 204 contains 7Kb of the alpha 2 collagen gene. This 7 Kb piece constitutes the 3' end of the gene. The same clone also contains 9 Kb of DNA that is immediately adjacent to the 3' end of the alpha 2 collagen gene. The cloned segment of the alpha 2 collagen gene is interrupted by 8 intervening sequences of various lengths. The coding sequences for collagen in this clone add up to approximately 1,800 bp, which correspond to about 1/3 of alpha 2 collagen mRNA. DNA sequence analysis of a small coding segment of lambda g COL 204 reveals a characteristic collagen type sequence which encodes for an amino acid sequence identical to a sequence found in calf alpha 2 collagen. The sequence of this region of the protein has not yet been determined for the chick alpha 2 collagen.     

Extensive homology between the carboxyl-terminal peptides of mouse alpha 1(IV) and alpha 2(IV) collagen

We have determined the complete primary structure for the carboxyl-terminal peptides of mouse alpha 1(IV) and alpha 2(IV) collagen; which have 229 and 227 amino acids, respectively. The amino acid sequences are 63% identical and conservatively substituted in 28 positions. A striking feature of these peptides is that the first half of each sequence is homologous with the second half, 37% in alpha 1(IV) and 36% in alpha 2(IV). These results suggest that the carboxyl-terminal peptides of type IV collagen are closely related in their structure and evolution. Presumably, they were first derived by internal duplication of a common ancestral DNA sequence which later, by gene duplication, gave rise to the two different but homologous carboxyl-terminal peptides of type IV collagen.     

Construction and characterization of cDNA clones encoding the 5' end of the chicken pro alpha 1(I) collagen mRNA

As a first step in isolating the 5' end of the chicken pro alpha 1(I) collagen gene, we constructed cDNA clones complementary to the 5' end of the pro alpha 1(I) mRNA using synthetic oligodeoxynucleotides complementary to a conserved region within the N-terminal telopeptide as primers. cDNA clones corresponding to the 5'-untranslated region, signal peptide, N-propeptide and telopeptide were identified based on homology with the human pro alpha 1(I) collagen protein sequence, and on hybridization to pro alpha 1(I) mRNA on Northern blots. A comparison of the nucleotide sequence of these clones with the sequence of the 5' end of the pro alpha 2(I) collagen mRNA confirms that there is 84% homology in a 49-bp region surrounding the translation start point, and shows that there is 70% homology in the nucleotide sequences encoding the N-propeptide triple helical region of the two type-I collagen chains.     

Nucleotide sequence coding for the human type IV collagen alpha 2 chain cDNA reveals extensive homology with the NC-1 domain of alpha 1 (IV) but not with the collagenous domain or 3'-untranslated region

We have isolated two overlapping cDNA clones that provide the complete nucleotide sequence coding for the NC-1 domain and 3'-untranslated region of the alpha 2 chain of human type IV collagen as well as a sequence encoding 232 residues of the collagenous domain. An extensive homology was observed between the sequences of the NC-1 domain of the alpha 1(IV) and alpha 2(IV) chains, but considerably less between the sequences encoding collagenous and 3'-untranslated regions. There were four interruptions in the collagenous sequence studied whereas the comparable region of the alpha 1(IV) chain had only two. A potential oligosaccharide attachment site was found in a 6-residue long interruption of the collagenous domain but none in the NC-1 domain.     

Human basement membrane collagen (type IV). The amino acid sequence of the alpha 2(IV) chain and its comparison with the alpha 1(IV) chain reveals deletions in the alpha 1(IV) chain

The cDNA and protein sequences of the N-terminal 60% of the alpha 2(IV) chain of human basement membrane collagen have been determined. By repeated primer extension with synthetic oligodeoxynucleotides and mRNA from either HT1080 cells or human placenta overlapping clones were obtained which cover 3414 bp. The derived protein sequence allows for the first time a comparison and alignment of both alpha chains of type IV collagen from the N terminus. This alignment reveals an additional 43 amino acid residues in the alpha 2(IV) chain as compared to the alpha 1(IV) chain. 21 of these additional residues form a disulfide-bridged loop within the triple helix which is unique among all known collagens.