Complete Structural Organization of the Human α1(V) Collagen Gene (COL5A1): Divergence from the Conserved Organization of Other Characterized Fibrillar Collagen Genes

Genes that encode the vertebrate fibrillar collagen types I–III have previously been shown to share a highly conserved intron/exon organization, thought to reflect common ancestry and evolutionary pressures at the protein level. We report here the complete intron/exon organization ofCOL5A1,the human gene that encodes the α1 chain of fibrillar collagen type V. The structure ofCOL5A1is shown to be considerably diverged from the conserved structure of the genes for fibrillar collagen types I–III.COL5A1has 66 exons, which is greater than the number of exons found in the genes for collagen types I–III. The increased number of exons is partly due to the increased size of the pro-α1(V) N-propeptide, relative to the sizes of the N-propeptides of the types I–III procollagen molecules. In addition, however, the increased number of exons is due to differences in the intron/exon organization of the triple-helix coding region ofCOL5A1compared to the organization of the triple-helix coding regions of the genes for collagen types I–III. Of particular interest is the increase of 54 bp exons in this region ofCOL5A1,strongly supporting the proposal that the triple-helix coding regions of fibrillar collagen genes evolved from duplication of a 54 bp primordial genetic element. Moreover, comparison of the structure ofCOL5A1to the highly conserved structure of the genes of collagen types I–III provides insights into the probable structure of the ancestral gene that gave rise to what appears to be two classes of vertebrate fibrillar collagen genes.     

Genomic organization of the human COL3A1 and COL5A2 genes: COL5A2 has evolved differently than the other minor fibrillar collagen genes.

We report here on the complete structure of the human COL3A1 and COL5A2 genes. Collagens III and V, together with collagens I, II and XI make up the group of fibrillar collagens, all of which share a similar structure and function; however, despite the similar size of the major triple-helical domain, the number of exons coding for the domain differs between the genes for the major fibrillar collagens characterized so far (I, II, and III) and the minor ones (V and XI). The main triple-helical domain being encoded by 49-50 exons, including the junction exons, in the COL5A1, COL11A1 and COL11A2 genes, but by 43-44 exons in the genes for the major fibrillar collagens. Characterization of the genomic structure of the COL3A1 gene confirmed its association with the major fibrillar collagen genes, but surprisingly, the genomic organization of the COL5A2 gene was found to be similar to that of the COL3A1 gene. We also confirmed that the two genes are located in tail-to-tail orientation with an intergenic distance of approximately 22 kb. Phylogenetic analysis suggested that they have evolved from a common ancestor gene. Analysis of the genomic sequences identified a novel single nucleotide polymorphism and a novel dinucleotide repeat. These polymorphisms should be useful for linkage analysis of the Ehlers-Danlos syndrome and related disorders.     

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.     

Structure of the Human Type IV CollagenCOL4A6Gene, Which Is Mutated in Alport Syndrome-Associated Leiomyomatosis

Basement membrane (type IV) collagen, a subfamily of the collagen protein family, is encoded by six distinct genes in mammals. Three of those,COL4A3, COL4A4,andCOL4A5,are linked with Alport syndrome (hereditary nephritis). Patients with leimoyomatosis associated with Alport syndrome have been shown to have deletions in the 5′ end of theCOL4A6gene, in addition to having deletions inCOL4A5(Zhouet al., Science261: 1167–1169, 1993). The humanCOL4A6gene is reported to be 425 kb as determined by mapping of overlapping YAC clones by probes for its 5′ and 3′ ends. In the present study we describe the complete exon/intron size pattern of the humanCOL4A6gene. The 12 λ phage clones characterized in the study spanned a total of 110 kb, including 85 kb of the actual gene and 25 kb of flanking sequences. The overlapping clones contained all 46 exons of the gene and all introns, except for intron 2. Since the total size of the exons and all introns except for intron 2 is about 85 kb, intron 2 must be about 340 kb. All exons of the gene were assigned toEcoRI restriction fragments to facilitate analysis of the gene in patients with leiomyomatosis associated with Alport syndrome. The exon size pattern ofCOL4A6is highly homologous with that of the human and mouseCOL4A2genes, with 27 of the 46 exons ofCOL4A6being identical in size between the genes.     

Assignment of the βB1 Crystallin Gene (CRYBB1) to Human Chromosome 22 and Mouse Chromosome 5

By using primers complementary to the rat βB1 crystallin gene sequence, we amplified exons 5 and 6 of the orthologous human gene (CRYBB1). The amplified human segments displayed greater than 88% sequence homology to the corresponding rat and bovine sequences.CRYBB1was assigned to the group 5 region in 22q11.2–q12.1 by hybridizing the exon 6 PCR product to somatic cell hybrids containing defined portions of human chromosome 22. The exon 5 and exon 6 PCR products ofCRYBB1were used to localize, by interspecific backcross mapping, the mouse gene (Crybb1) to the central portion of chromosome 5. Three other β crystallin genes (βB2(−1), βB3, and βA4) have previously been mapped to the same regions in human and mouse. We demonstrate that the βB1 and βA4 crystallin genes are very closely linked in the two species. These assignments complete the mapping and identification of the human and mouse homologues of the major β crystallins genes that are expressed in the bovine lens.     

Search for MHC-associated genes in human: five new genes centromeric to HLA-DP with yet unknown functions

Taking advantage of five mouse genomic or cDNA probes [KE5(probe 14), KE4 (probe11), KE3 (probe7), KE2 (probe5), and SET] mapped on the H-2K region in mouse, we have identified and localized homologues of these five genes in the human major histocompatibility complex region (HKE5, HKE4, HKE3, HKE2, and HSET, respectively). Cosmid cloning and pulsed field gel electrophoresis analyses indicated that a human homologous gene, HKE5, is located 10 kilobases (kb) centromeric of the 2(XI) collagen (COL11A2) gene followed by HKE4. HKE3, closely linked to HKE2, is located 170 kb centromeric of HKE4. Furthermore, HSET is located 50 kb centromeric of HKE2. This gene organization outside the DP subregion is completely identical to that of the mouse H-2K region centromeric of I-Pb 3, a mouse homologue of the DPB gene, except the lack of genes corresponding to the H-2K and -K2 genes in human.     

Genomic Structure of the Human Gene for Spinocerebellar Ataxia Type 2 (SCA2) on Chromosome 12q24.1

Spinocerebellar ataxia type 2 (SCA2) is a member of a group of neurodegenerative diseases that are caused by instability of a DNA CAG repeat. We report the genomic structure of theSCA2gene. Its 25 exons, encompassing approximately 130 kb of genomic DNA, were mapped onto the physical map of the region. Exonic sizes varied from 37 to 890 bp, and intronic sizes ranged from 323 bp to more than 15 kb. The CAG repeat was contained in the 5′ coding region of the gene in exon 1. Determination of the splice junction sequences indicated the presence of only one deviation from the GT-AG rule at the donor splice site of intron 9, which contained a GC instead of a GT dinucleotide. Exon 10, immediately downstream from this rare splice donor site, was alternatively spliced. Alternative splicing does not affect the reading frame and is predicted to encode an isoform containing 70 amino acids less.     

Complete Structure of the Human Gc Gene: Differences and Similarities between Members of the Albumin Gene Family

The sequence of the human Gc gene, including 4228 base pairs of the 5′-flanking region and 8514 base pairs of the 3′ flanking region (55,136 in total), was determined from five overlapping λ phage clones. The sequence spans 42,394 base pairs from the cap site to the polyadenylation site, and it reveals that the gene is composed of 13 exons, which are symmetrically placed within the three domains of the Gc protein. The first exon is partially untranslated, as is exon 12, which contains the termination codon TAG. Exon 13 is entirely untranslated, but contains the polyadenylation signal AATAAA. Ten central introns split the coding sequence between codon positions 2 and 3 and between codon positions 3 and 1 in an alternating pattern, exactly as has been observed in the structure of the albumin and α-fetoprotein genes. The Gc gene has several distinctive features which set it apart from the other members of the family. First, the gene is smaller by two exons, which results in a protein some 130 amino acids shorter than albumin or AFP. This decrease in size may result from the loss of two internal exons during the evolutionary history of the Gc gene. Second, exons 6, 8, 9, and 11 are smaller than their counterparts in albumin or AFP by a total of 8 codons (1, 4, 1, and 2, respectively). Although the mRNA and protein expressed from the Gc gene are significantly smaller, the gene itself is about 2.5 times larger than the other genes of the family. There are 13 interspersed DNA repeats within the human Gc gene which are absent from the same positions in the albumin or AFP genes, and hence must have been inserted after the triplication event(s) that gave rise to the gene family. Despite the differences, the Gc gene is nonetheless recognizable as a member of the albumin family.