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.     

R-banding and nonisotopic in situ hybridization: precise localization of the human type II collagen gene (COL2A1)

Summary A new mapping system, based on nonisotopic in situ hybridization combined with fluorescent staining of replicated prometaphase R-bands, is described. Replication of the bands is achieved by treatment of thymidinesynchronized cells with bromodeoxyuridine. The human COL2A1 gene was mapped to band 12q13.11–q13.12 in this manner, to illustrate the potential of the technique for improving the precision of chromosomal mapping and physical ordering of genes.     

Synteny between the loci for a novel FACIT-like collagen locus (D6S228E) and alpha 1 (IX) collagen (COL9A1) on 6q12-q14 in humans.

A 1.8-kb cDNA encoding portion of a novel collagenous chain was isolated from a human rhabdomyosarcoma cell line by cross-hybridization using a chicken type V collagen probe. Sequence analysis suggests that this chain belongs to the recently discovered group of collagens, termed the FACIT class of macromolecules. This cDNA was used to locate the corresponding gene (D6S228E) to chromosome 6, notably at position 6q12-q14. Interestingly, within this region of human chromosome 6 residues the alpha 1 (IX) collagen gene (COL9A1), a member of the FACIT group.     

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.     

Comparative cytogenetic mapping of COL14A1, the gene for human and mouse collagen XIV.

Utilizing the FISH technique, the gene for collagen XIV was mapped in the human and the mouse genome. The human gene (COL14A1) was assigned to chromosome bands 8q23-->q24.1. This assignment is in agreement with the localization of the undulin gene (UND), whose product has been suggested to be a variant of collagen XIV. The mouse gene (Col14a1) was assigned to chromosome 15 band D. Thus, collagen XIV represents another example of a gene that belongs to human/mouse homology group 90.     

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.     

Macrorestriction mapping of COL4A1 and COL4A2 collagen genes on human chromosome 13q34

The genes for the alpha-1 and alpha-2 chains of type IV collagen (COL4A1 and COL4A2) map to the same chromosomal band (13q34) and have a high degree of nucleotide homology. We have used pulsed field gel electrophoresis and cloned COL4A1 and COL4A2 DNA fragments as molecular probes to construct a 1200-kb macrorestriction map which encompasses both genes. The two genes are located within a 340-kb region with the 3' end of COL4A2 and the 5' region of COL4A1 separated by at least 100 kb but not more than 160 kb. These genes, therefore, are two members of a gene cluster on chromosome 13q34.     

Three novel collagen VI chains, alpha4(VI), alpha5(VI), and alpha6(VI)

We report the identification of three new collagen VI genes at a single locus on human chromosome 3q22.1. The three new genes are COL6A4, COL6A5, and COL6A6 that encode the alpha4(VI), alpha5(VI), and alpha6(VI) chains. In humans, the COL6A4 gene has been disrupted by a chromosome break. Each of the three new collagen chains contains a 336-amino acid triple helix flanked by seven N-terminal von Willebrand factor A-like domains and two (alpha4 and alpha6 chains) or three (alpha5 chain) C-terminal von Willebrand factor A-like domains. In humans, mRNA expression of COL6A5 is restricted to a few tissues, including lung, testis, and colon. In contrast, the COL6A6 gene is expressed in a wide range of fetal and adult tissues, including lung, kidney, liver, spleen, thymus, heart, and skeletal muscle. Antibodies to the alpha6(VI) chain stained the extracellular matrix of human skeletal and cardiac muscle, lung, and the territorial matrix of articular cartilage. In cell transfection and immunoprecipitation experiments, mouse alpha4(VI)N6-C2 chain co-assembled with endogenous alpha1(VI) and alpha2(VI) chains to form trimeric collagen VI molecules that were secreted from the cell. In contrast, alpha5(VI)N5-C1 and alpha6(VI)N6-C2 chains did not assemble with alpha1(VI) and alpha2(VI) chains and accumulated intracellularly. We conclude that the alpha4(VI)N6-C2 chain contains all the elements necessary for trimerization with alpha1(VI) and alpha2(VI). In summary, the discovery of three additional collagen VI chains doubles the collagen VI family and adds a layer of complexity to collagen VI assembly and function in the extracellular matrix.     

Comparative mapping of genes flanking the human chromosome 12 evolutionary breakpoint in the pig

Genes located on human chromosome 12 (HSA12) are conserved on pig chromosomes 5 and 14 (SSC5 and SSC14), with HSA12q23.3-->q24.11 harboring the evolutionary breakpoint between these chromosomes. For this study, pig sequence-tagged sites (STS) were developed for nine HSA12 genes flanking this breakpoint. Radiation hybrid (RH) mapping using the IMpRH panel revealed that COL2A1, DUSP6, KITLG, PAH and STAB2 map to SSC5, while PXN, PLA2G1B, SART3 and TCF1 map to SSC14. Polymorphisms identified in COL2A1, DUSP6, PAH, PLA2G1B and TCF1 were used for genetic linkage mapping and confirmed the map locations for these genes. Our results indicate that the HSA12 evolutionary breakpoint occurs between STAB2 and SART3 in a region spanning less than five million basepairs. These results refine the comparative map of the HSA12 evolutionary breakpoint region and help to further elucidate the extensive gene order rearrangements between HSA12 and SSC5 and 14.     

Multipoint linkage analysis in X-linked ocular albinism of the Nettleship-Falls type

Summary The genes that encode the alpha 1 (VI) and alpha 2 (VI) collagen chains, designated COL6A1 and COL6A2, map to human chromosomal band 21q22.3. Using pulsed-field gel electrophoresis and somatic cell hybrids, we found that COL6A1 and COL6A2 form a gene cluster on the most distal part of chromosome 21. Furthermore, we detected several DNA polymorphisms (both restriction site and VNTRs) associated with these loci. These polymorphisms make the COL6A1 and COL6A2 genes among the most informative markers on human chromosome 21.     

Modulation of transforming growth factor-beta (TGF-beta) signaling by endogenous sphingolipid mediators

Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that plays a critical role in tissue repair and fibrosis. Sphingolipid signaling has been shown to regulate a variety of cellular processes and has been implicated in collagen gene regulation. The present study was undertaken to determine whether endogenous sphingolipids are involved in the TGF-beta signaling pathway. TGF-beta treatment induced endogenous ceramide levels in a time-dependent manner within 5-15 min of cell stimulation. Using human fibroblasts transfected with a alpha2(I) collagen promoter/reporter gene construct (COL1A2), C(6)-ceramide (10 microm) exerted a stimulatory effect on basal and TGF-beta-induced activity of this promoter. Next, to define the effects of endogenous sphingolipids on TGF-beta signaling we employed ectopic expression of enzymes involved in sphingolipid metabolism. Sphingosine 1-phosphate phosphatase (YSR2) stimulated basal COL1A2 promoter activity and cooperated with TGF-beta in activation of this promoter. Furthermore, overexpression of YSR2 resulted in the pronounced increase of COL1A1 and COL1A2 mRNA levels. Conversely, overexpression of sphingosine kinase (SPHK1) inhibited basal and TGF-beta-stimulated COL1A2 promoter activity. These results suggest that endogenous ceramide, but not sphingosine or sphingosine 1-phosphate, is a positive regulator of collagen gene expression. Mechanistically, we demonstrate that Smad3 is a target of YSR2. TGF-beta-induced Smad3 phosphorylation was elevated in the presence of YSR2. Cotransfection of YSR2 with wild-type Smad3, but not with the phosphorylation-deficient mutant of Smad3 (Smad3A), resulted in a dramatic increase of COL1A2 promoter activity. In conclusion, this study demonstrates a direct role for the endogenous sphingolipid mediators in regulating the TGF-beta signaling pathway.     

Differential allelic expression of the type II collagen gene (COL2A1) in osteoarthritic cartilage.

Osteoarthritis (OA) is a common debilitating disease resulting from the degeneration of articular cartilage. The major protein of cartilage is type II collagen, which is encoded by the COL2A1 gene. Mutations at this locus have been discovered in several individuals with inherited disorders of cartilage. We have identified 27 primary OA patients who are heterozygous for sequence dimorphisms located in the coding region of COL2A1. These dimorphisms were used to distinguish the mRNA output from each of the two COL2A1 alleles in articular cartilage obtained from each patient. Three patients demonstrated differential allelic expression and produced < 12% of the normal level of mRNA from one of their COL2A1 alleles. The same allele shows reduced expression in all three patients, and this allele is more frequent in a well-defined OA population than in a control group, suggesting the possible existence of a rare COL2A1 allele that predisposes to OA.     

Are the nail-patella syndrome and the autosomal Goltz-like syndrome the phenotypic expressions of different alleles at the COL5A1 locus?

The COL5A1 gene, which encodes the pro 1(V) chain, was recently mapped to 9q34.3 in the same region as the nail-patella locus. This was taken as an indication that the nail-patella syndrome may be an inherited connective tissue disorder. We demonstrate COL5A1 heterozygous deletion and fibroblast under-expression of 1(V) chains in a girl with an unbalanced translocation resulting in 9q32qter monosomy. The patient presents dysplastic nails, a sign typical of nail-patella syndrome, but normal patella. Moreover, she has skin and bone disorders similar to those found in the Goltz syndrome. We suggest that monosomy for the COL5A1 gene is responsible for these connective tissue disorders. Accordingly, the nail-patella syndrome could be attributable to mutations inside the COL5A1 gene rather than to a deletion of it.