Physical mapping of mouse collagen genes on chromosome 10 by high-resolution FISH.

Fluorescence in situ hybridization (FISH) on mechanically stretched chromosomes (MSCs) and extended DNA fibers enables construction of high-resolution physical maps by accurate ordering and orienting genomic clones as well as by measuring physical lengths of gaps and overlaps between them. These high-resolution FISH targets have hitherto been used mainly in the study of the human genome. Here we have applied both MSCs and extended DNA fibers to the physical mapping of the mouse genome. At first, five mouse collagen genes were localized by metaphase-FISH: Col10a1 to chromosomal bands 10B1-B3; Col13a1 to 10B4; and Col6a1, Col6a2, and Col18a1 to 10B5-C1. The mutual order of the genes, centromere--Col10a1--Col13a1--Col6a2--Col6a1--Col18a1--telomere, was determined by FISH on metaphase chromosomes, MSCs, and extended DNA fibers. To our knowledge, this is the first time mouse metaphase chromosomes have been stretched and used as targets for FISH. We also used MSCs to determine the transcriptional orientations, telomere--5'-->3'--centromere, of both Col13a1 and Col18a1. With fiber-FISH, Col18a1, Col6a1, and Col6a2 were shown to be in a head-to-tail configuration with respective intergenic distances of about 350 kb and 90 kb. Comparison of our physical mapping results with the homologous human data reveals both similarities and differences concerning the chromosomal distribution, order, transcriptional orientations, and intergenic distances of the collagen genes studied.

Identification of eight novel 5'-exons in cerebral capillary malformation gene-1 (CCM1) encoding KRIT1.

Truncating mutations in the CCM1 gene encoding KRIT1 were recently found in patients affected by inherited cerebral capillary malformations, lesions that cause a wide variety of neurologic problems. However, CCM1 mutations have not been identified in all the families linked to CCM1. Here we demonstrate that the CCM1 gene contains eight additional exons which may thus encompass the missing mutations.

KRIT1 is mutated in hyperkeratotic cutaneous capillary-venous malformation associated with cerebral capillary malformation.

Hyperkeratotic capillary-venous malformations (HCCVMs) are rare cutaneous lesions that occur in a small subgroup of patients with cerebral capillary malformation (CCM). CCMs cause neurological problems that range from headaches to life-threatening intracranial bleeding. CCMs and HCCVMs have a similar histopathological appearance of dilated capillary-venous channels. Genetic linkage of inherited CCMs has been established to three chromosomal loci, 3q25. 2-27, 7p13-15 and 7q21-22. The first mutations were identified in the CCM1 gene (located on 7q21-22), which encodes KRIT1 protein (KREV1 interaction trapped 1), presumably a membrane-bound protein with signalling activity. Although KRIT1 is known to interact with KREV1/RAP1A, a Ras-family GTPase, the exact function of KRIT1 in the formation of cerebral capillaries and veins is poorly understood. In this study, we screened five families with CCM for mutations in the KRIT1 gene. In one of the families, CCMs co-segregated with HCCVMs. We identified a KRIT1Delta(G103)mutation in this family, suggesting that this rare form of the condition is also caused by mutations in the CCM1 gene and that KRIT1 is probably important for cutaneous vasculature. Interestingly, this deletion introduces the earliest stop codon among identified mutations, suggesting a possible correlation between the molecular alteration and the cutaneous phenotype. Another novel mutation, KRIT1(IVS2+2(T-->C)), was found in a family with only cerebral capillary-venous malformations.

Production of cartilage collagens during metaphyseal bone healing in the mouse.

Small defects of unfractured bone are believed to heal without a cartilaginous intermediate. We have determined the extent of cartilage production in an experimental model of metaphyseal bone repair involving defects in both cortical and cancellous bone, but no fracture. Northern analyses revealed the presence of mRNAs for type X and II collagens in the repair tissue. Immunohistology confirmed subperiosteal deposition of both collagen types adjacent to the defect. While the mRNAs for the two collagen types peaked by one week of defect healing, immunodetectable type X collagen was not observed until the second week. The data suggest that reactivity of periosteum and activation of chondrogenesis and subsequent endochondral ossification programs are involved in murine bone repair regardless of defect type.

Type X collagen, a natural component of mouse articular cartilage: association with growth, aging, and osteoarthritis.

OBJECTIVE: To perform a systematic study on the production and deposition of type X collagen in developing, aging, and osteoarthritic (OA) mouse articular cartilage. METHODS: Immunohistochemistry was employed to define the distribution of type X collagen and Northern analyses to determine the messenger RNA levels as an indicator of the synthetic activity of the protein. RESULTS: Type X collagen was observed in the epiphyseal and articular cartilage of mouse knee joints throughout development and growth. Type X collagen deposition in the transitional zone of articular cartilage became evident toward cessation of growth, at the age of 2-3 months. The most intense staining for type X collagen was limited to the tidemark, the border between uncalcified and calcified cartilage. Northern analysis confirmed that the type X collagen gene is also transcribed by articular cartilage chondrocytes. Intense immunostaining was observed in the areas of OA lesions, specifically, at sites of osteophyte formation and surface fibrillation. Type X collagen deposition was also seen in degenerating menisci. CONCLUSION: This study demonstrates that type X collagen is a natural component of mouse articular cartilage throughout development, growth, and aging. This finding and the deposition of type X collagen at sites of OA lesions suggest that type X collagen may have a role in providing structural support for articular cartilage.

Variability in the upstream promoter and intron sequences of the human, mouse and chick type X collagen genes.

The type X collagen gene is specifically expressed in hypertrophic chondrocytes during endochondral ossification. Transcription of the type X collagen gene by these differentiated cells is turned on at the same time as transcription of several other cartilage specific genes is switched off and before mineralization of the matrix begins. Analysis of type X collagen promoters for regulatory regions in different cell culture systems and in transgenic mice has given contradictory results suggesting major differences among species. To approach this problem, we have determined the nucleotide sequences of the two introns and upstream promoter sequences of the human and mouse type X collagen genes and compared them with those of bovine and chick. Within the promoter regions, we found three boxes of homology which are nearly continuous in the human gene but have interruptions in the murine gene. One of these interruptions was identified as a complex 1.9 kb repetitive element with homology to LINE, B1, B2 and long terminal repeat sequences. Regulatory elements of the human type X collagen gene are located upstream of the region where the repetitive element is inserted in the mouse gene, making it likely that the repetitive element is inserted between the coding region and regulatory sequences of the murine gene without interfering with its expression pattern. We also compared the sequences of the introns of both genes and found strong conservation. Comparisons of the mammalian sequences with promoter and first intron sequences of the chicken type X collagen gene revealed that only the proximal 120 nucleotides of the promoter were conserved, whereas all other sequences displayed no obvious homology to the murine and human sequences.

The mouse collagen X gene: complete nucleotide sequence, exon structure and expression pattern.

Overlapping genomic clones covering the 7.2 kb mouse alpha 1(X) collagen gene, 0.86 kb of promoter and 1.25 kb of 3'-flanking sequences were isolated from two genomic libraries and characterized by nucleotide sequencing. Typical features of the gene include a unique three-exon structure, similar to that in the chick gene, with the entire triple-helical domain of 463 amino acids coded by a single large exon. The highest degree of amino acid and nucleotide sequence conservation was seen in the coding region for the collagenous and C-terminal non-collagenous domains between the mouse and known chick, bovine and human collagen type X sequences. More divergence between the sequences occurred in the N-terminal non-collagenous domain. Similarity between the mammalian collagen X sequences extended into the 3'-untranslated sequence, particularly near the polyadenylation site. The promoter of the mouse collagen X gene was found to contain two TATAA boxes 159 bp apart; primer extension analyses of the transcription start site revealed that both were functional. The promoter has an unusual structure with a very low G + C content of 28% between positions -220 and -1 of the upstream transcription start site. Northern and in situ hybridization analyses confirmed that the expression of the alpha 1(X) collagen gene is restricted to hypertrophic chondrocytes in tissues undergoing endochondral calcification. The detailed sequence information of the gene is useful for studies on the promoter activity of the gene and for generation of transgenic mice.

Specific hybridization probes for mouse alpha 2(IX) and alpha 1(X) collagen mRNAs.

We have used polymerase chain reaction (PCR) technology and available cross-species sequence information to construct cDNA probes for mouse alpha 2(IX) and alpha 1(X) collagen transcripts. Sequencing confirmed the identification of the clones. Northern analysis proved sufficient divergence of the cloned sequences from other collagen transcripts: specific detection of the mouse 2.9 kb alpha 2(IX) and 3.3 kb alpha 1(X) collagen mRNAs was seen under normal hybridization and washing conditions.