Complete structure of the human COL11A2 gene: the exon sizes and other features indicate the gene has not evolved with genes for other fibriller collagens.
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Animals↗
Biomedical subjects
P Vandenberg
Publications and source records attributed to P Vandenberg.
The mouse col11a2 gene. Some transcripts from the adjacent rxr-beta gene extend into the col11a2 gene.
Type XI collagen is present in small amounts in cartilage, together with small amounts of type IX and type V collagens and large amounts of type II collagen. Here, primers based on the nucleotide sequences of partial human cDNAs and mouse genomic DNAs that were analyzed by other investigators were used to isolate a cDNA for the mouse col11a2 gene. Cosmid clones for the mouse col11a2 gene were isolated, and 12.4 kb of the nucleotide sequences were defined. Analysis of the genomic sequences identified three exons in the mouse gene that were recently shown to undergo alternative splicing (Tsumaki and Kimura, J. Biol. Chem. 270, 2372-2378, 1995; Zhidkova et al., J. Biol. Chem. 270, 94886-9493, 1995). In addition, analysis of the cosmid clones revealed that the 5' end of the mouse col11a2 gene was located head-to-tail with the mouse retinoic X receptor beta gene. RT-PCR assays demonstrated that some transcripts from the retinoic X receptor beta gene extend into the col11a2 gene. Therefore, there may be coordinate expression of the two genes.
Animals↗
The human COL11A2 gene structure indicates that the gene has not evolved with the genes for the major fibrillar collagens.
The human COL11A2 gene was analyzed from two overlapping cosmid clones that were previously isolated in the course of searching the human major histocompatibility region (Janatipour, M., Naumov, Y., Ando, A., Sugimura, K., Okamoto, N., Tsuji, K., Abe, K., and Inoko, H. (1992) Immunogenetics 35, 272-278). Nucleotide sequencing defined over 28,000 base pairs of the gene. It was shown to contain 66 exons. As with most genes for fibrillar collagens, the first intron was among the largest, and the introns at the 5'-end of the gene were in general larger than the introns at the 3'-end. Analysis of the exons coding for the major triple helical domain indicated that the gene structure had not evolved with the genes for the major fibrillar collagens in that there were marked differences in the number of exons, the exon sizes, and codon usage. The gene was located close to the gene for the retinoic X receptor beta in a head-to-tail arrangement similar to that previously seen with the two mouse genes (P. Vandenberg and D. J. Prockop, submitted for publication). Also, there was marked interspecies homology in the intergenic sequences. The amino acid sequences and the pattern of charged amino acids in the major triple helix of the alpha 2(XI) chain suggested that the chain can be incorporated into the same molecule as alpha 1(XI) and alpha 1(V) chains but not into the same molecule as the alpha 3(XI)/alpha 1(II) chain. The structure of the carboxyl-terminal propeptide was similar to the carboxyl-terminal propeptides of the pro alpha 1(XI) chain and pro alpha chains of other fibrillar collagens, but it was shorter because of internal deletions of about 30 amino acids.
Amino Acid Sequence↗
Mutations in type 1 procollagen that cause osteogenesis imperfecta: effects of the mutations on the assembly of collagen into fibrils, the basis of phenotypic variations, and potential antisense therapies.
Work by a large number of investigators over the last decade has established that over 90% of patients with osteogenesis imperfecta have mutations in one of the two genes for type I procollagen, that most unrelated probands have different mutations in the genes, and that the mutations found in most of the serious variants of the disease cause synthesis of abnormal pro alpha chains of the protein. The results have demonstrated that synthesis of structurally abnormal but partially functional pro alpha chains can interfere with folding of the central region of the protein into a triple-helical conformation, prevent processing of the N-terminal propeptides of procollagen, or produce subtle alterations in conformation that interfere with the self-assembly of the protein into collagen fibrils. One of the unsolved mysteries about the disease is why some mutations produce severe phenotypes, whereas very similar mutations produce mild phenotypes. Recent studies in transgenic mice suggest that nongenetic factors, such as stochastic events during development, may determine the severity of the disease phenotype produced by a specific mutation. Also, recent results raised the possibility that strategies of antisense gene therapy may be effective in treating the disease some time in the future. Specific inhibition of expression of a mutated collagen gene has been obtained with antisense oligonucleotides in cell culture experiments. However, there is no means of selective delivery of antisense oligonucleotides to the appropriate tissues.
Cells, Cultured↗
Molecular basis of heritable connective tissue disease.
The family of collagens represents a series of highly vulnerable gene-protein systems. This can be explained by the fact that both the folding of the pro alpha chains and the assembly of collagen monomers into fibrils highly depend on the principle of nucleated growth, in which every subunit of the system must have the correct structure. DNA analysis showed that over 90% of patients with OI have mutations in one of the two structural genes for type I procollagen, that many patients with severe chondrodysplasias apparently have mutations in the gene for type II procollagen, and that most patients with the potentially lethal type IV variant of Ehlers-Danlos syndrome have mutations in the gene for type III procollagen. The incidence of such genetic diseases varies from 1:100,000 births for lethal forms of OI to 1:25,000 births for mild forms (48). Mild chondrodysplasias such as the Stickler syndrome may have an incidence of 1:10,000 births. It is the subject of current investigation whether some mutations in the genes for type I, II, and III procollagen can also cause some of the common diseases of later onset, such as osteoporosis, osteoarthritis, or familial aneurysms. These genes have been demonstrated to be mutated in at least some subsets, and further analysis of the exceptionally large genes for most collagens is underway to resolve these questions. Rapid DNA analysis techniques, which are developed independently in several laboratories and in a concerted effort through the human genome project, will soon make it possible to screen a population for genetic defects and identify people at risk for developing connective tissue disease. As vulnerable as the collagen gene-protein system might be, the multimeric collagens may prove nevertheless to be accessible to gene therapy, as the suppression of defective alleles, e.g., through antisense strategies, may be much easier to accomplish than the gene augmentation necessary to correct other genetically determined diseases.
Collagen↗
An inbred line of transgenic mice expressing an internally deleted gene for type II procollagen (COL2A1). Young mice have a variable phenotype of a chondrodysplasia and older mice have osteoarthritic changes in joints.
Studies were carried out on a line of transgenic mice that expressed an internally deleted COL2A1 gene and developed a phenotype resembling human chondrodysplasias (Vandenberg et al. 1991. Proc. Natl. Acad. Sci. USA. 88:7640-7644. Marked differences in phenotype were observed with propagation of the mutated gene in an inbred strain of mice in that approximately 15% of the transgenic mice had a cleft palate and a lethal phenotype, whereas the remaining mice were difficult to distinguish from normal littermates. 1-d- and 3-mo-old transgenic mice that were viable showed microscopic signs of chondrodysplasia with reduced amounts of collagen fibrils in the cartilage matrix, dilatation of the rough surfaced endoplasmic reticulum in the chondrocytes, and decrease of optical path difference in polarized light microscopy. The transgenic mice also showed signs of disturbed growth as evidenced by lower body weight, lower length and weight of the femur, decreased bone collagen, decreased bone mineral, and decreased resistance of bone to breakage. Comparisons of mice ranging in age from 1 d to 15 mo demonstrated that there was decreasing evidence of a chondrodysplasia as the mice grew older. Instead, the most striking feature in the 15-mo-old mice were degenerative changes of articular cartilage similar to osteoarthritis.
Aging↗
The necessity of mission integration. A system develops processes to weave values into the life of the organization.
Essential to the future vitality and viability of a mission-driven organization is the integration of the mission into the organization's programs, policies, practices, and accountability. Holy Cross Health System (HCHS), South Bend, IN, launched an intensive educational effort with managers, staff members, and trustees to reinforce the basic belief that mission permeates all departments. Using the mission statements principles of fidelity, excellence, empowerment, and stewardship, HCHS leaders initiated a systemwide mission assessment and development effort. During assessment, each facilities' ad hoc team addressed and responded to the organization's mission standards on the basis of availability of personnel, size, facility, and particular circumstances. The assessment process called for interdisciplinary, institutional review teams to explore all aspects of mission activity. This process enabled HCHS to launch a systemwide educational effort about the importance and necessity of mission integration. HCHS then used the mission statement elements fidelity, excellence, empowerment, and stewardship to define new relationships of accountability.
Budgets↗
A better way to evaluate hospital capital expenditures.
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Accounting↗
Expression of a partially deleted gene of human type II procollagen (COL2A1) in transgenic mice produces a chondrodysplasia.
A minigene version of the human gene for type II procollagen (COL2A1) was prepared that lacked a large central region containing 12 of the 52 exons and therefore 291 of the 1523 codons of the gene. The construct was modeled after sporadic in-frame deletions of collagen genes that cause synthesis of shortened pro alpha chains that associate with normal pro alpha chains and thereby cause degradation of the shortened and normal pro alpha chains through a process called procollagen suicide. The gene construct was used to prepare five lines of transgenic mice expressing the minigene. A large proportion of the mice expressing the minigene developed a phenotype of a chondrodysplasia with dwarfism, short and thick limbs, a short snout, a cranial bulge, a cleft palate, and delayed mineralization of bone. A number of mice died shortly after birth. Microscopic examination of cartilage revealed decreased density and organization of collagen fibrils. In cultured chondrocytes from the transgenic mice, the minigene was expressed as shortened pro alpha 1(II) chains that were disulfide-linked to normal mouse pro alpha 1(II) chains. Therefore, the phenotype is probably explained by depletion of the endogenous mouse type II procollagen through the phenomenon of procollagen suicide.
Animals↗
Characterization of a type IV collagen major cell binding site with affinity to the alpha 1 beta 1 and the alpha 2 beta 1 integrins.
The aim of this investigation was to identify the domains of type IV collagen participating in cell binding and the cell surface receptor involved. A major cell binding site was found in the trimeric cyanogen bromide-derived fragment CB3, located 100 nm away from the NH2 terminus of the molecule, in which the triple-helical conformation is stabilized by interchain disulfide bridges. Cell attachment assays with type IV collagen and CB3 revealed comparable cell binding activities. Antibodies against CB3 inhibited attachment on fragment CB3 completely and on type IV collagen to 80%. The ability to bind cells was strictly conformation dependent. Four trypsin derived fragments of CB3 allowed a closer investigation of the binding site. The smallest, fully active triple-helical fragment was (150)3-amino acid residues long. It contained segments of 27 and 37 residues, respectively, at the NH2 and COOH terminus, which proved to be essential for cell binding. By affinity chromatography on Sepharose-immobilized CB3, two receptor molecules of the integrin family, alpha 1 beta 1 and alpha 2 beta 1, were isolated. Their subunits were identified by sequencing the NH2 termini or by immunoblotting. The availability of fragment CB3 will allow for a more in-depth study of the molecular interaction of a short, well defined triple-helical ligand with collagen receptors alpha 1 beta 1 and alpha 2 beta 1.
Amino Acid Sequence↗
Incremental change: a system reaction.
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Catchment Area, Health↗
Product yield in oxygenation of linoleate by soybean lipoxygenase: the value of the molar extinction coefficient in the spectrophotometric assay.
Two iodimetric methods and a gravimetric method were used to determine the spectrophotometric molar absorptivity of the purified product of lipoxygenase-catalyzed dioxygenation of linoleate (13-LS-hydroperoxy-cis,trans-9,11-octadecadienoate). Earlier determinations had led to the use of values varying from 24,000 to 28,000 M-1 cm-1 for epsilon at 235 nm. In the current work, the two iodimetric values (spectrophotometric and titrimetric) average 22,500, while gravimetric analysis of scrupulously purified material gives 22,900. Final 235-nm absorbancies for lipoxygenase runs over a wide range of linoleic acid concentrations up to 200 microM give a constant final percentage completion. If one assumes a 100% reaction, epsilon is 23,600. Each method has less than 1.5% standard error; the average of the three independent methods is 23,000 +/- 580 (2.5%), all being lower than the previous values. In the enzyme-catalyzed reaction of linoleate at less than 200 microM substrate, only 235-nm-absorbing material is formed. Above 200 microM linoleate, yields at 235 nm decrease and yields of materials absorbing at 280 nm increase (the latter is known to arise from lipoxygenase-catalyzed reaction of linoleyl hydroperoxide). Below 200 microM substrate, linoleate purified by HPLC produces only one HPLC-observable product, 13-linoleyl hydroperoxide. At higher substrate concentrations other HPLC peaks arise, again with higher wave-length absorptions. Spectrophotometric data using the epsilon determined here agree with those from the O2 electrode. It is concluded that at S less than 200 microM, saturating air, and sufficient enzyme, soybean lipoxygenase-1 produces a sole product and the reaction proceeds to completion.
Kinetics↗