Microbiota promotes recruitment and pro-inflammatory response of caecal macrophages during E. tenella infection.

BACKGROUND: Eimeria genus belongs to the apicomplexan parasite phylum and is responsible for coccidiosis, an intestinal disease with a major economic impact on poultry production. Eimeria tenella is one of the most virulent species in chickens. In a previous study, we showed a negative impact of caecal microbiota on the physiopathology of this infection. However, the mechanism by which microbiota leads to the physiopathology remained undetermined. Macrophages play a key role in inflammatory processes and their interaction with the microbiota during E. tenella infection have never been investigated. We therefore examined the impact of microbiota on macrophages during E. tenella infection. Macrophages were monitored in caecal tissues by immunofluorescence staining with KUL01 antibody in non-infected and infected germ-free and conventional chickens. Caecal cells were isolated, stained, analyzed and sorted to examine their gene expression using high-throughput qPCR. RESULTS: We demonstrated that microbiota was essential for caecal macrophage recruitment in E. tenella infection. Furthermore, microbiota promoted a pro-inflammatory transcriptomic profile of macrophages characterized by increased gene expression of NOS2, ACOD1, PTGS2, TNFα, IL1ß, IL6, IL8L1, IL8L2 and CCL20 in infected chickens. Administration of caecal microbiota from conventional chickens to germ-free infected chickens partially restored macrophage recruitment and response. CONCLUSIONS: Taken together, these results suggest that the microbiota enhances the physiopathology of this infection through macrophage recruitment and activation. Consequently, strategies involving modulation of the gut microbiota may lead to attenuation of the macrophage-mediated inflammatory response, thereby limiting the negative clinical outcome of the disease.

Circadian time series proteomics reveals daily dynamics in cartilage physiology.

OBJECTIVE: Cartilage in joints such as the hip and knee experiences repeated phases of heavy loading and low load recovery during the 24-h day/night cycle. Our previous work has shown 24 h rhythmic changes in gene expression at transcript level between night and day in wild type mouse cartilage which is lost in a circadian clock knock-out mouse model. However, it remains unknown to what extent circadian rhythms also regulate protein level gene expression in this matrix rich tissue. METHODS: We investigated daily changes of protein abundance in mouse femoral head articular cartilage by performing a 48-h time-series LC-MS/MS analysis. RESULTS: Out of the 1,177 proteins we identified across all time points, 145 proteins showed rhythmic changes in their abundance within the femoral head cartilage. Among these were molecules that have been implicated in key cartilage functions, including CTGF, MATN1, PAI-1 and PLOD1 & 2. Pathway analysis revealed that protein synthesis, cytoskeleton and glucose metabolism exhibited time-of-day dependent functions. Analysis of published cartilage proteomics datasets revealed that a significant portion of rhythmic proteins were dysregulated in osteoarthritis and/or ageing. CONCLUSIONS: Our circadian proteomics study reveals that articular cartilage is a much more dynamic tissue than previously thought, with chondrocytes driving circadian rhythms not only in gene transcription but also in protein abundance. Our results clearly call for the consideration of circadian timing mechanisms not only in cartilage biology, but also in the pathogenesis, treatment strategies and biomarker detection in osteoarthritis.

Early life stress in mice is a suitable model for Irritable Bowel Syndrome but does not predispose to colitis nor increase susceptibility to enteric infections.

Neonatal period is characterized by an immature intestinal barrier. Scattered evidence suggests that early life stressful events induce long lasting alterations of intestinal homeostasis mimicking Irritable Bowel Syndrome (IBS). Those observations highlighting defect of intestinal barrier by early life stress questioned its potential role as a risk factor for gastrointestinal disorders such as colitis and infections. In this study, we aimed to analyze if maternal separation (MS) in mice mimicks IBS main features. We next addressed whether MS could trigger or exacerbate colitis in genetically predisposed mice and/or enhance susceptibility to gastrointestinal infections in wild type mice. MS induced main features of IBS in adult wild type male mice i.e. intestinal hyperpermeability, visceral hypersensitivity, microbiota dysbiosis, bile acid malabsorption and low grade inflammation in intestine associated with a defect of Paneth cells and the ILC3 population. This breach in mucosal barrier functions in adults was associated with a systemic IgG response against commensal E. coli and increased IFNγ secretion by splenocytes. However, in IL10-/- mice, MS did not trigger nor worsen colitis. Furthermore, wild type mice submitted to MS did not show increase susceptibility to gastrointestinal infections (S. Typhimurium, L. monocytogenes or T. gondii) compared to controls. Altogether, our results identify MS in mice as a good experimental model for IBS mimicking all the main features. In addition, early life stress, even though it has long lasting consequences on intestinal homeostasis, does not constitute a facilitating factor to colitis in predisposed individuals nor to gastrointestinal infections in wild type mice.

A dominant TRPV4 variant underlies osteochondrodysplasia in Scottish fold cats.

OBJECTIVE: Scottish fold cats, named for their unique ear shape, have a dominantly inherited osteochondrodysplasia involving malformation in the distal forelimbs, distal hindlimbs and tail, and progressive joint destruction. This study aimed to identify the gene and the underlying variant responsible for the osteochondrodysplasia. DESIGN: DNA samples from 44 Scottish fold and 54 control cats were genotyped using a feline DNA array and a case-control genome-wide association analysis conducted. The gene encoding a calcium permeable ion channel, transient receptor potential cation channel, subfamily V, member 4 (TRPV4) was identified as a candidate within the associated region and sequenced. Stably transfected HEK293 cells were used to compare wild-type and mutant TRPV4 expression, cell surface localisation and responses to activation with a synthetic agonist GSK1016709A, hypo-osmolarity, and protease-activated receptor 2 stimulation. RESULTS: The dominantly inherited folded ear and osteochondrodysplasia in Scottish fold cats is associated with a p.V342F substitution (c.1024G>T) in TRPV4. The change was not found in 648 unaffected cats. Functional analysis in HEK293 cells showed V342F mutant TRPV4 was poorly expressed at the cell surface compared to wild-type TRPV4 and as a consequence the maximum response to a synthetic agonist was reduced. Mutant TRPV4 channels had a higher basal activity and an increased response to hypotonic conditions. CONCLUSIONS: Access to a naturally-occurring TRPV4 mutation in the Scottish fold cat will allow further functional studies to identify how and why the mutations affect cartilage and bone development.

Diagnosis and etiology of congenital muscular dystrophy.

OBJECTIVE: We aimed to determine the frequency of all known forms of congenital muscular dystrophy (CMD) in a large Australasian cohort. METHODS: We screened 101 patients with CMD with a combination of immunofluorescence, Western blotting, and DNA sequencing to identify disease-associated abnormalities in glycosylated alpha-dystroglycan, collagen VI, laminin alpha2, alpha7-integrin, and selenoprotein. RESULTS: A total of 45% of the CMD cohort were assigned to an immunofluorescent subgroup based on their abnormal staining pattern. Abnormal staining for glycosylated alpha-dystroglycan was present in 25% of patients, and approximately half of these had reduced glycosylated alpha-dystroglycan by Western blot. Sequencing of the FKRP, fukutin, POMGnT1, and POMT1 genes in all patients with abnormal alpha-dystroglycan immunofluorescence identified mutations in one patient for each of these genes and two patients had mutations in POMT2. Twelve percent of patients had abnormalities in collagen VI immunofluorescence, and we identified disease-causing COL6 mutations in eight of nine patients in whom the genes were sequenced. Laminin alpha2 deficiency accounted for only 8% of CMD. alpha7-Integrin staining was absent in 12 of 45 patients studied, and ITGA7 gene mutations were excluded in all of these patients. CONCLUSIONS: We define the distribution of different forms of congenital muscular dystrophy in a large cohort of mixed ethnicity and demonstrate the utility and limitations of current diagnostic techniques.

Molecular analysis for genetic counselling in amelogenesis imperfecta.

OBJECTIVE: To use molecular genetics to establish the mode of inheritance in a family with amelogenesis imperfecta. MATERIALS AND METHODS: The polymerase chain reaction was used to amplify exons of the amelogenin gene on the short arm of the X chromosome. RESULTS: A single base deletion mutation in exon 6 of the amelogenin gene was identified. This mutation was a single base deletion of a cytosine residue - 431delC - in codon 96 of exon 6, introducing a stop codon 30 codons downstream of the mutation in codon 126 of the exon. CONCLUSION: The firm establishment of an X-linked mode of inheritance affects the genetic counselling for this family.

Clinical and radiographic features of a family with autosomal dominant amelogenesis imperfecta with taurodontism.

This paper describes the clinical features of a family of four generations with autosomal dominant amelogenesis imperfecta with taurodontism (ADAIT). Considerable variation in phenotype was seen, both between individuals and within the dentition of some individuals. Many of the adults had received extensive dental restorative work. These findings re-enforce previous observations of variable phenotype in this and other forms of the condition and add to the argument for a revision of methods of classification. This history of this large family draws further attention to the restorative demands of this group of dental anomalies and, by their generous co-operation, will prove an invaluable help in the investigation by molecular genetic techniques of this disfiguring condition.

Biglycan and decorin bind close to the n-terminal region of the collagen VI triple helix.

The binding of native biglycan and decorin to pepsin-extracted collagen VI from human placenta was examined by solid phase assay and by measurement of surface plasmon resonance in the BIAcore(TM)2000 system. Both proteoglycans exhibited a strong affinity for collagen VI with dissociation constants (K(D)) of approximately 30 nm. Removal of the glycosaminoglycan chains by chondroitinase ABC digestion did not significantly affect binding. In coprecipitation experiments, biglycan and decorin bound to collagen VI and equally competed with the other, suggesting that biglycan and decorin bind to the same binding site on collagen VI. This was confirmed by electron microscopy after negative staining of complexes between gold-labeled proteoglycans and collagen VI, demonstrating that both biglycan and decorin bound exclusively to a domain close to the interface between the N terminus of the triple helical region and the following globular domain. In solid phase assay using recombinant collagen VI fragments, it was shown that the alpha2(VI) chain probably plays a role in the interaction.

The N-terminal N5 subdomain of the alpha 3(VI) chain is important for collagen VI microfibril formation.

Collagen VI assembly is unique within the collagen superfamily in that the alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains associate intracellularly to form triple helical monomers, and then dimers and tetramers, which are secreted from the cell. Secreted tetramers associate end-to-end to form the distinctive extracellular microfibrils that are found in virtually all connective tissues. Although the precise protein interactions involved in this process are unknown, the N-terminal globular regions, which are composed of multiple copies of von Willebrand factor type A-like domains, are likely to play a critical role in microfibril formation, because they are exposed at both ends of the tetramers. To explore the role of these subdomains in collagen VI intracellular and extracellular assembly, alpha 3(VI) cDNA expression constructs with sequential N-terminal deletions were stably transfected into SaOS-2 cells, producing cell lines that express alpha 3(VI) chains with N-terminal globular domains containing modules N9-N1, N6-N1, N5-N1, N4-N1, N3-N1, or N1, as well as the complete triple helix and C-terminal globular domain (C1-C5). All of these transfected alpha 3(VI) chains were able to associate with endogenous alpha 1(VI) and alpha 2(VI) to form collagen VI monomers, dimers, and tetramers, which were secreted. Importantly, cells that expressed alpha 3(VI) chains containing the N5 subdomain, alpha 3(VI) N9-C5, N6-C5, and N5-C5, formed microfibrils and deposited a collagen VI matrix. In contrast, cells that expressed the shorter alpha 3(VI) chains, N4-C5, N3-C5, and N1-C5, were severely compromised in their ability to form end-to-end tetramer assemblies and failed to deposit a collagen VI matrix. These data demonstrate that the alpha 3(VI) N5 module is critical for microfibril formation, thus identifying a functional role for a specific type A subdomain in collagen VI assembly.

Procollagen folding and assembly: the role of endoplasmic reticulum enzymes and molecular chaperones.

Procollagen assembly occurs within the endoplasmic reticulum, where the C-propeptide domains of three polypeptide alpha-chains fold individually, and then interact and trimerise to initiate folding of the triple helical region. This highly complex folding and assembly pathway requires the co-ordinated action of a large number of endoplasmic reticulum-resident enzymes and molecular chaperones. Disease-causing mutations in the procollagens disturb folding and assembly and lead to prolonged interactions with molecular chaperones, retention in the endoplasmic reticulum, and intracellular degradation. This review focuses predominantly on prolyl 1-hydroxylase, an essential collagen modifying enzyme, and HSP47, a collagen-specific binding protein, and their proposed roles as molecular chaperones involved in fibrillar procollagen folding and assembly, quality control, and secretion.

Proteasomal degradation of unassembled mutant type I collagen pro-alpha1(I) chains.

We have previously shown that type I procollagen pro-alpha1(I) chains from an osteogenesis imperfecta patient (OI26) with a frameshift mutation resulting in a truncated C-propeptide, have impaired assembly, and are degraded by an endoplasmic reticulum-associated pathway (Lamandé, S. R., Chessler, S. D., Golub, S. B., Byers, P. H., Chan, D., Cole, W. G., Sillence, D. O. and Bateman, J. F. (1995) J. Biol. Chem. 270, 8642-8649). To further explore the degradation of procollagen chains with mutant C-propeptides, mouse Mov13 cells, which produce no endogenous pro-alpha1(I), were stably transfected with a pro-alpha1(I) expression construct containing a frameshift mutation that predicts the synthesis of a protein 85 residues longer than normal. Despite high levels of mutant mRNA in transfected Mov13 cells, only minute amounts of mutant pro-alpha1(I) could be detected indicating that the majority of the mutant pro-alpha1(I) chains synthesized are targeted for rapid intracellular degradation. Degradation was not prevented by brefeldin A, monensin, or NH(4)Cl, agents that interfere with intracellular transport or lysosomal function. However, mutant pro-alpha1(I) chains in both transfected Mov13 cells and OI26 cells were protected from proteolysis by specific proteasome inhibitors. Together these data demonstrate for the first time that procollagen chains containing C-propeptide mutations that impair assembly are degraded by the cytoplasmic proteasome complex, and that the previously identified endoplasmic reticulum-associated degradation of mutant pro-alpha1(I) in OI26 is mediated by proteasomes.

Bethlem myopathy and engineered collagen VI triple helical deletions prevent intracellular multimer assembly and protein secretion.

Mutations in the genes that code for collagen VI subunits, COL6A1, COL6A2, and COL6A3, are the cause of the autosomal dominant disorder, Bethlem myopathy. Although three different collagen VI structural mutations have previously been reported, the effect of these mutations on collagen VI assembly, structure, and function is currently unknown. We have characterized a new Bethlem myopathy mutation that results in skipping of COL6A1 exon 14 during pre-mRNA splicing and the deletion of 18 amino acids from the triple helical domain of the alpha1(VI) chain. Sequencing of genomic DNA identified a G to A transition in the +1 position of the splice donor site of intron 14 in one allele. The mutant alpha1(VI) chains associated intracellularly with alpha2(VI) and alpha3(VI) to form disulfide-bonded monomers, but further assembly into dimers and tetramers was prevented, and molecules containing the mutant chain were not secreted. This triple helical deletion thus resulted in production of half the normal amount of collagen VI. To further explore the biosynthetic consequences of collagen VI triple helical deletions, an alpha3(VI) cDNA expression construct containing a 202-amino acid deletion within the triple helix was produced and stably expressed in SaOS-2 cells. The transfected mutant alpha3(VI) chains associated with endogenous alpha1(VI) and alpha2(VI) to form collagen VI monomers, but dimers and tetramers did not form and the mutant-containing molecules were not secreted. Thus, deletions within the triple helical region of both the alpha1(VI) and alpha3(VI) chains can prevent intracellular dimer and tetramer assembly and secretion. These results provide the first evidence of the biosynthetic consequences of structural collagen VI mutations and suggest that functional protein haploinsufficiency may be a common pathogenic mechanism in Bethlem myopathy.

Reliable and sensitive detection of premature termination mutations using a protein truncation test designed to overcome problems of nonsense-mediated mRNA instability.

The protein truncation test (PTT) is a mutation-detection method used to scan for premature termination (nonsense) mutations. PCR amplification of the DNA or mRNA source material is performed using forward primers containing a T7-promoter sequence and translation initiation signals such that the resultant products can be transcribed and translated in vitro to identify the smaller truncated protein products. mRNA is commonly used as the source material, but success of the PTT and other RNA-based mutation detection methods can be severely compromised by nonsense mutation-induced mRNA decay, a well-documented process that is often overlooked in mutation detection strategies. In this study, we develop an RNA-based PTT that overcomes the problem of mRNA decay by preincubating cells with cycloheximide to stabilise the mutant mRNA. The effectiveness of this method for mutation detection in abundant mRNAs was demonstrated in osteogenesis imperfecta fibroblasts by the protection of type I collagen (COL1A1) mRNA containing nonsense mutations that normally resulted in mutant mRNA degradation. Stabilisation of mutant mismatch repair gene (MLH1) mRNA was also observed in transformed lymphocytes from patients with hereditary nonpolyposis colorectal cancer (HNPCC). Importantly, our strategy also stabilised very low-level (or illegitimate) nonsense-containing transcripts in lymphoblasts from patients with Bethlem myopathy (COL6A1), familial adenomatous polyposis (APC), and breast cancer (BRCA1). The greatly increased sensitivity and reliability of this RT-PCR/PTT protocol has broad applicability to the many genetic diseases in which only blood-derived cells may be readily available for analysis.

Reduced collagen VI causes Bethlem myopathy: a heterozygous COL6A1 nonsense mutation results in mRNA decay and functional haploinsufficiency.

We have identified a new pathogenic mechanism for an inherited muscular dystrophy in which functional haploinsufficiency of the extracellular matrix protein collagen VI causes Bethlem myopathy. The heterozygous COL6A1 mutation results in a single base deletion from the mRNA and a premature stop codon. The mutant mRNA is unstable, subject to nonsense-mediated mRNA decay, and is almost completely absent both from patient fibroblasts and skeletal muscle, resulting in haploinsufficiency of the alpha1(VI) subunit and reduced production of structurally normal collagen VI. This is the first example of a muscular dystrophy caused by haploinsufficiency of a structural protein or member of the dystrophin-glycoprotein complex, and identifies collagen VI as a critical contributor to cell-matrix adhesion in skeletal muscle.

The role of the alpha3(VI) chain in collagen VI assembly. Expression of an alpha3(VI) chain lacking N-terminal modules N10-N7 restores collagen VI assembly, secretion, and matrix deposition in an alpha3(VI)-deficient cell line.

Collagen VI is a microfibrillar protein found in the extracellular matrix of virtually all connective tissues. Three genetically distinct subunits, the alpha1(VI), alpha2(VI), and alpha3(VI) chains, associate intracellularly to form triple-helical monomers, which then assemble into disulfide-bonded dimers and tetramers before secretion. Although sequence considerations suggest that collagen VI monomers composed of all three chains are the most stable isoform, the precise chain composition of collagen VI remains controversial and alternative assemblies containing only alpha1(VI) and alpha2(VI) chains have also been proposed. To address this question directly and study the role of the alpha3(VI) chain in assembly, we have characterized collagen VI biosynthesis and in vitro matrix formation by a human osteosarcoma cell line (SaOS-2) that is deficient in alpha3(VI) production. Northern analysis showed an abundance of alpha1(VI) and alpha2(VI) mRNAs, but no detectable alpha3(VI) mRNA was apparent in SaOS-2 cells. By day 30 of culture, however, small amounts of alpha3(VI) mRNA were detected, although the level of expression was still much less than alpha1(VI) and alpha2(VI). Collagen VI protein was not detected in SaOS-2 medium or cell layer samples until day 30 of culture, demonstrating that despite the abundant synthesis of alpha1(VI) and alpha2(VI), no stable collagen VI protein was produced without expression of alpha3(VI). The alpha1(VI) and alpha2(VI) chains produced in the absence of alpha3(VI) were non-helical and were largely retained intracellularly and degraded. The critical role of the alpha3(VI) chain in collagen VI assembly was directly demonstrated after stable transfection of SaOS-2 cells with an alpha3(VI) cDNA expression construct that lacked 4 of the 10 N-terminal type A subdomains. The transfected alpha3(VI) N6-C5 chains associated with endogenous alpha1(VI) and alpha2(VI) and formed collagen VI dimers and tetramers, which were secreted and deposited into an extensive network in the extracellular matrix. These data demonstrated that alpha3(VI) is essential for the formation of stable collagen VI molecules and subdomains N10-N7 are not required for molecular assembly.

In vitro expression analysis of collagen biosynthesis and assembly.

While the generalised pathway of collagen biosynthesis is well understood, the specific molecular interactions that drive chain recognition and assembly and the formation of tissue-specific extracellular supramolecular structures have not been elucidated. This review focuses on the use of in vitro collagen expression systems to explore some of these fundamental questions on the molecular basis of normal and mutant collagen assembly. Three in vitro expression/assembly systems are discussed. Firstly, a simple cell-free transcription/translation system to study the initial stages of collagen chain assembly. Secondly, a novel T7-driven high level expression system, using a recombinant vaccinia virus expressing T7 RNA polymerase, in transiently transfected cells which allows appropriate postranslational modification and collagen folding. Thirdly, the more complex questions of normal and mutant collagen extracellular matrix assembly are addressed by stable transfection and expression in cells which allow the formation of a 'tissue equivalent' matrix during long-term culture.

The type I collagen pro alpha 1(I) COOH-terminal propeptide N-linked oligosaccharide. Functional analysis by site-directed mutagenesis.

The C-propeptides of the pro alpha 1(I) and pro alpha 2(I) chains of type I collagen are each substituted with a single high-mannose N-linked oligosaccharide. Conservation of this motif among the fibrillar collagens has led to the proposal that the oligosaccharide has structural or functional importance, but a role in collagen biosynthesis has not been unambiguously defined. To examine directly the function of the pro alpha 1(I) C-propeptide N-linked oligosaccharide, the acceptor Asn residue was changed to Gln by site-directed mutagenesis. In transfected mouse Mov13 and 3T6 cells, unglycosylated mutant pro alpha 1(I) folded and assembled normally into trimeric molecules with pro alpha 2(I). In biosynthetic pulse-chase experiments mutant pro alpha 1(I) were secreted at the same rate as wild-type chains; however, following secretion, the chains were partitioned differently between the cell layer and medium, with a greater proportion of the mutant pro alpha 1(I) being released into the medium. This distribution difference was not eliminated by the inclusion of yeast mannan indicating that the high-mannose oligosaccharide itself was not binding to the matrix or the fibroblast surface after secretion. Subtle alterations in the tertiary structure of unglycosylated C-propeptides may have decreased their affinity for a cell-surface component. Further support for a small conformational change in the mutant C-propeptides came from experiments suggesting that unglycosylated pro alpha 1(I) chains were cleaved in vitro by the purified C-proteinase slightly less efficiently than wild-type chains. Mutant and normal pro alpha 1(I) were deposited with equal efficiency into the 3T6 cell accumulated matrix, thus the reduced cleavage by C-proteinase and altered distribution in the short pulse-chase experiments were not functionally significant in this in vitro extracellular matrix model system.

Endoplasmic reticulum-mediated quality control of type I collagen production by cells from osteogenesis imperfecta patients with mutations in the pro alpha 1 (I) chain carboxyl-terminal propeptide which impair subunit assembly.

A heterozygous single base change in exon 49 of COL1A1, which converted the codon for pro alpha 1(I) carboxyl-terminal propeptide residue 94 from tryptophan (TGG) to cysteine (TGT) was identified in a baby with lethal osteogenesis imperfecta (OI64). The C-propeptide mutations in OI64 and in another lethal osteogenesis imperfecta cell strain (OI26), which has a frameshift mutation altering the sequence of the carboxyl-terminal half of the propeptide (Bateman, J. F., Lamande, S. R., Dahl, H.-H. M., Chan, D., Mascara, T. and Cole, W. G. (1989) J. Biol. Chem. 264, 10960-10964), disturbed procollagen folding and retarded the formation of disulfide-linked trimers. Although assembly was delayed, the presence of slowly migrating, overmodified alpha 1(I) and alpha 2(I) chains indicated that mutant pro alpha 1(I) could associate with normal pro alpha 1(I) and pro alpha 2(I) to form pepsin-resistant triple-helical molecules, a proportion of which were secreted. Further evidence of the aberrant folding of mutant procollagen in OI64 and OI26 was provided by experiments demonstrating that the endoplasmic reticulum resident molecular chaperone BiP, which binds to malfolded proteins, was specifically bound to type I procollagen and was coimmunoprecipitated in the osteogenesis imperfecta cells but not control cells. Experiments with brefeldin A, which inhibits protein export from the endoplasmic reticulum, demonstrated that unassembled mutant pro alpha 1(I) chains were selectively degraded within the endoplasmic reticulum resulting in reduced collagen production by the osteogenesis imperfecta cells. This biosynthetic deficiency was reflected in the inability of OI64 and OI26 cells to produce a substantial in vitro collagenous matrix when grown in the continuous presence of ascorbic acid to allow collagen matrix formation. Both these carboxyl-terminal propeptide mutants showed a marked reduction in collagen accumulation to 20% (or less) of control cultures, comparable to the reduced collagen content of tissues from OI26.

Genomic sequence of mouse COL1A1 encoding the collagen propeptides.

The nucleotide sequences of the mouse pro alpha 1(I) gene regions coding for the N- and C-propeptides is reported. The exon-intron structure was highly homologous to human COL1A1 and the deduced amino acid sequences of the N- and C-propeptides showed 67% and 91% identity with the human sequence. This gene sequence information will allow the production of specific gene mutations by site-directed mutagenesis to study the structure and function of these important propeptide domains.