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The complete sequence of the cDNA for the pro alpha 1 (I) chain of mouse type I procollagen is presented. The encoded amino acid sequence shows 96% identity to the human pro alpha 1 (I) collagen chain.

Over 11,000 bp of previously undefined sequences of the human COL2A1 gene were defined. The results made it possible to compare the intron structures of a highly complex gene from man and mouse. Surprisingly, the sizes of the 53 introns of the two genes were highly conserved with a mean difference of 13%. After alignment of the sequences, 69% of the intron sequences were identical. The introns contained consensus sequences for the binding of over 100 different transcription factors that were conserved in the introns of the two genes. The first intron of the gene contained 80 conserved consensus sequences and the remaining 52 introns of the gene contained 106 conserved sequences for the binding of transcription factors. The 5'-end of intron 2 in both genes had a potential for forming a stem loop in RNA transcripts.

Cells from transgenic mice expressing a human mini-gene for collagen I were used as markers to follow the fate of mesenchymal precursor cells from marrow that were partially enriched by adherence to plastic, expanded in culture, and then injected into irradiated mice. Sensitive PCR assays for the marker collagen I gene indicated that few of the donor cells were present in the recipient mice after 1 week, but 1-5 months later, the donor cells accounted for 1.5-12% of the cells in bone, cartilage, and lung in addition to marrow and spleen. A PCR in situ assay on lung indicated that the donor cells diffusely populated the parenchyma, and reverse transcription-PCR assays indicated that the marker collagen I gene was expressed in a tissue-specific manner. The results, therefore, demonstrated that mesenchymal precursor cells from marrow that are expanded in culture can serve as long-lasting precursors for mesenchymal cells in bone, cartilage, and lung. They suggest that cells may be particularly attractive targets for gene therapy ex vivo.

Collagen fibrils resemble smectic, liquid crystals in being highly ordered axially but relatively disordered laterally. In some connective tissues, x-ray diffraction reveals three-dimensional crystallinity in the molecular packing within fibrils, although the continued presence of diffuse scatter indicates significant underlying disorder. In addition, several observations from electron microscopy suggest that the molecular packing is organized concentrically about the fibril core. In the present work, theoretical equatorial x-ray diffraction patterns for a number of models for collagen molecular packing are calculated and compared with the experimental data from tendon fibrils. None of the models suggested previously can account for both the crystalline Bragg peaks and the underlying diffuse scatter. In addition, models in which any of the nearest-neighbor, intermolecular vectors are perpendicular to the radial direction are inconsistent with the observed radial orientation of the principal approximately 4 nm Bragg spacing. Both multiple-start spiral and concentric ring models are devised in which one of the nearest-neighbor vectors is along the radial direction. These models are consistent with the radial orientation of the approximately 4 nm spacing, and energy minimization results in radially oriented crystalline domains separated by disordered grain boundaries. Theoretical x-ray diffraction patterns show a combination of sharp Bragg peaks and underlying diffuse scatter. Close agreement with the observed equatorial diffraction pattern is obtained. The concentric ring model is consistent with the observation that the diameters of collagen fibrils are restricted to discrete values.

Two different mutations were found in two unrelated probands with lethal chondrodysplasias, one with achondrogenesis type II and the other with the less severe phenotype of hypochondrogenesis. The mutations in the COL2A1 gene were identified by denaturing gradient gel electrophoresis analysis of genomic DNA followed by dideoxynucleotide sequencing and restriction site analysis. The proband with achondrogenesis type II had a heterozygous single-base mutation that substituted aspartate for glycine at position 310 of the alpha 1(II) chain of type II procollagen. The proband with hypochondrogenesis had a heterozygous single-base mutation that substituted serine for glycine at position 805. Type II collagen extracted from cartilage from the probands demonstrated the presence of type I collagen and a delayed electrophoretic mobility, indicating post-translational overmodifications. Analysis of CNBr peptides showed that, in proband 1, the entire peptides were overmodified. Examination of chondrocytes cultured in agarose or alginate indicated that there was a delayed secretion of type II procollagen. In addition, type II collagen synthesized by cartilage fragments from the probands demonstrated a decreased thermal stability. The melting temperature of the type II collagen containing the aspartate-for-glycine substitution was reduced by 4 degrees C, and that of the collagen containing the serine-for-glycine substitution was reduced by 2 degrees C. Electron microscopy of the extracellular matrix from the chondrocyte cultures showed a decreased density of matrix and the presence of unusually short and thin fibrils. Our results indicate that glycine substitutions in the N-terminal region of the type II collagen molecule can produce more severe phenotypes than mutations in the C-terminal region. The aspartate-for-glycine substitution at position 310, which was associated with defective secretion and a probable increased degradation of collagen, is the most destabilizing mutation yet reported in type II procollagen.

Inconsistent data have been reported on the size of the promoter that is necessary for high levels of tissue-specific expression of the COL1A1 gene for type I procollagen. Some of the inconsistencies may be traced to the use of reporter gene constructs. Therefore, we prepared transgenic mice with modifications of the intact gene engineered so that the level of expression of the transgene could be assayed both as mRNA and protein that were similar to the products from the endogenous COL1A1 gene. The results with a mini-COL1A1 gene lacking 41 internal exons and introns indicated that the first intron and 90% of the 3'-untranslated region were not essential for tissue-specific expression. In a hybrid COL1A1/COL2A1 construct, a 1.9-kilobase 5'-fragment from the COL1A1 gene that contained only 476 of the promoter was linked to a promoterless 29.5-kilobase fragment of the human COL2A1 gene for type II procollagen. The hybrid COL1A1/COL2A1 construct was expressed as both mRNA and protein in tissues that normally synthesize type I procollagen but not type II procollagen. Apparently, 476 base pairs of the promoter are sufficient to drive tissue-specific expression of the COL1A1 gene and totally inappropriate expression of the COL2A1 gene.

The collagen superfamily of proteins now contains at least 19 proteins formally defined as collagens and an additional ten proteins that have collagen-like domains. The most abundant collagens form extracellular fibrils or network-like structures, but the others fulfill a variety of biological functions. Some of the eight highly specific post-translational enzymes involved in collagen biosynthesis have recently been cloned. Over 400 mutations in 6 different collagens cause a variety of human diseases that include osteogenesis imperfecta, chondrodysplasias, some forms of osteoporosis, some forms of osteoarthritis, and the renal disease known as the Alport syndrome. Many of the disease phenotypes have been produced in transgenic mice with mutated collagen genes. There has been increasing interest in the possibility that the unique post-translational enzymes involved in collagen biosynthesis offer attractive targets for specifically inhibiting excessive fibrotic reactions in a number of diseases. A number of experiments suggest it may be possible to inhibit collagen synthesis with oligo-nucleotides or antisense genes.

The enzymes procollagen C- and N-proteinases specifically cleave carboxyl- and amino-terminal propeptides of procollagens. After cleavage of the propeptides, the resulting collagens self-assemble into fibrils. In most previous experiments with the enzymes, the substrate was monomeric type I procollagen. Here we have prepared aggregates of type I procollagen from chick embryo tendons by using 1 to 100 micrograms/ml of 500-kDa dextran sulfate or 3 to 5% (w/v) polyethylene glycol (M(r) 3350). Aggregation of the substrate with dextran sulfate increased its rate of cleavage by purified or crude C-proteinase from chick embryo tendons 10- to 15-fold. Aggregation of the substrate with 25 to 100 microgram/ml of dextran sulfate increased the rate of cleavage by purified N-proteinase about 4-fold. The rate of cleavage by crude N-proteinase was enhanced only about 2-fold, apparently because of partial precipitation of the enzyme by dextran sulfate. Using polyethylene glycol to aggregate the substrate increased the rate of cleavage by procollagen C-proteinases 5- to 20-fold. Aggregation with polyethylene glycol also increased the rate of cleavage by purified procollagen N-proteinases 2- to 5-fold. With crude N-proteinase, the rate of cleavage was increased only 1.5-fold. The results suggest that the rate of cleavage of the substrate by both enzymes is increased by the aggregation of the substrate itself by dextran sulfate or polyethylene glycol. The increased rates of cleavage seen after aggregation of substrate can be used to develop more sensitive assays for the enzymic activities.

A line of transgenic mice have been investigated that expressed moderate levels of an internally deleted human gene for the pro alpha (I) chain of type I procollagen. These mice expressed the gene at approximately 50% that of the endogenous gene. The gene construct was modeled after a sporadic in-frame deletion of the human gene that produced a lethal variant of osteogenesis imperfecta by causing biosynthesis of shortened pro alpha (I) chains. Periera et al. (1993) reported extensive fracturing in these mice with femurs that were shorter in length and bone that had decreased ash weight, mineral, and collagen content. These workers demonstrated an increased brittleness in bone using biomechanical measurements. The functional consequences of these mutant genes were examined in both transgenic and in normal littermate mice to determine if a valid model at the ultrastructural and analytical level had been produced for OI. X-ray microanalysis of bone mineral demonstrated a significantly lower calcium-to-phosphorus (Ca/P) molar ratio in transgenic mouse bone than in normal littermates; this was a feature of human OI bone. Fourier transform infrared spectroscopy confirmed that the mineral present was apatitic in nature despite the lower Ca/P molar ratio. Alizarin red skeletal staining showed the presence of multiple fracture calluses on the ribs and on the long bones of some of the transgenic mice, this was not seen on normal littermates. No light microscopic differences were observed between normal and transgenic mice; however, many ultrastructural correlates with human OI were observed in the transmission electron microscope. Anomalous fibrils associated with type I collagen, and an amorphous calcified material was observed lining the cartilage, extending beyond the lamina limitans in young transgenic mice.

Recently two techniques have been reported which use arbitrarily primed RT-PCR amplification of cDNA fragments from subsets of mRNAs to detect cDNA fragments from differentially expressed mRNAs. Here we report a simple and rapid PCR-based protocol to both detect and isolate cDNA fragments of up to 3000 base pairs from differentially expressed genes in two easy steps. To generate cDNAs from most mRNAs, the first step consisted of reverse transcription using a fully degenerated 6-mer oligonucleotide as primer. The second step consisted of PCR amplification of internal regions of the cDNAs with two or three longer primers with arbitrary but defined sequences. DNA fragments were easily displayed by agarose gel electrophoresis and then excised for direct use in cloning, sequencing, and Northern blot analysis. By repeating the PCR amplification (second step) on the same cDNA templates (first step) ten times with different sets of primers, over 170 discrete cDNA fragments were obtained from a single tissue. By combining the two-step procedure with 3'-RNA-anchored cDNA extension, additional DNA fragments can be generated from the same mRNA. The new procedure was used here to define 3600 bp of a new brain-specific mRNA.

A series of antisense oligonucleotides (ASOs) were synthesized and tested to define the best target sites within an RNA transcript of collagen for effective inhibition of expression. The test system consisted of mouse NIH 3T3 fibroblasts that were stably transfected with a human minigene for procollagen I so that the cells simultaneously synthesized full-length mouse pro alpha 1 (I) chains and internally deleted human pro alpha 1 (I) chains. The sequences of the transcripts from both genes were compared, and a series of 28 ASOs were designed to target sites in which there were at least two base differences within a 20-nucleotide sequence between the human and mouse transcripts. Six of the ASOs specifically decreased the levels of pro alpha 1 (I) chain synthesized from the human gene without a decrease in the levels of pro alpha 1 (I) chains from the mouse endogenous gene. The most effective ASOs reduced the intracellular levels of human pro alpha 1 (I) chains relative to the mouse pro alpha 1 (I) chains to 37-67% of the control values. Combined addition of two effective ASOs or a second administration of the same effective ASO did not produce any additive effect. The results did not support previous suggestions that the best target sites for ASOs were sequences around initiation codons for translation, at intron-exon boundaries, or in single-stranded loops in hairpin structures. Also, the results did not support previous suggestions that the most effective ASOs are those with the highest affinities for their target sequences. Instead, the most consistent pattern in the data was that the most effective ASOs were those targeted to sequences that were predicted to form clustered double-stranded structures in RNA transcripts.

Previous studies demonstrated that type II procollagen is synthesized by HT-1080 cells that are stably transfected with constructs of the human COL2A1 gene that contain the promoter and 5' end of either the COL2A1 gene or the human COL1A1 gene. Since the host HT-1080 cells were from a human tumor line that synthesizes type IV collagen but not type II or type I procollagen, the results suggested that the constructs were integrated near active enhancers or promoters. Here, however, we demonstrate that a 33-kb construct of the COL2A1 gene containing a 5' fragment from the same gene was inserted into both alleles of the endogenous COL2A1 gene on chromosome 12, apparently by homologous recombination by a nonconservative pathway. In contrast, a similar construct of the COL2A1 gene in which the 5' end was replaced with a 1.9-kb fragment from the 5' end of the COL1A1 gene was inserted into both alleles of the locus for the COL1A1 gene on chromosome 17. Therefore, targeted insertion of the gene construct was not directed by the degree of sequence homology. Instead, it was directed by the relatively short 5' fragment from the COL1A1 gene that contained the promoter and the initially transcribed sequences of the gene. After insertion, both gene constructs were expressed from previously inactive loci.

Previously, transgenic mice were prepared that developed a lethal phenotype of fragile bones because they expressed an internally deleted mini-gene for the pro alpha 1(I) chain of human type I procollagen. The shortened pro alpha 1(I) chains synthesized from the human transgene bound to and produced degradation of normal pro alpha 1(I) chains synthesized from the normal mouse alleles. Here we assembled an antisense gene that was similar to the internally deleted COL1A1 minigene but the 3' half of the gene was inverted so as to code for an antisense RNA. Transgenic mice expressing the antisense gene had a normal phenotype, apparently because the antisense gene contained human sequences instead of mouse sequences. Two lines of mice expressing the antisense gene were bred to two lines of transgenic mice expressing the mini-gene. In mice that inherited both genes, the incidence of the lethal fragile bone phenotype was reduced from 92% to 27%. The effects of the antisense gene were directly demonstrated by an increase in the ratio of normal mouse pro alpha 1(I) chains to human mini-pro alpha 1(I) chains in tissues from mice that inherited both genes and had a normal phenotype. The results raise the possibility that chimeric gene constructs that contain intron sequences and in which only the second half of a gene is inverted may be particularly effective as antisense genes.

We developed a robot to carry out standard Sanger dideoxyribonucleotide sequencing reactions efficiently and with minimal human intervention. A commercial robot was adapted to our design and specifications, and we programmed it to perform up to 240 sequencing reactions in a single unattended run of 7 h. The robot configuration can be easily altered to allow 480 reactions to be performed in an unattended run of 14 h. The special features of our robot include cooled reagent reservoirs and cooled chambers for storage of DNA templates and completed reactions as well as reproducible aspiration of small volumes by using a sensing algorithm. The robot has successfully performed over 3500 DNA reactions in about 30 separate runs in our DNA core facility.

Recent work has demonstrated that more than 90% of patients with osteogenesis imperfecta (OI) have mutations in the gene for either the pro alpha 1 (I) chain or the gene for the pro alpha 2 (I) chain of type I procollagen. Three molecular mechanisms have explained the devastating effects of these mutations. In addition, the data provide several general conclusions about mutations in collagen genes. Studies in cell cultures with antisense oligonucleotides and studies in transgenic mice with an antisense gene raised the possibility that several strategies eventually may be developed to treat OI by converting severe forms of the disease to milder forms.

A new dominant mutation in the COL2A1 gene was found in a 38-week-old fetus with hypochondrogenesis. Denaturing gradient gel electrophoresis was used to analyze all 44 exons coding for the triple-helical domain of COL2A1 gene and the corresponding exon-intron boundaries. The technique detected a new sequence variation in exon 35. Sequencing of exon 35 demonstrated a single base mutation that converted the codon for glycine at position 604 to a codon for alanine. Electrophoresis of pepsin-digested collagen extracted from the diseased cartilage showed a doublet band of the alpha 1(II) chain of type II collagen and the presence of alpha 1(I) and alpha 2(I) chains of type I collagen. Two-dimensional analysis of cyanogen bromide peptides from the type II collagen revealed post-translational overmodification of peptides CB12, CB11, CB8, and CB10.5, whereas peptide CB9.7 migrated normally. Microscopic examination of cartilage showed that the mutation altered the organization of the growth plate. Also, articular chondrocytes contained large cisternae of rough endoplasmic reticulum. The density of the extracellular matrix was reduced, and the intensity of the staining with an antibody to type II collagen was diminished. In contrast, a significant staining with an antibody to type I collagen was observed. In situ hybridization with cRNA probes revealed a significant level of alpha 1(I) mRNA in the cytoplasm of the patient's chondrocytes. The signal for alpha 1(II) mRNA was about the same in control samples. The results indicated, therefore, that the genes for both type I and type II procollagens were simultaneously expressed in chondrocytes from the patient.

Procollagen N-proteinase (EC 3.4.24.14) is the enzyme that specifically cleaves the NH2-terminal propeptides from type I procollagen. Two forms of N-proteinase with apparent molecular sizes of 300 and 500 kDa were found in partially purified preparations from fetal bovine tendon extracts. The 500-kDa form of enzyme was purified 16,000-fold with a recovery of 8% from the extracts of the tendons by six purification steps. The purified enzyme was a neutral, Ca(2+)-dependent proteinase (5-10 mM) that was inhibited by metal chelators. The 500-kDa enzyme contained unreduced polypeptides of 58, 125, 170, and 190 kDa which were separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Electron microscopic study indicated that the enzyme molecules were generally globular and had diameters of 33 +/- 4 nm. Other properties of the 500-kDa enzyme were: 1) the Km for type I procollagen is 35 nM at pH 7.5 and 35 degrees C, and the kcat is 290 h-1; 2) the activation energy for reaction with type I procollagen is 10,050 cal mol-1; 3) the isoelectric point is 3.8; 4) the enzyme cleaves the NH2-terminal propeptides of type II procollagen as well as type I procollagen but not of type III procollagen; and 5) the enzyme specifically cleaves a -Pro-Gln- bond in the pro-alpha 1(I) chain and an -Ala-Gln- bond in the pro-alpha 2(I) chain. The bovine N-proteinase with a mass of 300 kDa was found to be similar to the 500-kDa enzyme and appeared to be a degraded form of the 500-kDa enzyme generated during purification. The N-proteinase from fetal bovine skin extracts also contained 300-kDa and 500-kDa enzyme forms.

Procollagen I was isolated from cultured skin fibroblasts from a proband who was homozygous for a mutation in the COL1A2 gene that substituted a serine codon for a glycine codon at position 661 of the alpha 2(I) chain. The procollagen I was cleaved to pCcollagen I by procollagen N-proteinase and the pCcollagen I was used as a substrate for assay of self-assembly of collagen I into fibrils. The mutated pCcollagen I was cleaved to collagen I by procollagen C-proteinase at the same rate as control pCcollagen I. However, self-assembly of the mutated collagen I had a lag period that was 15-fold greater than the lag period observed with normal collagen I under the same conditions. Also, self-assembly of the mutated collagen I had a propagation rate of about one-fourth of the propagation rate of normal collagen I. In addition, the critical concentration for fibril assembly was slightly increased. Rotary shadowing electron microscopy of the mutated procollagen I did not reveal any increased flexibility of the triple helix as was seen previously with two mutated procollagens I in which there were substitutions of cysteine for glycine residues in the alpha 1(I) chain (Vogel, B. E., Doelz, R., Kadler, K. E., Hojima, Y., Engel, J., and Prockop, D. J. (1988) J. Biol. Chem. 263, 19249-19255; Lightfoot, S. J., Holmes, D. F., Brass, A., Grant, M. E., Byers, P. H., and Kadler, K. E. (1992) J. Biol. Chem. 267, 25521-25528). However, morphometric analysis by dark-field light microscopy and electron microscopy showed that the fibrils formed from the mutated collagen I appeared thicker in diameter than the fibrils formed from the normal collagen I. Comparison of the results with similar data on four mutated procollagens previously studied raised the possibility that mutations which markedly increase the critical concentration of fibril assembly produce more severe phenotypes than mutations which change other parameters of fibril assembly.

A recently developed recombinant system for synthesis of human procollagen II by stably transfected host cells was used to prepare adequate amounts of protein to study the self-assembly of collagen II into fibrils. The procollagen II was cleaved to pCcollagen II by procollagen N-proteinase (EC 3.4.24.14), the pCcollagen II was chromatographically purified, and the pCcollagen II was then used as a substrate to generate collagen II fibrils by cleavage with procollagen C-proteinase. The kinetics for assembly of collagen II fibrils were similar to those observed previously for the self-assembly of collagen I in that a distinct lag phase was observed followed by a sigmoidal propagation phase. However, under the same experimental conditions, the lag time for assembly of collagen II fibrils was 5-6-fold longer, and the propagation rate for collagen II fibrils was about 30-fold lower than for collagen I fibrils. The relatively long lag time for the assembly of collagen II into fibrils made it possible to demonstrate that most of the conversion of pCcollagen II to collagen II occurred in the solution phase. The critical concentration at 37 degrees C for collagen II was about 50-fold greater than the critical concentration for collagen I. The Gibbs free energy change for the assembly of collagen II into fibrils was -40 kJ/mol, a value that was about 14 kJ/mol less than the free energy change for collagen I and about the same as the free energy change for the homotrimer of collagen I. Dark-field light microscopy and negative-staining electron microscopy demonstrated that the collagen II fibrils were thin and formed network-like structures. The results demonstrated, therefore, that the structural information of the monomer is sufficient to explain the characteristically small diameters and arcade-like geometry of collagen II fibrils found in cartilage and other tissues.

Phenotype variability and incomplete penetrance are frequently observed in human monogenic diseases such as osteogenesis imperfecta. Here an inbred strain of transgenic mice expressing an internally deleted gene for the pro alpha 1(I) chain of type I procollagen (COL1A1) was bred to wild type mice of the same strain so that the inheritance of a fracture phenotype could be examined in a homogeneous genetic background. To minimize the effects of environmental factors, the phenotype was evaluated in embryos that were removed from impregnated females 1 d before term. Examination of stained skeletons from 51 transgenic embryos from 11 separate litters demonstrated that approximately 22% had a severe phenotype with extensive fractures of both long bones and ribs, approximately 51% had a mild phenotype with fractures of ribs only, and approximately 27% had no fractures. The ratio of steady-state levels of the mRNA from the transgene to the level of mRNA from the endogenous gene was the same in all transgenic embryos. The results demonstrated that the phenotypic variability and incomplete penetrance were not explained by variations in genetic background or levels in gene expression. Instead, they suggested that phenotypic variation is an inherent feature of expression of a mutated collagen gene.

Procollagen C- and N-proteinases specifically cleave the C- and N-terminal extension propeptides of type I, II and III procollagen molecules. The collagen molecules generated by the enzymes self-assemble into collagen fibrils. We previously observed the inhibition of these enzymes purified from chick tendons by several divalent metals. Here the inhibitory effects of CdCl2, CuCl2, ZnCl2, NiCl2, CoCl2 and Hg(C2H3O2)2 have been studied in detail using crude or purified C- and N-proteinases from chick tendons and sterna. CdCl2 was a strong inhibitor of C-proteinases from both sources, and the inhibition was independent of enzyme purity (I50 = 10-16 microM). In contrast, CuCl2 and ZnCl2 were inhibitory only of purified C-proteinase. With the N-proteinase, CuCl2 was a strong inhibitor, and the inhibition was independent of the purity of the enzyme preparation used (I50 = 14-40 microM). On the other hand, CdCl2 was a moderate inhibitor, and ZnCl2 was a strong inhibitor only of the purified N-proteinase (I50 = 8-17 microM). NiCl2 inhibited crude and purified N-proteinase from sternum (I50 = 23-29 microM) but not from tendon. These results suggest, therefore, that the accumulation of some of these metals in the body may cause suppression of collagen fibril formation in tissues.

Apparently because the biosynthetic pathways involve eight or more highly specific post-translational enzymes, it has been difficult to obtain expression of genes for fibrillar collagens in recombinant systems. Here two constructs of the human gene for procollagen II (COL2A1) were prepared, one with about 0.5 kb of a promoter for a procollagen I gene (COL1A1) and the other with about 4 kb of the promoter for the procollagen II gene. The constructs, together with a neomycin-resistant gene, were transfected into a human tumour cell line (HT1080) that synthesizes the collagen IV found in basement membranes, but does not synthesize any fibrillar collagen. About two per 100 clones resistant to the neomycin analogue G418 synthesized and secreted human procollagen II. Milligram quantities of the recombinant procollagen II were readily isolated from the cultured medium. The recombinant procollagen II had the expected amino acid sequence as defined by nucleotide sequencing of mRNA-derived cDNA and the expected amino acid composition as defined by analysis of procollagen II that was converted into collagen II by digestion with procollagen N- and C-proteinases. Also, analysis of the carbohydrate content indicated that there was glycosylation of some of the hydroxylysine residues but no evidence of post-translational overmodification of the residues. In addition, the protein was shown to have a native conformation as assayed by a series of protease digestions. No essential differences were found between clones transfected with the COL2A1 gene construct containing the COL1A1 promoter and the similar construct containing the COL2A1 promoter in terms of number of clones synthesizing recombinant procollagen II and the levels of expression. With both constructs, the expression of the COL2A1 gene was closely related to copy number. The results demonstrated therefore that it is not essential to use a promoter for a gene normally expressed in a host cell in order to obtain gene copy-number-dependent expression of an exogenous collagen gene in stably transfected cells.

A search for mutations in the gene for type II procollagen (COL2A1) was carried out in a family with late-onset spondyloepiphyseal dysplasia resulting in short sature, restricted mobility and severe pain in joints, deforming arthritis in the hips, and claudication. Analysis of the HindIII and VNTR polymorphisms at the COL2A1 gene in the family raised the possibility that the gene cosegregated with the disease. Screening for mutations in the COL2A1 gene using PCR-denaturing gradient get electrophoresis suggested a sequence variation in exon 19 of one allele of the COL2A1 gene in the proband. Direct sequencing of the PCR products for exon 19 revealed a single base mutation that converted the codon of -GGT- for glycine at alpha 1-247 to -AGT-, a codon for serine. The mutant that converted the present in all affected family members, but absent in nonaffected members and in a group of 50 unrelated healthy individuals. It was also absent in 20 unrelated patients with chondrodysplasia and 30 unrelated patients with early-onset osteoarthritis.

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.

We performed detailed DNA sequencing analysis on type III collagen cDNA from 58 patients with either intracranial artery aneurysms or cervical artery dissections. The 58 patients were of seven different nationalities; among the patients were three pairs of relatives, so that 55 were unrelated, and of these, 29 had at least one blood relative with either an intracranial artery aneurysm or a cervical artery dissection. The age of the patients at the time of diagnosis ranged from 15 to 68 years (mean +/- SD = 40.3 +/- 11.0). The study group consisted of 25 males and 33 females. The analysis covered 3,232 nucleotides of significant (nonredundant) sequences per allele; therefore, we analyzed as many as 355,520 nucleotides. Mutations in the coding sequences for the triple-helical domain of type III collagen were excluded in 40 individuals with intracranial aneurysms and 18 individuals with cervical artery dissections. Direct sequencing of polymerase chain reaction products allowed mutations to be excluded with a high degree of confidence. Mutations that markedly decreased expression from one allele were also excluded in 42 of the 58 individuals, since the presence of both bases at one or more polymorphic sites in the 42 patients showed that two alleles were transcribed. The results indicated that mutations in the gene for type III procollagen (COL3A1) are not a common cause of either intracranial artery aneurysms or cervical artery dissections.

Several techniques have recently been developed to detect single-base mismatches in DNA heteroduplexes that contain one strand of wild-type and one strand of mutated DNA. Here we tested the hypothesis that an appropriate system of mildly denaturing solvents can amplify the tendency of single-base mismatches to produce conformational changes, such as bends in the double helix, and thereby increase the differential migration of DNA heteroduplexes and homoduplexes during gel electrophoresis. The best separations of heteroduplexes and homoduplexes were obtained with a standard 6% polyacrylamide gel polymerized in 10% ethylene glycol/15% formamide/Tris-taurine buffer. As predicted by the hypothesis of solvent-induced bends, when the concentration of either ethylene glycol or formamide was increased, the differential migration decreased. Also, single-base mismatches within 50 bp of one end of a heteroduplex did not produce differential migration. Sixty of 68 single-base mismatches in a series of PCR products were detected in some 59 different sequence contexts. The eight mismatches not detected were either within 50 bp of the nearest end of the PCR product or in isolated high-melting-temperature domains. Therefore, it was possible to predict in advance the end regions and sequence contexts in which mismatches may be difficult to detect. The procedure can be applied to any PCR products of 200-800 bp and requires no special equipment or preparation of samples.

Direct sequencing of polymerase chain reaction (PCR)-amplified genomic DNA from a patient with spondyloepiphyseal dysplasia and precocious osteoarthritis revealed a single-base change in exon 11 of the type II procollagen gene (COL2A1), which produces an Arg-->Cys mutation in one allele. The proband is a member of a large Chilean kindred presenting with chondrodysplasia of the hips, knees, shoulders, elbows, and spine associated with severe, early-onset osteoarthritis. All affected individuals exhibit mildly short stature; in addition, five out of seven affected family members display shortened metacarpals or metatarsals. DNA from affected and unaffected family members was PCR-amplified and analysis of restriction digests of the products determined that the mutation segregated with the disease with a lod score of 2.2 at zero recombination. The mutation, which resides in the triple-helical region of type II procollagen at amino acid position 75, is the second example of an Arg-->Cys mutation in the COL2A1 gene in heritable cartilaginous disease and is the first example of a point mutation in the amino terminal region of the alpha 1(II) chain, that results in a spondyloepiphyseal dysplastic phenotype.

A construct of the human gene for procollagen II (COL2A1) was prepared with an internal deletion of 5 kilobases that removed 12 exons coding for 291 amino acids from near the NH2 terminus of the triple helix. The construct was then used to transfect stably a human tumor cell line (HT-1080), and clones secreting internally deleted pro alpha 1(II) chain of procollagen II were isolated. The protein was purified, and the thermal stability of the triple-helical domain was assayed by brief protease digestion. The thermal stability of the internally deleted protein was the same as that of intact collagen II even though the triple helix was 39% shorter. Additionally, the thermal stability of the collagenase A fragment was the same as that of the collagenase A fragment of normal collagen II even though it was 38% shorter. Analysis of the results suggested that the thermal stabilities of large fragments of collagen II depended primarily on their contents of -Gly-Pro-Hyp-triplets corrected for length.

Previous reports have provided inconsistent data as to the cis-regulatory elements that are essential for correct expression of the gene for the pro alpha 1 (I) chain of type I procollagen (COL1A1) in the many tissues in which the protein is synthesized. Here, two internally deleted minigene versions of the human COL1A1 gene were used to prepare transgenic mice. The constructs made it possible to test regulatory sequences in the normal context of the gene. Also, in contrast to the reporter genes used in previous experiments, the constructs made it possible to assay quantitatively expression of the exogenous genes relative to expression of the endogenous COL1A1 gene, both as mRNA and as protein. The average level of expression of the minigenes varied among three transgenic lines, but the ratio of expression of the minigenes to expression of the endogenous gene was the same in all transgenic mice of a given line. Within the same line, the ratio of expression was essentially the same in nine or more tissues in which expression of the endogenous gene varied widely. Also, the ratio of expression within a given line was the same in 15-day-old embryos and in mice ranging in age from 4 days to 4 months. In addition, the ratio remained constant during repair of a surgical wound. The results demonstrated, therefore, that the minigene constructs with about 2.3 kb of the promoter region and about 2 kb of the 3'-flanking region contained all of the sequences necessary for correct expression of the genes in a tissue-specific and development-specific manner.(ABSTRACT TRUNCATED AT 250 WORDS)

A recombinant system was developed for the production of homotrimeric type I collagen in stably transfected HT1080 cells. A DNA construct (COL1A1-CMV) was prepared that contained the cDNA for the human COL1A1 gene under the transcriptional control of the promoter and enhancer of the immediate early gene of CMV. The construct, which also contained a neomycin-resistance gene, was transfected into HT1080 cells, a human fibrosarcoma cell line that synthesizes type IV collagen but does not normally synthesize any of the fibrillar collagens. Cells derived from the neomycin-resistant transfectants were then screened using a polyclonal antibody specific for human pro alpha 1(I) chains in order to identify clones that secreted high levels of the pro alpha(I) chain of type I procollagen. About 2% of neomycin-resistant clones secreted procollagen that consisted of a homotrimer of pro alpha 1(I) chains. The procollagen was post-translationally over-modified as judged by slower migration on SDS-polyacrylamide gel electrophoresis of the pro alpha 1(I) chains compared to pro alpha 1(I) chains of normal type I procollagen. The procollagen was triple helical as assayed by protease digestion with a variable cleavage at 38 degrees C and a thermal transition of both the intact and partially cleaved protein of about 41 degrees C. The system provides a method of expressing genes for fibrillar procollagens so that fully recombinant proteins are generated and easily isolated.

A series of oligonucleotide primers was designed to generate polymerase chain reaction products that contained exons 6 to 49 of the human gene for type II procollagen (COL2A1) and that could be used to detect sequence variations by denaturing gradient gel electrophoresis. To improve the sensitivity of the analysis, GC clamps were introduced into one primer of each pair. The procedure successfully detected 10 neutral single-base variations in the gene. In addition, the procedure detected a single-base deletion in exon 43 that introduced a premature termination codon in exon 44 and caused the Stickler syndrome (arthro-ophthalmopathy) in one family. The mutation is the fourth mutation in the COL2A1 gene shown to cause the Stickler syndrome. The mutation is similar to the first three mutations causing the disease in that they also introduced premature termination signals. Since only one mutation introducing a premature termination codon was found in the course of defining 120 or more mutations in type I and III procollagens, the results suggest that such mutations may have a special relationship to the Stickler syndrome.

A search for mutations in the gene for type II procollagen (COL2A1) was carried out in affected members of a family with early-onset cataracts, lattice degeneration of the retina, and retinal detachment. They had no symptoms suggestive of involvement of nonocular tissues, as is typically found in the Stickler syndrome. The COL2A1 gene was amplified with PCR, and the products were analyzed by denaturing gradient gel electrophoresis. The results suggested a mutation in one allele for exon 10. Sequencing of the fragment demonstrated a single-base mutation that converted the codon for glycine at position alpha 1-67 to aspartate. The mutation was found in three affected members of the family available for study but not in unaffected members or 100 unrelated individuals. Comparison with previously reported mutations suggested that mutations introducing premature termination codons in the COL2A1 gene are a frequent cause of the Stickler syndrome, but mutations in the COL2A1 gene that replace glycine codons with codons for bulkier amino acid can produce a broad spectrum of disorders that range from lethal chondrodysplasias to a syndrome involving only ocular tissues, similar to the syndrome in the family originally described by Wagner in 1938.

Detailed DNA sequencing of the triple-helical domain of type III procollagen was carried out on cDNA prepared from 54 patients with aortic aneurysms. The 43 male and 11 female patients originated from 50 different families and five different nationalities. 43 patients had at least one additional blood relative who had aneurysms. Five overlapping asymmetric PCR products, covering all the coding sequences of the triple-helical domain of type III procollagen, were sequenced with 28 specific sequencing primers. Analysis of the sequencing gels revealed only two nucleotide changes that altered the structure of the protein. One was a substitution of threonine for proline at amino acid position 501 and its functional importance was not clearly established. The other was a substitution of arginine for an obligatory glycine at amino acid position 136. In 40 of the 54 patients, detection of a polymorphism in the mRNA established that both alleles were expressed. The results indicate that mutations in type III procollagen are the cause of only about 2% of aortic aneurysms.

Skin fibroblasts from a proband with mild osteogenesis imperfecta (type I) synthesized normal pro alpha 2(I) chains and shortened pro alpha 2(I) chains of type-I procollagen. The type-I collagen that contained the shortened alpha 2(I) chains was thermally unstable in that it was cleaved at 30 degrees C by a mixture of trypsin and chymotrypsin. The mutation generating the shortened pro alpha 2(I) chains was shown to be a deletion of 19 base pairs from +4 to +22 of intron 13 of the COL1A2 gene by sequencing of genomic DNA and allele-specific oligonucleotide hybridization. The same mutation was found in the proband's affected father. Probe-protection experiments with S1 nuclease demonstrated that about 88% of the RNA transcripts from the mutated allele were spliced by exon skipping from exon 12 to exon 14 and that about 12% of the RNA transcripts were normally spliced. There was no evidence for use of cryptic splice sites, even though two cryptic splice sites had more favorable statistical scores and delta G degree 37 values than the new site that was created by the mutation and that was used for splicing of 12% of the transcripts into a normal mRNA. Comparison of the results with observations on 17 previously reported mutations that produced in-frame deletions of amino acids from the triple-helical domain of type-I collagen indicated that deletions in the N-terminal half of the alpha 2(I) chain tended to produce milder phenotypes than similar deletions elsewhere in the alpha 1(I) or alpha 2(I) chains.

A line of transgenic mice was prepared that expressed moderate levels of an internally deleted human gene for the pro alpha 1(I) chain of type I procollagen. The gene construct was modeled after a sporadic in-frame deletion of the human gene that produced a lethal variant of osteogenesis imperfecta by causing biosynthesis of shortened pro alpha 1(I) chains. 89 transgenic mice from the line were examined. About 6% had a lethal phenotype with extensive fractures at birth, and 33% had fractures but were viable. The remaining 61% of the transgenic mice had no apparent fractures as assessed by x ray examination on the day of birth. Brother-sister matings produced eight litters in which approximately 40% of the mice had the lethal phenotype, an observation indicating that expression of the exogenous gene was more lethal in putative homozygous mice from the line. Examination of femurs from the transgenic mice indicated that the bones were significantly shorter in length and had a decrease in wet weight, mineral content, and collagen content. However, there was no statistically significant change in the mineral to collagen ratio. Biomechanical measurements on femurs from the mice at 6 wk indicated a decrease in force and energy to failure. There was also a decrease in strain to failure and an increase in Young's modulus of elasticity, observations indicating increased brittleness of bone matrix. The results suggested that the transgenic mice may be an appropriate model for testing potential therapies for osteogenesis imperfecta. They may also be a useful model for studying osteoporosis.

Mutations in the genes coding for the pro alpha 1 and pro alpha 2 chains of type I procollagen have been found in many patients with osteogenesis imperfecta (OI), a heritable disorder of connective tissue. The severity of the disease varies between families and even among members of the same family. This phenotypic variability covers a spectrum extending from very mild forms that cannot be easily detected to perinatally lethal forms. One explanation for this phenotypic variability is the nature of the mutation in the type I procollagen genes. Another explanation is mosaicism. Here we report on 2 families with propositi who have OI, whereas their mothers had a milder form of the disease. In one family, the molecular defect was previously shown to be a substitution of alpha 1(904) by cysteine [Constantinou et al., 1990]. The biochemical phenotype was characterized by significant post-translational overmodification of the mutated type 1 collagen molecules which also had a 3-4 degrees C decrease in their thermal unfolding. Also, secretion of the procollagen into the culture media was delayed. In the second family, the proposita's muscle fibroblasts synthesized and secreted type I procollagen molecules that were highly over-modified along the entire length of their triple-helical domain. Cells from the mother also synthesized normal and over-modified protein, although the amount of over-modified protein was less than that synthesized by her daughter's cells. The exact molecular defect has not yet been defined.(ABSTRACT TRUNCATED AT 250 WORDS)

A series of antisense oligonucleotides were developed to inhibit specifically expression of a mutated exogenous gene for collagen without inhibiting expression of an endogenous gene for the same protein. The test system consisted of mouse NIH 3T3 cells that were stably transfected with an internally deleted construct of the human gene for the pro alpha 1(I) chain of type I procollagen [Olsen et al. (1991) J. Biol. Chem. 266, 1117]. The target site was a region at the 3' end of exon 1 and the first few nucleotides of intron 1 of the exogenous human gene that differed in sequence by nine nucleotides from the sequence of the endogenous mouse gene. Expression of the two genes was assayed by Western blot with cross-reacting antibodies and by steady-state levels of mRNAs. None of the oligonucleotides were effective in concentrations up to 25 microM when administered without any carrier. However, when administered with 5 or 10 micrograms/mL lipofectin, one of the oligonucleotides in concentrations of 0.1-0.2 microM inhibited expression of the exogenous gene from 50% to 80% without significant inhibition of expression of the endogenous gene. Also, a missense version of the same oligonucleotide had no significant effect, and the inhibition observed with the most effective oligonucleotide was abolished by a single base change. Time course experiments indicated that, after a 4-h treatment, inhibition appeared at 8 h and persisted for at least 22 h.(ABSTRACT TRUNCATED AT 250 WORDS)

A series of protocols were developed for a commercially available automated workstation to prepare samples for amplification of DNA by the polymerase chain reaction (PCR) on automated thermal cyclers, to load PCR products onto agarose electrophoretic gels and to carry out dideoxynucleotide sequencing of DNA templates. The protocols and the software programs developed reduced by two-thirds the time required to carry out the procedures manually. The programs and protocols also improved the quality and the consistency of both the PCR products and the sequencing reactions.

Genetic linkage analyses suggest that mutations in type II collagen may be responsible for Stickler syndrome, or arthro-ophthalmopathy (AO), in many families. In the present study oligonucleotide primers were developed to amplify and directly sequence eight of the first nine exons of the gene for type II procollagen (COL2A1). Analysis of the eight exons in 10 unrelated probands with AO revealed that one had a single-base mutation in one allele that changed the codon of -CGA- for arginine at amino acid position alpha 1-9 in exon 7 to a premature termination signal for translation. The second mutation found to cause AO was, therefore, similar to the first in that both created premature termination signals in the COL2A1 gene. Since mutations producing premature termination signals have not previously been detected in genes for fibrillar collagens, the results raise the possibility that such mutations in the COL2A1 gene are a common cause of AO.

Cultured skin fibroblasts were examined from two probands with type II (lethal) osteogenesis imperfecta. One proband had a single base mutation which converted the glycine codon at position alpha 1-244 in the alpha 1(I) chain of procollagen I into a cysteine codon whereas the other had a similar mutation that converted the glycine codon at position alpha 2-787 of the alpha 2(I) chain into a cysteine codon. Both mutations produced post-translational overmodification of procollagen I. The Cys alpha 1-244 mutation, however, had a minimal effect on the thermal stability or secretion of the protein whereas the Cys alpha 2-787 mutation markedly decreased the thermal stability and, apparently as a result, essentially none of the mutated protein was secreted. The results provide clear exceptions to two previous generalizations about the position-specificity of glycine substitutions in procollagen I.

Sequenase, Taq DNA polymerase and Bst DNA polymerase were tested for sequencing of DNA on microtiter plates using dried down reagents. Several parameters were investigated to expedite the drying process while minimizing damage to the enzyme. Sequenase did not tolerate drying very well, and frequently generated sequences with weak signals and many sites of premature termination. With Taq DNA polymerase it was possible to obtain sequences of good quality. However, there was considerable variation of results between experiments and between batches of microtiter plates. Bst DNA polymerase generated sequences of excellent quality. It was stable for more than a week in dried-down state at -20 degrees C and at least overnight at room temperature. The method described here using Bst DNA polymerase is well suited for laboratory robots and workstations that typically employ 96-well microtiter plates.

A single-base mutation in intron 37 of the gene for type III procollagen (COL3A1) was found in a proband with the type IV variant of Ehlers-Danlos syndrome. Probe-protection experiments with S1 nuclease and RNA from fibroblasts incubated at 37 degrees C demonstrated that about 35% of the total mRNA or about 70% of the mRNA from mutated allele was spliced by exon skipping. The effects of the mutation were temperature-sensitive in that the amount of RNA from the mutated allele that was spliced by exon skipping was 87.1 +/- 7.7% at 31 degrees C, 70.1 +/- 6.5% at 37 degrees C, and 85.4 +/- 11.1% at 42 degrees C. The effects of temperature on aberrant RNA splicing were, therefore, the reverse of those reported for four previous mutants in collagen genes. The increase in abnormal RNA splicing when the temperature was raised from 31 degrees to 37 degrees C seen with previously reported mutants suggested that RNA-RNA hybridization of U1snRNA to the 5'-splice site in the substrate may be limiting in the processing of transcripts from the mutated alleles, since RNA-RNA hybridizations become less favorable at higher temperatures. The decrease in abnormal RNA splicing seen here when the temperature was raised from 31 degrees to 37 degrees C suggested that protein-RNA or protein-protein binding steps become rate limiting with the G+5 mutation in intron 37 of the COL3A1 gene.

Uniparental disomy for chromosome 7 has been described previously in two individuals with cystic fibrosis. Here, we describe a third case that was discovered because the proband was homozygous for a mutation in the COL1A2 gene for type I procollagen, although his mother was heterozygous and his father did not have the mutation. Phenotypically, the proband was similar to the two previously reported cases with uniparental disomy for chromosome 7, in that he was short in stature and growth retarded. Paternity was assessed with five polymorphic markers. Chromosome 7 inheritance in the proband was analyzed using 12 polymorphic markers distributed along the entire chromosome. Similar analysis of the proband's two brothers established the phase of the alleles at the various loci, assuming minimal recombination. The proband inherited only maternal alleles at five loci and was homozygous at all loci examined, except one. He was heterozygous for an RFLP at the IGBP-1 locus at 7p13-p12. The results suggest that the isodisomy was not complete because of a recombination event involving the proximal short arms of two maternal chromosomes. In addition, the phenotype of proportional dwarfism in the proband suggests imprinting of one or more growth-related genes on chromosome 7.

Previous observations established that pNcollagen III copolymerized with collagen I and decreased the diameter of the fibrils formed (Romanic, A.M., Adachi, E., Kadler, K.E., Hojima, Y., and Prockop, D.J. (1991) J. Biol. Chem. 266, 12703-12709). Here, procollagen I alone or mixtures of procollagen I and pCcollagen I were incubated with procollagen C-proteinase to generate pNcollagen I or mixtures of pNcollagen I and collagen I. The results confirmed previous reports that pNcollagen I assembles into sheet-like structures. They also demonstrated that polymerization of pNcollagen I exhibits a lag period and propagation phase similar to those seen with other protein self-assembly systems. In addition, the results demonstrated that pNcollagen I formed true copolymers with collagen I in that the presence of pNcollagen I increased the lag time, decreased the propagation rate, and increased the concentration of collagen I in solution at equilibrium. Copolymerization of pNcollagen I with collagen I, however, differed in two features from copolymerization of pNcollagen III with collagen I. One was that, in confirmation of previous work, copolymerization of pNcollagen I with collagen I markedly altered the circularity of the fibrils formed. The second difference was that the copolymerization increased the concentration in solution at equilibrium of pNcollagen I whereas copolymerization with collagen I was previously shown to decrease the concentration in solution of pNcollagen III. The increase in concentration in solution of pNcollagen I was explicable either by the assembly of soluble oligomers of pNcollagen I and collagen I, or by subtle changes in the activities of pNcollagen I and collagen I in the solid-phase. Comparison with previous data with pNcollagen III indicated that although pNcollagen I and pNcollagen III copolymerize with collagen I, there are marked differences in the two kinds of copolymers.

Collagen fibrils generated in vitro at 37 degrees C by enzymic removal of C-terminal propeptides from type I pC-collagen (an intermediate in the normal processing of type I procollagen to collagen containing the C-terminal propeptides but not the N-terminal propeptides) display shape polarity, with one tip fine tapered and the other coarse tapered. Mass measurements by scanning transmission electron microscopy show that the mass per unit length along both kinds of tip increases roughly linearly over distances of approximately 100 D periods from the fibril end [D (axial periodicity) = 67 nm]. The fine tips of fibrils of widely differing lengths exhibit near-identical mass distributions, the mass in all cases increasing at the rate of approximately 17 molecules per D period, irrespective of fibril length. Coarse tips display less regular behavior. These results show that (i) the shape of a fine tip is not conical but resembles more closely a paraboloid of revolution, and (ii) for this shape to be maintained throughout growth, accretion (rate of mass uptake per unit area) cannot everywhere be the same on the surface of the tip but must decrease as the diameter increases. To a first approximation, accretion alpha (diameter)-1.

A model was developed to account for the recent observations indicating that type I collagen fibrils assembled in vivo grow from symmetrical pointed tips. The essential features of the model are (i) a distinctive structural nucleus forms at each end of a growing fibril and growth of the fibril then proceeds by propagation of the two structural nuclei, (ii) the two structural nuclei have similar spiral or helical conformations, and (iii) assembly of each structural nucleus requires two kinds of specific binding steps defined as 3.4 D-period and 0.4 D-period overlaps, but propagation of the nucleus requires only the 3.4 D-period binding step.

A proband with arterial ruptures and skin changes characteristic of the type IV variant of Ehlers-Danlos syndrome was found to have a single-base mutation in the type III procollagen gene, which converted the codon for glycine at amino acid position 1018 to a codon for aspartate. (Amino acid positions are numbered by the standard convention in which the first glycine of the triple-helical domain of an alpha chain is number 1. The numbers of positions in the alpha 1(III) chains can be converted to positions in the human pro alpha(III) chain by adding 167.) Nucleotide sequencing of overlapping PCR products in which the two alleles were distinguished demonstrated that the mutation of glycine 1018 was the only mutation that changed the primary structure of type III procollagen. The glycine substitution markedly decreased the amount of type III procollagen secreted into the medium by cultured skin fibroblasts from the proband. It is surprising that the same mutation was found in about 94% of the peripheral blood leukocytes from the proband's asymptomatic 72-year-old mother. Other tissues from the mother contained the mutated allele; it was present in 0%-100% of different samples of hair cells and in about 40% of cells from the oral epithelium. Therefore, the mother was a mosaic for the mutation. Since the mutated allele was present in cells derived from all three germ layers, the results indicated that the mutation arose by the late blastocyst stage of development. The results also indicate that assays of blood leukocytes do not always reveal mosaicism or predict phenotypic involvement of tissues, such as blood vessels, that are derived from the same embryonic cells as are leukocytes.

Type I collagen, the most abundant structural protein in vertebrates, is comprised of two alpha 1(I) chains and one alpha 2(I) chain. Fibroblasts from a proband with osteogenesis imperfecta, however, were shown to synthesize a type I procollagen that was a homotrimer of pro alpha 1(I) chains. The absence of pro alpha 2(I) chains in the procollagen provided a unique opportunity to assess the role of the alpha 2(I) chain in collagen fibrillogenesis by examining the self-assembly de novo of the homotrimeric collagen generated in vitro. The results demonstrated that the fibrils formed by the homotrimeric collagen had an asymmetric banding pattern similar to fibrils of normal heterotrimeric type I collagen. However, the efficiency for self-assembly of the homotrimer into fibrils was markedly reduced in that the critical concentration at 37 degrees C was 40-fold greater than for self-assembly of the heterotrimeric molecule. A van't Hoff-type plot of the data was used to determine values for delta G, delta H and delta S. The values indicated the self-assembly of the homotrimer is similar to self-assembly of the heterotrimer in that the process is entropy driven. The process is, however, less favorable in that the delta G value was 10 kJ/mol less negative. The results suggest that the presence of the alpha 2(I) chain in type I collagen helps drive the self-assembly process, probably because the alpha 2(I) chain is more hydrophobic than the alpha 1(I) chain and, therefore, smaller amounts of structured water may be lost during self-assembly of the homotrimer than during self-assembly of the heterotrimer.(ABSTRACT TRUNCATED AT 250 WORDS)

Liver fibrosis was induced in rats both with carbon tetrachloride and dimethylnitrosamine. Assays were performed on steady-state levels of messenger RNAs in the liver for several collagens and basement membrane components. The results indicated marked increases in the steady-state levels of messenger RNA for type I collagen, type III collagen, type IV collagen and the B2 component of laminin. In the same animals, immunoassays were performed for serum levels of the N-terminal propeptide of type III procollagen and the 7S fragment of type IV collagen. The results demonstrated an increase in the serum levels of 7S fragment that occurred early and closely paralleled the increase in the steady-state levels of messenger RNA for the alpha 1(IV) chain of type IV collagen. In contrast, no significant increase was seen in the serum levels of the N-propeptide of type III procollagen. The results suggest that immunoassays for 7S fragment of type IV collagen in serum are a more sensitive index for liver cell damage and fibrosis than assays for the N-propeptide of type III procollagen. The results suggest that greater attention should be paid to assays of 7S fragments in assessing hepatic fibrosis in man.