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D J Prockop

Publications and source records attributed to D J Prockop.

At least 109 records · Page 6Linked to original sources
PubMed1995

Transgenic mice with targeted inactivation of the Col2 alpha 1 gene for collagen II develop a skeleton with membranous and periosteal bone but no endochondral bone.

Homologous recombination in embryonic stem cells was used to prepare transgenic mice with an inactivated Col2a1 gene for collagen II, the major protein component of the extracellular matrix of cartilage. Heterozygous mice had a minimal phenotype. Homozygous mice developed into fetuses that were delivered vaginally but died either just before or shortly after birth. The cartilage in the mice consisted of highly disorganized chondrocytes with a complete lack of extracellular fibrils discernible by electron microscopy. There was no endochondrial bone or epiphyseal growth plate in long bones. However, many skeletal structures such as the cranium and ribs were normally developed and mineralized. The results demonstrate that a well-organized cartilage matrix is required as a primary tissue for development of some components of the vertebrate skeleton, but it is not essential for others.

Animals↗
PubMed1995

The human COL11A2 gene structure indicates that the gene has not evolved with the genes for the major fibrillar collagens.

The human COL11A2 gene was analyzed from two overlapping cosmid clones that were previously isolated in the course of searching the human major histocompatibility region (Janatipour, M., Naumov, Y., Ando, A., Sugimura, K., Okamoto, N., Tsuji, K., Abe, K., and Inoko, H. (1992) Immunogenetics 35, 272-278). Nucleotide sequencing defined over 28,000 base pairs of the gene. It was shown to contain 66 exons. As with most genes for fibrillar collagens, the first intron was among the largest, and the introns at the 5'-end of the gene were in general larger than the introns at the 3'-end. Analysis of the exons coding for the major triple helical domain indicated that the gene structure had not evolved with the genes for the major fibrillar collagens in that there were marked differences in the number of exons, the exon sizes, and codon usage. The gene was located close to the gene for the retinoic X receptor beta in a head-to-tail arrangement similar to that previously seen with the two mouse genes (P. Vandenberg and D. J. Prockop, submitted for publication). Also, there was marked interspecies homology in the intergenic sequences. The amino acid sequences and the pattern of charged amino acids in the major triple helix of the alpha 2(XI) chain suggested that the chain can be incorporated into the same molecule as alpha 1(XI) and alpha 1(V) chains but not into the same molecule as the alpha 3(XI)/alpha 1(II) chain. The structure of the carboxyl-terminal propeptide was similar to the carboxyl-terminal propeptides of the pro alpha 1(XI) chain and pro alpha chains of other fibrillar collagens, but it was shorter because of internal deletions of about 30 amino acids.

Amino Acid Sequence↗
PubMed1995

Conservation of the sizes of 53 introns and over 100 intronic sequences for the binding of common transcription factors in the human and mouse genes for type II procollagen (COL2A1).

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.

Animals↗
PubMed1995

Cultured adherent cells from marrow can serve as long-lasting precursor cells for bone, cartilage, and lung in irradiated mice.

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.

Animals↗
PubMed1995

Substitution of aspartic acid for glycine at position 310 in type II collagen produces achondrogenesis II, and substitution of serine at position 805 produces hypochondrogenesis: analysis of genotype-phenotype relationships.

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.

Amino Acid Sequence↗
PubMed1995

Tissue-specific expression of the gene for type I procollagen (COL1A1) in transgenic mice. Only 476 base pairs of the promoter are required if collagen genes are used as reporters.

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.

Animals↗
PubMed1995

Bone fragility in transgenic mice expressing a mutated gene for type I procollagen (COL1A1) parallels the age-dependent phenotype of human osteogenesis imperfecta.

An inbred strain of transgenic mice that expressed a mutated gene for type I procollagen and that developed spontaneous fractures was used to study the effects of age on the phenotype of fragile bones. The mutated gene has been shown to cause depletion of type I collagen in the transgenic mice because it generated shortened pro alpha 1(I) chains that bound to and produced degradation of normal pro alpha 1(I) chains synthesized from the endogenous mouse COL1A1 gene. For this study, femurs from transgenic mice ranging in age from 0.5-24 months were examined. The results demonstrated that the level of expression of the transgene was independent of age. Femurs from the transgenic mice were more fragile than controls at 0.5 and 1.5 months, they were biomechanically normal at 6 months, and then they were more fragile at 24 months. The normal biomechanical properties of the bones from the transgenic mice at 6 months were accompanied by periosteal thickening of the bones together with an increase in the collagen content that was not associated with a proportional increase in mineral content. The results indicated that the effects of age, mechanical stress, and hormonal action produced a biological compensation for the mutated gene by either increasing collagen synthesis of bone, decreasing collagen degradation, or both. The biological compensation was apparently lost by 24 months when the outer diameters of the femurs were again less than in controls, the cortical thickness was about the same as in controls, and both the collagen and mineral contents were less than controls. The results demonstrated that bone fragility in the transgenic mice paralleled the age-dependent phenotype of human osteogenesis imperfecta. Therefore the transgenic mice appeared to be useful models for osteogenesis imperfecta. They also may be useful models for some forms of osteoporosis.

Aging↗
PubMed1995

Radial packing, order, and disorder in collagen fibrils.

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.

Animals↗
PubMed1995

Collagens: molecular biology, diseases, and potentials for therapy.

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.

Animals↗
PubMed1994

A rapid and simple PCR-based method for isolation of cDNAs from differentially expressed genes.

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.

Base Sequence↗
PubMed1994

Specific inhibition of expression of a human collagen gene (COL1A1) with modified antisense oligonucleotides. The most effective target sites are clustered in double-stranded regions of the predicted secondary structure for the 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.

3T3 Cells↗
PubMed1994

Targeted insertions of two exogenous collagen genes into both alleles of their endogenous loci in cultured human cells: the insertions are directed by relatively short fragments containing the promoters and the 5' ends of the genes.

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.

3T3 Cells↗
PubMed1994

Partial rescue of a lethal phenotype of fragile bones in transgenic mice with a chimeric antisense gene directed against a mutated collagen gene.

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.

Animals↗
PubMed1994

Mutation in the COL2A1 gene in a patient with hypochondrogenesis. Expression of mutated COL2A1 gene is accompanied by expression of genes for type I procollagen in chondrocytes.

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.

Adult↗
PubMed1994

Characterization of type I procollagen N-proteinase from fetal bovine tendon and skin. Purification of the 500-kilodalton form of the enzyme from bovine tendon.

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.

Amino Acid Sequence↗