Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.5 (RNase)
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This study compares the synthesis of mutant type I collagen in cultured dermal fibroblasts and trabecular osteoblasts that were isolated from a patient with moderately severe osteogenesis imperfecta (type IV). Previous study of this patient's dermal fibroblasts revealed a 2000 dalton deletion located in cyanogen bromide peptide 4 of alpha 2(I)-collagen. The phenotype of the bone cell cultures was defined by a 3-4 day logarithmic phase doubling time, predominantly type I collagen production over type III and alkaline phosphatase activity 13.5 times dermal fibroblast levels. The current study revealed that both fibroblasts and osteoblasts synthesized a normal and a shortened alpha 2(I) chain, each as the product of separate alleles. Following pepsin treatment of the procollagens, a shortened alpha 1(I) chain was also seen in both cell types. Cyanogen bromide peptide mapping of osteoblast alpha-chains demonstrated the same deletions in the cyanogen bromide peptide 4 as observed in the fibroblast cyanogen bromide maps. PAGE analysis of oligonucleotide-specific cDNA that was reverse transcribed from RNA isolated from fibroblasts and osteoblasts also demonstrated the presence of two bands, one the normal size of alpha 2(I) cDNA and a second species that was smaller by 54 base pairs. Sequencing of polymerase chain reaction-amplified cDNA fragments revealed an in-frame deletion of exon 12. This finding was confirmed by the RNase protection method. Genomic DNA sequencing detected a T----G point mutation in the second position of the 5' splice donor site of intron 12. Therefore, in this patient with osteogenesis imperfecta there was no qualitative alteration in the osteoblast-specific expression of this mutant alpha 2(I)-collagen allele compared to dermal fibroblasts.
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PMID:Expression of mutant alpha (I)-procollagen in osteoblast and fibroblast cultures from a proband with osteogenesis imperfecta type IV. 164 48

We have developed a strategy for the detection, localization and sequence determination of point mutations in the mRNA coding for the alpha 1(I) and alpha 2(I) chains of type I collagen. Point mutations are detected by RNase A cleavage of mismatches in RNA/RNA hybrids. The mRNAs coding for the fibrillar collagens present special problems for hybrid analysis because of their large size and their GC-rich and repetitive sequences. We have generated a series of overlapping antisense riboprobes covering the entire pro alpha 1(I) and pro alpha 2(I) mRNAs. Uniformly labelled normal antisense riboprobes are hybridized with the total fibroblast RNA of patients with possible mutations in type I collagen. Mismatches in the resulting RNA/RNA hybrids are cleaved with RNase A and the labelled riboprobe cleavage products are examined electrophoretically. The sensitivity and specificity of the system were demonstrated by the detection and localization of a known point mutation in the codon for alpha 1(I) glycine 988 (1). DNA for sequencing the mutations localized by hybrid analysis may be obtained by either (1) generation of a fibroblast cDNA library and isolation of both alleles by plaque screening, or (2) a more rapid method using first strand cDNA synthesis from poly (A+)-mRNA, followed by PCR amplification of the mutation-containing region of the DNA/RNA hybrid. This strategy for detection and isolation has wide application not only for mutations causing connective tissue disorders, but also for mutations in other large and repetitive genes. We have used this strategy for the detection and sequencing of a point mutation in alpha 2(I) mRNA associated with a case of lethal osteogenesis imperfecta. The G----A point mutation in the codon for alpha 2(I) glycine residue 805 results in the substitution of an aspartic acid at this position and is consistent with the proband's collagen protein data.
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PMID:Detection of point mutations in type I collagen by RNase digestion of RNA/RNA hybrids. 169 2

A proband with lethal osteogenesis imperfecta has been investigated for the causative defect at the levels of collagen protein, mRNA, and DNA. Analysis of type I collagen synthesized by the proband's fibroblasts showed excessive post-translational modification of alpha 1(I) chains along the entire length of the helix. Oververmodification of alpha chains could be prevented by incubation of the cells at 30 rather than 37 degrees C, and the thermal stability of the triple helix, as determined by protease digestion, was normal. RNase A cleavage of RNA:RNA hybrids formed between the proband's mRNA and antisense RNA derived from normal pro-alpha 1(I) chain cDNA clones was used to locate an abnormality to exon 43 of the proband's pro-alpha 1(I) collagen gene (COL1A1). The nucleotide sequence of the corresponding gene region showed, in one allele, the deletion of 9 base pairs, not present in either parent, within a repeating sequence of exon 43. The mutation causes the loss of one of three consecutive Gly-Ala-Pro triplets at positions 868-876, but does not otherwise disrupt the Gly-X-Y sequence. Procollagen processing in fibroblast cultures and susceptibility of the mutant collagen I to cleavage with vertebrate collagenase were normal, indicating that the slippage of collagen chains by one Gly-X-Y triplet does not abolish amino-propeptidase and collagenase cleavage sites. How the mutation produces the lethal osteogenesis imperfecta phenotype is not entirely clear; the data suggest that the interaction of alpha chains immediately prior to helix formation may be affected.
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PMID:A 9-base pair deletion in COL1A1 in a lethal variant of osteogenesis imperfecta. 193 61

Growth factor-depleted Swiss 3T3 cells responded to basic fibroblast growth factor (bFGF) with a burst of mitogenesis and with a rapid and marked increase in thrombospondin (TS) mRNA levels. mRNA levels for the alpha 1 chain of type I collagen and for fibronectin were unaffected. At early times following stimulation (0-2 h), "superinduction" of TS mRNA by inhibition of protein synthesis with cycloheximide was not observed, and the increase in TS mRNA could be attributed primarily to an increase in transcription rate of the TS gene. However, at later times (4-8 h) the combination of cycloheximide and bFGF superinduced TS mRNA levels, suggesting the existence of a labile inhibitor of transcription or a short-lived RNase that might be produced in response to prolonged treatment with bFGF. In contrast to its stimulatory effect on 3T3 cells, bFGF did not stimulate the proliferation of mouse muscle BC3H1 cells nor did it cause an increase in TS mRNA levels, but BC3H1 cells do respond to bFGF by inhibition of myogenic differentiation. We propose, on the basis of these and other findings, that TS facilitates the progression of some anchorage-dependent cells through the cell cycle.
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PMID:Thrombospondin gene expression is associated with mitogenesis in 3T3 cells: induction by basic fibroblast growth factor. 221 38

A method for detecting a wide variety of mutations within type I collagen has been developed and evaluated on a series of patients with osteogenesis imperfecta. RNA, extracted from the nuclear and cytoplasmic compartment of cultured fibroblasts from affected individuals, is hybridized with antisense single-stranded cDNA to the alpha 1(I) mRNA. The hybrid is digested with RNase A and T1 under varying degrees of stringency. The resistant RNA bands are separated by electrophoresis in agarose, transferred to nitrocellulose, and hybridized with antisense cRNA colinear with the protecting probe. This approach is capable of detecting previously defined mutations such as 252-base pair deletion and a 1-base pair mismatch within the alpha 1(I) mRNA. The method appears to be particularly useful in detecting abnormalities of RNA processing that behave as an insert or deletion within the mature mRNA. The procedure should be generally applicable for the identification and localization of any mutation within an entire gene if the gene is expressed as an RNA and a complete cDNA for the mRNA is available.
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PMID:Detection of mutations in human type I collagen mRNA in osteogenesis imperfecta by indirect RNase protection. 254 16

We have identified a point mutation in one alpha 1(I) collagen allele (COL1A1) of a child with the type IV osteogenesis imperfecta phenotype. When compared to parental and control samples, skin fibroblasts of the proband synthesized two populations of type I collagen molecules. One population was normal; the other was delayed in secretion and electrophoretic migration due to post-translational overmodification. Two-dimensional gel electrophoresis of the CNBr peptides demonstrated a gradient of overmodification beginning near the carboxyl-terminal CB peptides. This predicts that the mutation delaying helix formation is near the carboxyl-terminal end of one of the component chains of type I collagen. The mRNA of the patient was probed with overlapping antisense riboprobes to type I collagen cDNA. Cleavage of a mismatch in RNA/RNA hybrids of RNase A allowed the location of the mutation to a 225-base pair region of alpha 1(I) cDNA. The mismatch was not present in RNA/RNA hybrids from either parent. This region of both alpha 1(I) alleles of the patient was isolated by screening a lambda ZAP cDNA library. Sequence determination of both alleles demonstrated a single nucleotide change, G----A, resulting in the substitution of a serine for a glycine at amino acid residue 832. This point mutation occurs in the coding region for alpha 1(I) CB6 and is concordant with the protein data. The finding of a glycine substitution in an alpha 1(I) chain of a patient with the milder type IV osteogenesis imperfecta phenotype requires modification of current molecular models for types II and IV osteogenesis imperfecta.
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PMID:Osteogenesis imperfecta type IV. Detection of a point mutation in one alpha 1(I) collagen allele (COL1A1) by RNA/RNA hybrid analysis. 274 20

Calf aortic smooth muscle cell cultures produce both type III and type I collagen. Polyadenylated mRNA species purified from these cells direct the synthesis of prepro-alpha 1(III), prepro-alpha 1(I), and prepro-alpha 2(I) in a rabbit reticulocyte cell-free system. These polypeptides were identified by specific immunoprecipitation, cyanogen bromide peptide mapping, and bacterial collagenase digestion. Lower molecular weight collagenase susceptible polypeptides were also produced in translation reactions incubated under conditions optimized for incorporation of radiolabeled amino acids. Their presence did not appear to result from ribonuclease or protease involvement or from premature termination. Increasing the Mg2+ concentration in the translation system significantly reduced the production of these lower molecular weight species. Pulse-chase experiments indicate that the time required for completion of full length preprocollagen at the high Mg2+ concentration is greatly decreased compared to the low concentration. Additional experiments suggest that the incomplete collagen polypeptides result from pausing of ribosome movement during elongation. The relative synthesis of type III and type I chains was examined as a function of mRNA concentration in the cell-free system. At levels of RNA above saturation, the relative production of type III decreased with respect to type I. These data suggest that the ability of the alpha 1(III) mRNA to initiate translation is less efficient than the mRNAs of alpha 1(I) and alpha 2(I).
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PMID:Cell-free translation of calf type III collagen. Effect of magnesium on ribosome movement during elongation. 661 53

The relationship between the clinical severity of osteogenesis imperfecta (OI) and the location and type of amino acid substitution in type I collagen is not identical for mutations in the alpha 1(I) and alpha 2(I) chains. Furthermore, the alpha 2(I) chain, once thought to be associated with moderate forms of OI, has now been associated with approximately as many lethal as non-lethal cases. We describe two novel substitutions for glycine in the alpha 2(I) chain, one associated with a lethal phenotype in twins and the other with a moderate non-lethal phenotype. The type I collagen of all probands was characterized electrophoretically by two populations of alpha chains, one normal and one with delayed migration. Cyanogen bromide peptides of the overmodified alpha 1(I) chains revealed delayed migration of all peptides except CB6. The indicated target region of alpha 1(I) and alpha 2(I) cDNA of the probands was analyzed by RNA-DNA hybrid analysis with RNase A digestion. All probands had mismatches in the region of alpha 2(I) coding for amino acids 642-912. The lethal phenotype was associated with a G-->A mutation, resulting in Gly706-->serine; the non-lethal mutation was a G-->T change resulting in Gly676-->valine. Both mutations occurred de novo in the probands; parental leukocyte DNA was normal. In conjunction with the previously described exon deletions and point mutations in alpha 2(I), these mutations define five alternating non-lethal/lethal regions along the chain and support a regional, as opposed to a gradient, model of OI pathophysiology. These mutations in particular help to define a lethal/non-lethal junction at about alpha 2(I) amino acid 700.
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PMID:Two additional cases of osteogenesis imperfecta with substitutions for glycine in the alpha 2(I) collagen chain. A regional model relating mutation location with phenotype. 769 12

This study tested the hypothesis that the remodeling processes of adult periodontal ligament (PDL) reiterate the cellular and molecular events that occur sequentially during development. Type XII collagen has been implicated in the three-dimensional organization of the PDL extracellular matrix, and its expression has been restricted to the terminally differentiated stages. This study focused on the examination of the temporal and spatial expression of type XII collagen during experimental PDL remodeling in the rat. The temporal expressions of types I and XII collagen mRNAs were examined by RNA transfer blot and RNase protection assays, respectively, and were found to be relatively stable in the control group throughout the experimental period. In the tooth movement group, the expression of type I collagen increased at 72 hours and sustained the high level of expression at one week, while an increase in the expression of type XII collagen was first noted at the one-week period. The temporal activation of types I and XII collagen expression in the remodeling occurred in a pattern similar to that found during the development of the PDL. The spatial expression of type XII collagen mRNA was examined by in situ hybridization in the one-week-tooth-movement specimens. Labeled cells, which were more evident in the tension side, typically exhibited a spindle shape and were surrounded by the mature PDL matrix. Our data suggest that the type XII collagen expression may be closely associated with the functional regeneration of the PDL.
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PMID:Temporal and spatial expressions of type XII collagen in the remodeling periodontal ligament during experimental tooth movement. 787 23

Serine for glycine substitutions in type I collagen have been described in seven cases of lethal type II osteogenesis imperfecta (OI), and six cases of nonlethal OI. We describe here two cases of moderately severe type IV OI with serine substitutions at alpha 1(I) Gly352 and alpha 2(I) Gly922, respectively. In both cases, G-->A point mutations were detected by RNase A cleavage of RNA/RNA and RNA/DNA hybrids. These cases extend the location for serine substitutions producing the moderately severe OI phenotype to the alpha 2(I) chain and the amino-terminal end of the alpha 1(I) chain. Their location supports a regional model of OI pathophysiology for serine substitutions. The proband with alpha 2(I) Gly922-->Ser has both normal and overmodified forms of both type I collagen chains. The overmodified form has delayed migration of all CNBr peptides. Helix thermal stability is decreased 4 degrees C. The fibroblast collagen protein and RNA of her unaffected parents are normal. However, the father was demonstrated to be a mosaic carrier using leukocyte DNA. The fibroblasts of the proband whose serine substitution is at alpha 1(I) Gly352 synthesize type I procollagen chains with delayed electrophoretic migration; normally migrating forms are difficult to detect. Only alpha 1(I) CB 8 displayed delayed migration. Helix thermal stability is reduced 2 degrees C. Parental genomic DNA was normal.
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PMID:Serine for glycine substitutions in type I collagen in two cases of type IV osteogenesis imperfecta (OI). Additional evidence for a regional model of OI pathophysiology. 809 76


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