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1.
Biochem Biophys Res Commun ; 227(2): 576-80, 1996 Oct 14.
Article in English | MEDLINE | ID: mdl-8878555

ABSTRACT

The effect of the presence of the regions 5' and 3' of the hIL-5 gene on the expression of recombinant hIL-5 in CHO-cells was investigated. The 3.2 kb hIL-5 gene-fragment was cloned and four different dexamethasone-inducible rhIL-5 mammalian expression-vectors were constructed, each containing a different part of the gene-fragment. Results indicated that deletion of the region 5' to the gene increases production three-fold whereas a four-fold decrease in production was observed with deletion of the region 3' to the gene. Deletion of both regions increased rhIL-5 production by only one and a half-fold. These results indicate that there are elements in a 928 bp region 3' to the gene, including the 3'-NTR, that have an enhancing or stabilizing effect on expression of hIL-5 in CHO-cells.


Subject(s)
Interleukin-5/biosynthesis , Interleukin-5/genetics , Sequence Deletion , Animals , CHO Cells , Cloning, Molecular , Cricetinae , Gene Expression , Humans , Mammals , Plasmids , Recombinant Proteins/biosynthesis , Regulatory Sequences, Nucleic Acid , Transfection
2.
Matrix Biol ; 15(2): 66-7; author reply 70, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8837006
3.
Clin Chim Acta ; 230(1): 1-8, 1994 Oct 14.
Article in English | MEDLINE | ID: mdl-7850987

ABSTRACT

Two patients, diagnosed with 3-methylglutaconic aciduria, who presented with diverse clinical and metabolic manifestations, were studied. Glycine conjugation as a possible detoxification mechanism in these two patients yielded negative results. Carnitine conjugates were however detected. 3-Methylglutarylcarnitine was observed in the urine of both patients, while one of the patients excreted detectable quantities of 3-methylglutaconylcarnitine, a previously unknown metabolite. The presence of this metabolite in urine samples from one patient and the apparent correlation between administered carnitine and the conjugate excretion profile seems to indicate that carnitine may play an important role in future therapeutic programmes.


Subject(s)
Acidosis/urine , Carnitine/analogs & derivatives , Glutarates/urine , Acidosis/drug therapy , Carnitine/therapeutic use , Carnitine/urine , Chromatography, Gas , Female , Fibroblasts/enzymology , Fibroblasts/metabolism , Glycine/analogs & derivatives , Glycine/urine , Humans , Hydro-Lyases/metabolism , Infant , Male , Spectrometry, Mass, Fast Atom Bombardment
4.
Clin Chim Acta ; 230(1): 91-9, 1994 Oct 14.
Article in English | MEDLINE | ID: mdl-7850997

ABSTRACT

The identity of two formerly novel citric acid analogues, homocitric acid and methylhomocitric acid, in urine samples from patients with propionic acidaemia was confirmed by gas chromatography and mass spectrometry. Authentic reference substances were synthesized using a Reformatskii reaction. Homocitric acid and methylhomocitric acid were detected as minor metabolites in the urine samples from propionyl coenzyme A carboxylase deficient individuals. It was shown that these substances can be formed by the citrate synthase condensation reaction of alpha-ketoglutarate with acetyl coenzyme A and propionyl coenzyme A, respectively.


Subject(s)
Acidosis/urine , Citrate (si)-Synthase/metabolism , Propionates/urine , Tricarboxylic Acids/urine , Acidosis/diagnosis , Carboxy-Lyases/deficiency , Female , Gas Chromatography-Mass Spectrometry , Humans , Infant, Newborn , Ketoglutaric Acids/chemistry , Male , Methylmalonyl-CoA Decarboxylase , Reference Standards , Tricarboxylic Acids/chemical synthesis
5.
J Inherit Metab Dis ; 17(6): 738-47, 1994.
Article in English | MEDLINE | ID: mdl-7707698

ABSTRACT

The absolute separation of the four stereoisomeric configurations of methylcitric acid can be achieved on a nonchiral stationary phase SE30 capillary column using the corresponding O-acetylated (tri-(-)-2-butyl ester derivatives. Identification of the separated isomers was done using methylcitric acid produced by si-citrate synthase and methylcitrate synthase of Candida lipolitica. si-Citrate synthase produces the (2S,3S)-, (2S,3R)- and a small amount of the (2R,3S)-isomers. Methylcitrate synthase produces the (2R,3S)-isomer, indicating that this enzyme is more stereospecific than the animal citrate synthase enzyme. The (2R,3R)-isomer may act as an inhibitor of aconitase.


Subject(s)
Citrate (si)-Synthase/metabolism , Citrates/chemistry , Citrates/biosynthesis , Gas Chromatography-Mass Spectrometry , Stereoisomerism
6.
Connect Tissue Res ; 29(1): 1-11, 1993.
Article in English | MEDLINE | ID: mdl-8339541

ABSTRACT

The molecular basis for Osteogenesis Imperfecta in a large kindred with a highly variable phenotype was identified by sequencing the mutant pro alpha 1 (I) protein, cDNA and genomic DNA from the proband. Fibroblasts from different affected individuals all synthesize both normal Type I procollagen molecules and abnormal Type I procollagen molecules in which one or both pro alpha 1 (I) chain(s) contain a cysteine residue within the triple helical domain. Protein studies of the proband localized the mutant cysteine residue to the alpha 1 (I) CB 8 peptide. We now report that cysteine has replaced glycine at triple helical residue 175 disrupting the invariant Gly-X-Y structural motif required for perfect triple helix formation. The consequences include post-translational overmodification, decreased thermal stability, and delayed secretion of mutant molecules. The highly variable phenotype in the present kindred cannot be explained solely on the basis of the cysteine for glycine substitution but will require further exploration.


Subject(s)
Collagen/chemistry , Collagen/genetics , Cysteine/analysis , Glycine/analysis , Osteogenesis Imperfecta/genetics , Amino Acid Sequence , Base Sequence , Chromatography, High Pressure Liquid , Collagen/metabolism , Cysteine/metabolism , DNA/genetics , Female , Fibroblasts/metabolism , Glycine/metabolism , Humans , Middle Aged , Molecular Sequence Data , Mutation/genetics , Osteogenesis Imperfecta/metabolism , Phenotype , Procollagen/analysis , Procollagen/chemistry , Procollagen/genetics , Protein Processing, Post-Translational
7.
J Inherit Metab Dis ; 16(5): 837-43, 1993.
Article in English | MEDLINE | ID: mdl-8295398

ABSTRACT

Prenatal diagnosis was performed in a family affected by generalized glutathione synthetase deficiency. The disorder is transmitted by autosomal recessive inheritance. The first child born in this family died of the disorder at 6 weeks of age. Prenatal diagnosis was performed in two subsequent pregnancies. Amniotic fluid samples were collected by amniocentesis in the 16th and 17th weeks of pregnancy, respectively. In the case of the second pregnancy the concentration of 5-oxoproline in the amniotic fluid was measured by stable isotope dilution, while both stable isotope dilution and glutathione synthetase activity measurements were employed in the prenatal analysis of the third pregnancy. The 5-oxoproline concentration in the second pregnancy was even lower than that of the controls and in the case of the third pregnancy the results fell within the control range. The second pregnancy resulted in the birth of a clinically healthy girl, and the outcome of 5-oxoproline concentration in a urine sample taken just after birth confirmed the unaffected state. The third pregnancy resulted in the birth of a healthy boy at term, and the 5-oxoproline concentration in his urine and the glutathione synthetase activity in haemolysates were determined. The results confirmed that this infant was also unaffected and he apparently had two normal alleles for the enzyme.


Subject(s)
Glutathione Synthase/deficiency , Metabolism, Inborn Errors/diagnosis , Prenatal Diagnosis , Amniotic Fluid/chemistry , Chromatography, High Pressure Liquid , Female , Gas Chromatography-Mass Spectrometry , Humans , Infant , Infant, Newborn , Male , Metabolism, Inborn Errors/genetics , Pedigree , Pregnancy , Pyrrolidonecarboxylic Acid/analysis
8.
Biochem J ; 257(2): 439-45, 1989 Jan 15.
Article in English | MEDLINE | ID: mdl-2649075

ABSTRACT

Fibroblasts from two lethal variants of osteogenesis imperfecta were shown to synthesize increased amounts of type IV procollagen. Previous studies established that one of these variants had a non-functional allele for the pro alpha 2 chain of type I procollagen, whereas the other pro alpha 2(I) allele contained a mutation leading to synthesis of shortened pro alpha 2(I) chains. In the two variants, the relative level of mRNA for pro alpha 1(IV) was 31 and 42% of the level of mRNA for pro alpha 1(I) chains. A value of less than 2% was found for a third lethal and four non-lethal variants of osteogenesis imperfecta. Immunofluorescent staining of fibroblasts from the two variants synthesizing increased amounts of type IV procollagen indicated that a homogeneous population of cells synthesized both type IV and type I procollagen. The results suggest that mutations in the type I procollagen genes that result in osteogenesis imperfecta can be associated with increased expression of the genes for type IV procollagen.


Subject(s)
Osteogenesis Imperfecta/genetics , Procollagen/genetics , Basement Membrane , Blotting, Northern , Cells, Cultured , Fibroblasts/metabolism , Fluorescent Antibody Technique , Gene Expression Regulation , Humans , Mutation , Procollagen/metabolism , RNA, Messenger/analysis , Skin/metabolism
9.
J Biol Chem ; 262(31): 15151-7, 1987 Nov 05.
Article in English | MEDLINE | ID: mdl-2822714

ABSTRACT

A chimeric gene was constructed in which sequences between 253 base pairs (bp) upstream of the start of transcription of the human pro-alpha 1(I) collagen gene and 117 bp downstream of that site were fused to the human alpha 1-globin gene, at a site immediately upstream of the globin initiation codon. Expression of this and subsequent chimeric gene constructions was investigated in microinjected Xenopus laevis oocytes. Presence of 253 bp of pro-alpha 1(I) collagen gene promoter sequences, containing a CAAT box and a TATA box, resulted in a relatively modest level of expression of the linked globin sequences. Lengthening of the pro-alpha 1(I) collagen gene promoter region to include 2400 bp of upstream sequences, increased transcription of the marker gene to some extent. Strong activation of transcription was obtained when a 782-bp fragment of the first intron of the collagen gene was introduced in chimeric constructions containing only 240 bp of the collagen promoter. An enhancing effect of the intron segment on expression of the marker gene was observed from downstream or upstream positions relative to the initiation site of transcription. Sequencing of the intron segment revealed the presence of four decanucleotide consensus sites for binding of the constitutive transcription factor Sp1, as well as an enhancer "core" motif. Our experiments also showed that the cis-acting region strongly enhance the activity of the pro-alpha 1(I) promoter in transfected fibroblasts. On the basis of these observations we conclude that the segment broadly located between +700 and +1300 in the first intron of the human pro-alpha 1(I) collagen gene contains cis-acting sequences with an enhancer effect on transcription of the gene.


Subject(s)
Genes , Introns , Procollagen/genetics , Transcription, Genetic , Base Sequence , Chimera , DNA Restriction Enzymes , Humans
11.
J Biol Chem ; 261(19): 9056-64, 1986 Jul 05.
Article in English | MEDLINE | ID: mdl-3722186

ABSTRACT

Synthesis of procollagen was examined in skin fibroblasts from a patient with a moderately severe autosomal dominant form of osteogenesis imperfecta. Proteolytic removal of the propeptide regions of newly synthesized procollagen, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, revealed the presence of type I collagen in which two alpha 1(I) chains were linked through interchain disulfide bonds. Fragmentation of the disulfide-bonded alpha 1(I) dimers with vertebrate collagenase and cyanogen bromide demonstrated the presence of a cysteine residue in alpha 1(I)CB8, a fragment containing amino acid residues 124-402 of the alpha 1(I) collagen chain. Cysteine residues are not normally found in the triple-helical domain of type I collagen chains. The heterozygous nature of the molecular defect resulted in the formation of three kinds of type I trimers: a normal type with normal pro-alpha(I) chains, a type I trimer with one mutant pro-alpha 1(I) chain and two normal chains, and a type I trimer containing two mutant pro-alpha 1(I) chains and one normal pro-alpha 2(I) chain. The presence of one or two mutant pro-alpha 1(I) chains in trimers of type I procollagen was found to reduce the thermal stability of the protein by 2.5 and 1 degree C, respectively. In addition to post-translational overmodification, procollagen containing one mutant pro-alpha 1(I) chain was also cleared more slowly from cultured fibroblasts. The most likely explanation for these disruptive changes in the physical stability and secretion of the mutant procollagen is that a cysteine residue is substituted for a glycine in half of the pro-alpha 1(I) chains synthesized by the patient's fibroblasts.


Subject(s)
Cysteine , Osteogenesis Imperfecta/genetics , Procollagen/biosynthesis , Skin/metabolism , Chymotrypsin , Cyanogen Bromide , Disulfides/analysis , Fibroblasts/metabolism , Genes, Dominant , Humans , Kinetics , Macromolecular Substances , Osteogenesis Imperfecta/metabolism , Peptide Fragments/analysis , Procollagen/genetics , Protein Conformation , Trypsin
12.
Nucleic Acids Res ; 13(7): 2207-25, 1985 Apr 11.
Article in English | MEDLINE | ID: mdl-2987845

ABSTRACT

Using a cDNA probe specific for the bovine Type II procollagen, a series of overlapping genomic clones containing 45 kb of contiguous human DNA have been isolated. Sequencing of a 54 bp exon, number 29, provided direct evidence that the recombinant clones bear human Type II collagen sequences. Localization of the 5' and 3' ends of the gene indicated that the human Type II collagen gene is 30 kb in size. This value is significantly higher than that of the homologous avian gene. The segregation of a polymorphic restriction site in informative families conclusively demonstrated that the Type II gene is found in a single copy in the human haploid genome. Finally, sequencing of a triple helical domain exon has confirmed that a rearrangement leading to the fusion of two exons occurred in the pro alpha 1(I) gene, following the divergence of the fibrillar collagens.


Subject(s)
DNA/isolation & purification , Procollagen/genetics , Amino Acid Sequence , Cloning, Molecular , DNA Restriction Enzymes/metabolism , Humans , Nucleic Acid Hybridization
13.
Nucleic Acids Res ; 13(8): 2815-26, 1985 Apr 25.
Article in English | MEDLINE | ID: mdl-2582365

ABSTRACT

A bovine cDNA library constructed from fetal cartilage RNA was screened with a pro alpha 1(II) collagen specific chicken cDNA. A recombinant clone (Bc 7), with an insert of 1 kb, was identified and shown to contain sequences exhibiting 85% homology with the chicken pro alpha 1(II) collagen C-propeptide. Interspecies comparison strongly suggested that one potential glycosylation site present in the avian C-propeptide is not utilized, since this site is absent in the bovine chain. In addition, two overlapping genomic clones (Pal 3 and Pal 4) were isolated and partially characterized. These clones span 23 kb of DNA and contain approximately 17 kb of the pro alpha 1(II) calf gene. Sequencing of exon 1 has determined the length of the 3' untranslated region and the exact location of the polyadenylation attachment site.


Subject(s)
Collagen/genetics , DNA/analysis , Amino Acid Sequence , Animals , Cartilage/analysis , Cattle , Chickens , Cloning, Molecular , DNA Restriction Enzymes/metabolism , Nucleic Acid Hybridization , Poly A/metabolism , RNA/metabolism , RNA, Messenger
14.
J Biol Chem ; 258(23): 14385-9, 1983 Dec 10.
Article in English | MEDLINE | ID: mdl-6689020

ABSTRACT

Homologous DNA fragments were prepared from cloned cDNAs for the pro-alpha 1(I) and pro-alpha 2(I) chains of human type I procollagen. The DNA fragments were then used to develop a dot blot hybridization assay for mRNAs for pro-alpha 1(I) and pro-alpha 2(I) chains in skin fibroblasts. In normal fibroblasts, the ratio of the steady state levels of the two mRNAs was 1.94 +/- 0.34 S.D. The ratio for the rates of synthesis of the two pro-alpha chains in the same cells was 1.84 +/- 0.13 S.D. Since the two ratios were essentially the same, the results indicated that the mRNAs for the two chains are translated at about the same rates. Therefore, there is no need to invoke translational control or more complex mechanisms to explain synthesis of pro-alpha 1(I) and pro-alpha 2(I) chains in a stoichiometry of 2:1. The dot blot hybridization assay was also used to examine the levels of the mRNAs in fibroblasts from several variants of osteogenesis imperfecta. In two of the variants, the ratios of the steady state levels of mRNAs for pro-alpha 1(I) and pro-alpha 2(I) chains were 3.05 and 2.52, respectively. In the same fibroblasts, the ratios for the rates of synthesis of the two chains were 2.99 +/- 0.43 and 2.45 +/- 0.16, respectively. Therefore, even though the ratios of the levels of the two mRNAs in the fibroblasts were abnormal, the two mRNAs were still translated at the same rates, and there was no evidence of differential regulation at the translational level.


Subject(s)
Gene Expression Regulation , Osteogenesis Imperfecta/genetics , Procollagen/genetics , Protein Biosynthesis , RNA, Messenger/metabolism , DNA/metabolism , Humans , Kinetics , Nucleic Acid Hybridization
15.
J Biol Chem ; 258(16): 10128-35, 1983 Aug 25.
Article in English | MEDLINE | ID: mdl-6309769

ABSTRACT

Three overlapping genomic clones covering 28 kilobases of the human pro-alpha 2(I) collagen gene have been isolated from a lambda phage library. The analysis of 12 introns and 12 exons in the 3' end region has shown that the human gene has a structure remarkably similar to that reported for the homologous chicken gene. One large intron, in the alpha-chain domain, contains an AluI sequence flanked by short direct repeats; a second AluI sequence is present 4 kilobases downstream from the termination codon. The analysis of the exon coding for the 3'-untranslated region has revealed that the pro-alpha 2(I) collagen gene transcribes at least four different mRNAs in cultured fibroblasts. The colinearity and exact location of the termini of these transcripts was determined by Northern blots, R-looping analysis, S1 protection, and DNA sequencing. The ends of two transcripts are closely preceded by the canonical polyadenylation signal (AAUAAA), whereas two of its variations (AUUAAA and AUUAA) precede the ends of the other two transcripts.


Subject(s)
Poly A/analysis , Procollagen/genetics , Base Sequence , DNA/analysis , Endonucleases/metabolism , Fibroblasts/analysis , Humans , Microscopy, Electron , Nucleic Acid Hybridization , Single-Strand Specific DNA and RNA Endonucleases
16.
J Biol Chem ; 258(12): 7721-8, 1983 Jun 25.
Article in English | MEDLINE | ID: mdl-6863261

ABSTRACT

Synthesis of type I procollagen was examined in skin fibroblasts from a proband with a lethal variant of osteogenesis imperfecta. The fibroblasts synthesized shortened pro-alpha 2(I) chains and these shortened chains accounted for all the pro-alpha 2(I) chains synthesized by the cells. In addition, there was a decrease in the relative rate of synthesis of pro-alpha 2(I) chains. Fragmentation of the shortened pro-alpha 2(I) chains with vertebrate collagenase and cyanogen bromide demonstrated that the shortening was in alpha 2(I)-CB3,5A, a fragment from about the middle of the chain containing amino acid residues 361 to 775. Based on the relative mobility in electrophoretic gels, the shortening was about 20 amino acid residues. The decreased synthesis of pro-alpha 2(I) chains was demonstrated by an increase in the ratio for the rates of synthesis of pro-alpha 1(I):pro-alpha 2(I) chains. It was associated with an increase in the ratio of mRNAs for pro-alpha 1(I):pro-alpha 2(I) in the cells. Fibroblasts from the father also demonstrated a decreased synthesis of pro-alpha 2(I) chains as reflected by an increase in the ratio of newly synthesized pro-alpha 1(I):pro-alpha 2(I) chains. No shortened pro-alpha 2(I) chains were seen in fibroblasts from either the father or the mother. The observations suggested that the proband inherited a nonfunctioning pro-alpha 2(I) gene from her father and that the gene for the shortened pro-alpha 2(I) chain probably arose from a sporadic mutation.


Subject(s)
Osteogenesis Imperfecta/metabolism , Procollagen/genetics , Cells, Cultured , Female , Fetus , Fibroblasts/metabolism , Genetic Variation , Humans , Kinetics , Macromolecular Substances , Molecular Weight , Osteogenesis Imperfecta/genetics , Peptide Fragments/analysis , Phenotype , Pregnancy , Skin/metabolism
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