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A Dubart

Publications and source records attributed to A Dubart.

At least 37 records · Page 2Linked to original sources
PubMed1987

Structure of the gene for human uroporphyrinogen decarboxylase.

Uroporphyrinogen decarboxylase, the fifth enzyme of the heme biosynthetic pathway, is an housekeeping enzyme whose activity is enhanced during erythropoietic differentiation. We have previously shown that this increased activity was in part accounted for by an enhanced transcription of the gene in erythropoietic tissues. To elucidate further the tissue specific regulation of an housekeeping gene we have isolated the human URO-D gene and determined its organization. The cloned gene comprises 10 exons spread over 3 Kb. Two transcriptional start sites were determined and analysis of 900 bp of the 5' flanking region suggests a very simple structural organization for the URO-D gene promoter. We also show that this gene is functional when transfected into mouse fibroblasts, and that its promoter is sensitive to a viral enhancer.

Animals↗
PubMed1986

Molecular cloning and complete primary sequence of human erythrocyte porphobilinogen deaminase.

We have cloned and sequenced a cDNA clone coding for human erythrocyte porphobilinogen deaminase. It encompasses the translated region, part of the 5' and the 3' untranslated regions. The deduced 344 amino acid sequence is consistent with the molecular weight and the partial amino-acid sequence of the NH2 terminal of the purified erythrocyte enzyme. Southern analysis of human genomic DNA shows that its gene is present as a single copy in the human genome and Northern analysis demonstrates the presence of a single size species of mRNA in erythroid and non-erythroid tissues and in several cultured cell lines. Quantitative determinations indicate that the amount of PBG-D mRNA is modulated both by the erythroid nature of the tissue and by cell proliferation, probably at the transcriptional level.

Amino Acid Sequence↗
PubMed1986

Molecular cloning and nucleotide sequence of a complete human uroporphyrinogen decarboxylase cDNA.

We have cloned and sequenced a full-length cDNA coding for human uroporphyrinogen decarboxylase. The deduced 367-amino acid sequence is consistent with the molecular weight, the partial amino acid sequence of cyanogen bromide peptides, and the total amino acid composition of the purified enzyme. Southern analysis of human genomic DNA shows that its gene is present as a single copy in the human genome, and Northern analysis demonstrates the presence of a single size species of mRNA in erythroid and non-erythroid tissues and in several cultured cell lines. We have also demonstrated that the level of uroporphyrinogen decarboxylase mRNA is markedly increased in tissues or cell lines of erythroid origin and that this is due to a tissue-specific transcriptional activation of the uroporphyrinogen decarboxylase gene.

Amino Acid Sequence↗
PubMed1986

Molecular cloning and sequencing of the human erythrocyte 2,3-bisphosphoglycerate mutase cDNA: revised amino acid sequence.

The human erythrocyte 2,3-bisphosphoglycerate mutase (BPGM) is a multifunctional enzyme which controls the metabolism of 2,3-diphosphoglycerate, the main allosteric effector of haemoglobin. Several cDNA banks were constructed from reticulocyte mRNA, either by conventional cloning methods in pBR322 and screening with specific mixed oligonucleotide probes, or in the expression vector lambda gt 11. The largest cDNA isolated contained 1673 bases [plus the poly(A) tail], which is slightly smaller than the size of the intact mRNA as estimated by Northern blot analysis (approximately 1800 bases). This cDNA encodes for a protein of 258 residues; the protein yielded 34 tryptic peptides which were subsequently isolated by h.p.l.c. Our nucleotide sequence data were entirely confirmed by the amino acid composition of these tryptic peptides and reveal several major differences from the published sequence; the revised amino acid sequence of human BPGM is presented. These findings represent the first step in the study of the expression and regulation of this enzyme as a specific marker of the erythroid cell line.

Amino Acid Sequence↗
PubMed1986

Assignment of human uroporphyrinogen decarboxylase (URO-D) to the p34 band of chromosome 1.

A cDNA probe corresponding to mRNA encoding human uroporphyrinogen decarboxylase (URO-D) was used to determine the chromosomal localization of the URO-D gene in the human genome. In agreement with previous studies, we have found that the locus for URO-D is located on chromosome 1 in hybrid cell mapping panels. The use of in situ hybridization allowed us to map the URO-D locus to band 1p34.

Animals↗
PubMed1984

Cell-free translation of messenger RNA for human bisphosphoglyceromutase.

mRNAs prepared from different human tissues were translated in a cell-free reticulocyte lysate system and, when present, the neosynthesized bisphosphoglyceromutase ( BPGM ) was specifically isolated by immunoprecipitation and analyzed by polyacrylamide gel electrophoresis. Analysis of the translation products showed that bisphosphoglyceromutase was synthesized in vitro with its mature molecular weight and messenger RNA specifying the synthesis of BPGM exhibited a sedimentation coefficient of 12 S in human reticulocytes. This synthesis seems to be highly tissue specific since we could not evidence any synthesis of this enzyme using mRNA obtained from non erythroid tissue. The BPGM synthesis represents 0.1% of the total neosynthesized non heme proteins in human reticulocyte and ten times less (0.01%) in human fetal liver.

Animals↗
PubMed1984

Cell-free translation of human uroporphyrinogen decarboxylase mRNAs.

Uroporphyrinogen decarboxylase was synthesized in a reticulocyte lysate cell-free system under the direction of messenger RNAs isolated from human fetal liver and from human reticulocytes. The enzyme was specifically isolated by immuno affinity chromatography. Analysis of the translation products showed that uroporphyrinogen decarboxylase was synthesized in vitro with its mature molecular weight. This enzyme represented 0.04% of the total neosynthesized proteins under the direction of fetal liver mRNA and about ten times less (0.005%) with reticulocyte mRNA.

Anemia, Sickle Cell↗
PubMed1984

Isolation and identification of a cDNA clone coding for rat uroporphyrinogen decarboxylase.

We have cloned and identified a DNA sequence complementary to the mRNA of uroporphyrinogen decarboxylase ( UroDCase ) from rat. This mRNA is a minor species (0.1%) of the total mRNA from anemic rat spleen. Poly(A)+ mRNA was enriched for UroDCase mRNA to 20% purity by a very efficient procedure involving two successive steps of preparative gel electrophoresis under various denaturing conditions. cDNA prepared from partially purified UroDCase mRNA (1% purity) was cloned in the Pst I site of pBR322 by using the homopolymeric G-C tailing method. Primary screening of 500 clones from this cDNA library was performed with a cDNA probe complementary to highly purified mRNA for UroDCase (20% purity) and UroDCase cDNA clones were finally identified by hybrid-selected translation. The rat cDNA clones obtained hybridize to human UroDCase mRNA. This will permit the isolation of the corresponding human gene and molecular analysis of porphyria cutanea tarda, the commonest type of porphyria.

Animals↗
PubMed1984

Molecular cloning of a cDNA sequence complementary to porphobilinogen deaminase mRNA from rat.

A cDNA clone containing sequences complementary to the mRNA coding for anemic rat spleen porphobilinogen deaminase (EC 4.3.1.8) has been isolated. A cDNA library was prepared from partially purified mRNA (1% purity). This library was then screened by colony hybridization, using a cDNA probe derived from porphobilinogen deaminase mRNA further enriched (10-20% purity) by gel electrophoresis in the presence of methylmercury hydroxide. Colonies hybridizing with the probe were analyzed by hybrid-selected translation using anemic rat spleen mRNA. Four recombinant plasmids containing porphobilinogen deaminase cDNA sequences were identified by specific immunoprecipitation of the translational product from hybrid-selected mRNA. Porphobilinogen deaminase mRNA was shown to contain 1800 bases by blot hybridization analysis. The cloned cDNA sequence consists of 1500 bases. Hybridization analysis of poly(A)+ RNA from uninduced and induced mouse erythroleukemic cells indicated that induction to erythroid differentiation by dimethyl sulfoxide results in a 10-fold increase of porphobilinogen deaminase mRNA. The rat cDNA clones hybridize to the corresponding sequences encoding human porphobilinogen deaminase. This property will be useful for isolation of human gene(s) and further characterization of the molecular lesion(s) responsible for acute intermittent porphyria.

Ammonia-Lyases↗
PubMed1982

Lasting Hb F reactivation and Hb A2 reduction induced by the treatment of Hodgkin's disease in a woman heterozygous for beta-thalassemia and the Swiss type of the heterocellular hereditary persistence of Hb F.

A remarkable augmentation of Hb F and a reduction of Hb A2 were observed in a Sicilian woman during and after a course of treatment for Hodgkin's disease. An inverse correlation between the proportion of Hb F and Hb A2 was found over an 8-year period, as well as in populations of red blood cells fractionated by density gradient. She exhibited two genetic defects, the Swiss type of heterocellular hereditary persistence of fetal hemoglobin and a beta-thalassemia trait, which were confirmed by the study of the hemoglobin synthesis and by a family study. The lasting reactivation of Hb F synthesis is attributable to the interaction of several acquired and inherited factors.

Adult↗
PubMed1981

[Antenatal diagnosis of sickle-cell anaemia by DNA analysis of amniotic fluid cells. A preliminary study in the French West-Indies (author's transl)].

The genetic polymorphism previously reported to be associated with the sickle-cell (beta S) gene in black U.S.A. citizens was studied in the population of two French West-Indies islands in order to evaluate its potential application to the antenatal diagnosis of sickle-cell anaemia. The polymorphism consists of a change in the DNA sequences located near the 3' end of the beta globin gene. The change can be detected by means of the restriction endonuclease Hpa I. When cellular DNA is digested with this enzyme, the beta globin gene is contained in a DNA fragment measuring either 7.6 or 13.0 kilobases (kb). In 70% of SS homozygous subjects in Martinique and 57% in Guadeloupe the beta S gene was carried by a 13.0 kb DNA fragment, whereas the normal beta A gene was carried by a 7.6 kb DNA fragment. This polymorphism would make it possible to detect the foetal beta S gene in the DNA of amniotic fluid cells by linkage analysis.

Amniotic Fluid↗
PubMed1981

Isoelectric focusing of human hemoglobins.

Isoelectric focusing on thin layer of acrylamide gel has been used for the characterization of 79 different variants of hemoglobin A. This method has replaced the cellulose acetate electrophoresis in clinical laboratories in Martinique, Guadeloupe and Creteil for the detection of abnormal hemoglobins in populations at risk. Up to now 15,000 samples from adults have been evaluated. In addition this method has been used for the screening of 7,000 cord blood samples and for the prenatal diagnosis of severe hemoglobinopathies.

Adult↗
PubMed1981

New techniques for the prenatal diagnosis of hemoglobinopathies.

Two new techniques have been devised for the prenatal diagnosis of hemoglobinopathies performed on fetal blood samples. Isoelectric focusing (IEF) of hemoglobins was compared to the classical chromatography of labelled globin chains for 51 fetal blood samples, 40 being obtained for prenatal diagnosis of hemoglobinopathies, in Paris. In all cases the two methods provided identical results. Adult hemoglobin was quantitatively evaluated. In addition blood samples obtained in other centers after abortion of 22 fetuses homozygous for beta thalassemia did not exhibited measureable amounts of Hb A by IEF. The fetal blood must be free of maternal contamination. If present maternal red blood cells can be completely eliminated by selective lysis using the 0RSKOV reaction. A chromatography of hemoglobins on Biorex 70 has been devised very recently to overcome the two limitations of IEF: First the contamination of fetal blood samples by maternal cells, and second the impossibility to evaluate Hb A when present in proportion below 1%. The chromatography of hemoglobins on Biorex 70 is performed in 75 minutes with 0.1 mg of hemoglobin present in membrane free hemolysate. The optical density recording allows to evaluate .5% of Hb A and to detect .1% of Hb A. In addition, the radioactivity profile of the chromatography can be determined. It has to be used in case of maternal contamination.

Anemia, Sickle Cell↗
PubMed1981

Beta-thalassemia and sickle cell disease in culture of early erythroid precursors: hemoglobin synthesis and ultrastructural study.

Hemoglobin synthesis was studied in culture of early erythroid precursors (BFU-E) from the blood of nine patients exhibiting sickle cell anemia and of 14 with various types of beta-thalassemia. The results concerning gamma gene expression and plating efficiency in heterozygotes for sickle cell anemia were similar to those of normal adults (gamma/alpha = 0.05; 65 BFU-E colonies/10(6) plated cells) while, in contrast, homozygotes for sickle cell disease exhibited average values higher than the controls (gamma/alpha = 0.18; 80 BFU-E colonies/10(6) plated cells). However, the results were very heterogeneous from one subject to another. In heterozygotes for beta-thalassemia, gamma gene expression and plating efficiency were both slightly higher than those for normal individuals (gamma/alpha = 0.095; 129 BFU-E colonies/10(6) plated cells). In patients homozygous for beta-thalassemia, a marked increase in plating efficiency and gamma-chain synthesis was constantly observed (gamma/alpha = 0.41; 221 BFU-E colonies/10(6) plated cells). The high proportion of gamma chain synthesis was not related to a positive selection of F cells, since the gamma/alpha ratio remained constant during the in vitro erythroid maturation. Furthermore, a major increase in free alpha chain proteolysis can be ruled out, since the beta/alpha ratio was of the same order of magnitude in culture and in freshly drawn cells. Thus, the increased Hb F synthesis in vitro was the consequence of a true stimulation of gamma gene expression, which permitted partial correction of the globin chain imbalance. Ultrastructural studies in two homozygotes for beta-thalassemia showed a marked decrease in the abnormalities of the erythroblasts derived from erythroid precursors in vitro in comparison to those from fresh bone marrow samples. In particular, Heinz bodies were much less numerous and a high frequency of mature erythroblasts and reticulocytes was observed in culture. These results support the view that, in sickle cell anemia and beta-thalassemia, a high potential for gamma gene expression exists and can be expressed in culture.

Anemia, Sickle Cell↗
PubMed1981

Study of hemoglobin synthesis by affinity chromatography on Sepharose haptoglobin.

An affinity chromatography system, using haptoglobin bound covalently to Sepharose 4B, has been developed to purify human hemoglobin from soluble non-heme proteins. Agarose-haptoglobin specifically binds hemoglobin. Globin chains were eluted from the agarose-haptoglobin after removal of the heme. This method has allowed accurate measurements of globin chain synthesis in blood and bone marrow samples from adults and in culture of early erythroid precursors present in adult blood or bone marrow.

Bone Marrow↗
PubMed1980

Fetal hemoglobin synthesis in culture of early erythroid precursors (BFU-E) from the blood of normal adults.

BFU-E from the blood of 14 normal adults have been grown by the plasma clot technique. The hemoglobins synthesized in burst colonies were purified from other proteins by affinity chromatography on Sepharose-haptoglobin. The radioactivity incorporated in the globin chains was estimated by CM-cellulose chromatography in urea. The number of bursts scored at the 14th day of culture fluctuated between 50-130 (average 86, s: 29) for 10(6) mononuclear plated cells. A constant reactivation of fetal hemoglobin was found (from 1.4% to 11%, mean value 5.8%, s:3.07), but was lower than previously described, mainly because of the highly selective purification of Hb. This reactivation of fetal hemoglobin was not dependent upon the concentration of erythropoietin (from 1 U/ml to 6 U/ml) nor on the purity of the erythropoietin preparations (from 6 U/mg of protein to 70 000U/mg of protein). In addition, the same subject exhibited a constant proportion of Hb F synthesized in culture over a period of time up to 6 months. A positive correlation exists between the proportion of Hb F in culture and that of F cells present in the blood, with the exception of two subjects. Such findings suggest that Hb F in culture is a characteristic of each individual and that this reactivation often represents an amplification of the Hb F synthesis in vivo.

Adult↗
PubMed1980

Globin-chain affinity chromatography on Sepharose-haptoglobin: a new method of study of hemoglobin synthesis in reticulocytes, in bone marrow and in colonies of erythroid precursors.

In the present work we have developed an affinity chromatography system, using haptoglobin bound covalently to Sepharose 4B, to purify hemoglobin from soluble non-heme proteins. Sepharose-haptoglobin specifically binds hemoglobin. It exhibits the same characteristics in its interactions with hemoglobin and alpha or beta hemoglobin chains as does haptoglobin in solution. Globin chains can be eluted from the Sepharose-haptoglobin after removal of the heme. This method has allowed accurate measurements of globin-chain synthesis in blood and bone marrow samples and in culture of early erythroid precursors.

Adult↗