Nucleotide sequence of the mouse ferritin H chain gene.
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Alleles↗
Biomedical subjects
C Beaumont
Publications and source records attributed to C Beaumont.
The mouse porphobilinogen deaminase gene. Structural organization, sequence, and transcriptional analysis.
The porphobilinogen deaminase gene encodes the third enzyme of the heme biosynthetic pathway. This gene is expressed in a tissue-specific manner and gives rise to two isoenzymatic forms encoded by mRNA species differing in their 5' extremity. Recent studies in human demonstrated that the tissue-specific expression of the porphobilinogen deaminase gene is determined in erythropoietic cells, by the utilization of a specific promoter situated 3' to the housekeeping promoter used in other cell types. This results, through differential splicing, in the mutually exclusive presence of either exon 1 or exon 2 in mature mRNAs. Here, we report the cloning and sequencing of the porphobilinogen deaminase gene from mouse. The overall organization of the mouse gene is similar to that of the human one. In the housekeeping promoter, only a short stretch of homology is found including two potential Sp1 binding sites; in contrast, more extensive similarity appears in the erythroid-specific promoter including two motifs also found in globin gene, a CACCC box, and a recently described Ery F1 consensus binding sequence. We derived a set of single-stranded probes corresponding to different parts of the mouse gene to carry out a detailed analysis of the transcriptional unit in various cell types, using a run-on transcription assay on isolated nuclei. In liver cells, the first (non-erythropoietic) exon is more actively transcribed than parts of the gene situated downstream, suggesting that the elongation of transcripts is blocked within the 5' part of the first intron. In erythropoietic cells, the downstream promoter becomes activated; surprisingly, the initiation of transcription is also enhanced from the upstream (housekeeping) promoter and most of the transcripts initiated at the housekeeping promoter stop downstream of the first exon, between the two promoters.
Amino Acid Sequence↗
A point mutation G----A in exon 12 of the porphobilinogen deaminase gene results in exon skipping and is responsible for acute intermittent porphyria.
We have determined the mutation in a patient with acute intermittent porphyria. The mRNA coding for porphobilinogen deaminase was reverse transcribed then the cDNA was enzymatically amplified in vitro. Upon sequencing of a polymerase chain reaction product of abnormal size we found that this fragment lacked exon 12 of the gene. We analysed a genomic fragment containing exon 12 and determined that the patient was heterozygous for a point mutation G A at the last position of exon 12. We propose that this base change is responsible for an abnormal processing of the mutant allele such that exon 12 is missing in the mature mRNA. The resulting aberrant mRNA encodes a truncated protein which is inactive but stable and can be detected using antibodies directed against the normal enzyme.
Acute Disease↗
Transcriptional regulation of ferritin H and L subunits in adult erythroid and liver cells from the mouse. Unambiguous identification of mouse ferritin subunits and in vitro formation of the ferritin shells.
Ferritin H and L subunits present cell-specific features of structure, function, and transcriptional regulation. Mouse Friend erythroleukemia cells offer an interesting model to analyze the erythroid-specific expression of ferritin genes for comparison with the liver, an iron-storing tissue. cDNA clones for mouse ferritin H and L subunits have been isolated and sequenced. The two subunits have very similar calculated masses, 20.9 and 20.6 kDa for H and L, respectively. Electrophoretic analysis of the subunits encoded by the cDNA 1) allows unambiguous identification of mouse ferritin subunits; 2) clearly shows that mouse H and L chains can make heteropolymers in vitro; and 3) demonstrates that, at least in vitro, free subunits can coexist with subunits polymerized into complete shells. The mouse ferritin gene family displays a variable degree of complexity, ranging from three homologous sequences for the H genes to 10-14 homologous loci for the L genes. Transcription of ferritin genes exhibits tissue-specific difference. Nuclear transcriptional run-off experiments show that the L gene is more actively transcribed in the liver than in Friend erythroleukemia cells at different stages of maturation. The accumulation of the H subunit mRNA which results from dimethyl sulfoxide induction of Friend cells is the consequence of an increase in the transcription rate of the H gene. However, the H gene mRNA is transcribed at a similar rate in the liver and in induced Friend cells although 5-fold more mRNA accumulates in these cells. Therefore, there is a tissue-specific regulation of mouse ferritin expression at both the transcription and mRNA stability levels.
Animals↗
Ferritin synthesis in differentiating Friend erythroleukemic cells.
We have investigated the regulation of ferritin synthesis during induction of Friend erythroleukemic cells by dimethyl sulfoxide. Northern blot analysis shows that mouse ferritin H and L mRNAs each contain approximately 1.1 kilobases. The levels of both mRNAs increase after addition of dimethyl sulfoxide in a biphasic manner. After a sharp rise in the first 6 h, the levels decline and then rise again over the next 90 h. These increases in mRNA levels reflect increased transcription of both mRNAs. Analysis of ferritin subunit synthesis surprisingly showed no corresponding increase in the rate of protein synthesis, suggesting that the additional mRNA was not in functional polysomes. These studies also indicated a novel processing of mouse ferritin H subunits. H subunits appear to be synthesized as a precursor of approximately 22,500. This form is not present in mature shells. Pulse-chase experiments indicated that the precursor is first processed to an intermediate form of 20,000 and then to the 18,000 component found in functional shells.
Animals↗
Tissue-specific expression of porphobilinogen deaminase. Two isoenzymes from a single gene.
Porphobilinogen deaminase (hydroxymethylbilane synthase; EC 4.3.1.8), the third enzyme of the heme biosynthetic pathway, catalyzes the stepwise condensation of four porphobilinogen units to yield hydroxymethylbilane, which is in turn converted to uroporphyrinogen III by cosynthetase. We compared the apparent molecular mass of porphobilinogen deaminase from erythropoietic and from non-erythropoietic cells by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immune-blotting. The results indicate that two isoforms of porphobilinogen deaminase can be distinguished and differ by 2000 Da. Analysis of cell-free translation products directed by mRNAs from human erythropoietic spleen and from human liver demonstrates that the two isoforms of porphobilinogen deaminase are encoded by distinct messenger RNAs. We cloned and sequenced cDNAs complementary to the non-erythropoietic form of porphobilinogen deaminase encoding RNA. Comparison of these sequences to that of human erythropoietic mRNA [Raich et al. (1986) Nucleic Acids Res. 14, 5955-5968] revealed that the two mRNA species differ by their 5' extremity. From the mRNA sequences we could deduce that an additional peptide of 17 amino acid residues at the NH2 terminus of the non-erythropoietic isoform of porphobilinogen deaminase accounts for its higher molecular mass. RNase mapping experiments demonstrate that the two porphobilinogen deaminase mRNAs are distributed according to a strict tissue-specificity, the erythropoietic form being restricted to erythropoietic cells. We propose that a single porphobilinogen deaminase gene is transcribed from two different promoters, yielding the two forms of porphobilinogen deaminase mRNAs. Our present finding may have some relevance for further understanding the porphobilinogen deaminase deficiency in certain cases of acute intermittent porphyria with an enzymatic defect restricted in non-erythropoietic cells.
Ammonia-Lyases↗
Porphyria cutanea tarda and HLA-linked hemochromatosis. Evidence against a systematic association.
This study was designed to test the hypothesis that a hemochromatosis allele is implicated in the expression of porphyria cutanea tarda. HLA phenotypes were determined in 69 porphyria cutanea tarda patients, 42 of which had the sporadic type (normal erythrocyte uroporphyrinogen decarboxylase activity) and 27 unrelated patients who had the familial type (diminished erythrocyte uroporphyrinogen decarboxylase activity). The incidence of HLA antigen A3, a marker of the hemochromatosis allele, was identical in the sporadic patients (23.8%), in the familial patients (22.2%), and in the controls (24.5%). Furthermore, no clinical difference could be found between A3 and non-A3 patients. These results demonstrate no systematic association between hemochromatosis and porphyria cutanea tarda in the population studied. Another HLA-linked gene, however, could be implicated in the expression of the disease as HLA antigen DR7 presented an incidence statistically different (p less than 0.05) between sporadic (16.6%) and familial patients (43%).
Adult↗
Equal value, equal pay.
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Economics, Nursing↗
[Improvement of the assay of serum ferritin using an international standard solution].
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Ferritins↗
Uroporphyrinogen decarboxylase structural mutant (Gly281----Glu) in a case of porphyria.
Uroporphyrinogen decarboxylase deficiency in man is responsible for familial porphyria cutanea tarda and hepatoerythropoietic porphyria. A recent study of a family with hepatoerythropoietic porphyria showed that the enzyme defect resulted from rapid degradation of the protein in vivo. Cloning and sequencing of a complementary DNA for the mutated gene revealed that the mutation was due to the replacement of a glycine residue by a glutamic acid residue at position 281. This base change leads to a protein that is very rapidly degraded in the presence of cell lysate. Characterization of the mutation will allow comparison of this defect in a homozygous patient with defects in other patients with familial porphyria cutanea tarda.
Amino Acid Sequence↗
Porphobilinogen deaminase is unstable in the absence of its substrate.
Porphobilinogen deaminase is induced during the dimethyl sulfoxide-mediated differentiation of Friend erythroleukemia cells. We have previously shown that when succinylacetone, a potent inhibitor of porphobilinogen formation, is present during the differentiation process, the induction of the enzyme is apparently suppressed. Here, we provide evidence that, in this condition, porphobilinogen deaminase is synthesized normally but does not accumulate as a consequence of an accelerated turnover. The normal half-life of the protein is 24 h but decreases to 10 h when the formation of its substrate is impaired by succinylacetone. We propose that when the enzyme is covalently bound to its substrate, a normal step in this enzymatic reaction, it is protected from proteolytic degradation, and we show that this new finding is relevant to the human disorder acute intermittent porphyria.
Ammonia-Lyases↗
Resistance to benzimidazole of Haemonchus contortus utkalensis in sheep on Martinique.
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Animals↗
Molecular analysis of uroporphyrinogen decarboxylase deficiency in a family with two cases of hepatoerythropoietic porphyria.
In order to determine the molecular basis of uroporphyrinogen (URO) decarboxylase deficiency responsible for hepatoerythropoietic porphyria (HEP) and familial porphyria cutanea tarda, we used a human URO decarboxylase cDNA to analyze the organization and expression of the URO decarboxylase gene in lymphoblastoid cells from normal individuals and from two patients with HEP. We could detect neither deletions nor rearrangements in the URO decarboxylase gene. Synthesis, processing, and cell-free translation of the specific transcripts appeared to be normal. The half-life of the abnormal protein was 12 times shorter than that of the normal enzyme. The results indicate that the enzyme defect is due to a rapid degradation of the protein in vivo. This study is the first to provide information regarding the molecular mechanism responsible for the URO decarboxylase deficiency in HEP.
Carboxy-Lyases↗
Sexual activity after coronary bypass surgery.
Although successful rehabilitation of coronary artery bypass surgery (CABS) patients should include consideration of their sexuality, there is a paucity of data regarding their sexual activity (SA). One hundred thirty-four patients were interviewed in regard to the impact of surgery on their sexuality and the relation of SA to their work status. Eighty-four of the 92 previously sexually active patients and two of the inactive ones resumed SA. Sexual dissatisfaction prior to surgery was a negative factor (p less than .05), while return to work, in the group that was working before, was positive (p less than .05) for resumption of SA. The average time before resumption of SA after CABS was 7.8 weeks. Thirty-nine percent of patients decreased the frequency of SA. Seventeen percent of patients and 35 percent of their partners expressed fear of resumption of SA. Twenty-three percent of patients had symptoms during intercourse. The couples who resumed sexual activity had a closer emotional relationship (p less than .02). Two-thirds of the patients received sexual instructions, but in only 20 percent of the cases did the physician himself initiate discussion. Although after CABS patients fare much better in regard to SA when compared to myocardial infarction patients reported in other studies, CABS does not provide a net gain in SA and sexual functioning. Comprehensive sexual counseling is still not being adequately addressed.
Adult↗
Accumulation of porphobilinogen deaminase, uroporphyrinogen decarboxylase, and alpha- and beta-globin mRNAs during differentiation of mouse erythroleukemic cells. Effects of succinylacetone.
We have monitored, during the dimethyl sulfoxide (Me2SO)-induced differentiation of MEL cells, the accumulation of mRNAs encoding two enzymes of the heme biosynthetic pathway, namely porphobilinogen deaminase and uroporphyrinogen decarboxylase. Our results demonstrate that the induction of these two enzymes is accounted for by a coordinate increase in their corresponding mRNAs, as estimated by hybridization with specific cloned cDNA probes. These events occur early during the differentiation process and precede the accumulation of alpha- and beta-globin mRNAs. Blocking the heme biosynthetic pathway with succinylacetone does not appear to modify the Me2SO-mediated increase of porphobilinogen deaminase and uroporphyrinogen decarboxylase mRNAs although succinylacetone has been shown to prevent the induction of immunoreactive porphobilinogen deaminase as well as its enzymatic activity (Beaumont, C., Deybach, J. C., Grandchamp, B., Da Silva, V., de Verneuil, H., and Nordmann, Y. (1984) Exp. Cell Res. 154, 474-484). Heme depletion resulting from the presence of succinylacetone in the culture medium reduces the extent of the Me2SO-mediated accumulation of alpha- and beta-globin mRNAs, and this effect is reversed by the addition of 10 microM exogenous hemin. Although the presence of succinylacetone prevents hemoglobinization of MEL cells, it does not prevent MEL cells from losing their proliferative capacity when treated with Me2SO.
Ammonia-Lyases↗
Hereditary hepatic porphyria with delta aminolevulinate dehydrase deficiency: immunologic characterization of the non-catalytic enzyme.
Immunoreactive delta-aminolevulinate dehydrase (ALA-D) was measured in lysates from two porphyric patients with ALA-D deficiency (enzyme activities were below 2% of the normal level). By using two different immunologic methods, we found a cross-reactive immunologic material (CRIM+) which corresponded to 20% and 33% of the control level. Therefore the molecular basis that accounts for the deficiency of ALA-D in these patients is a structurally modified enzyme. The methods used to determine the molecular weight (by Western blotting) and the isoelectric point (by chromatofocusing) of the mutants did not show any difference by comparison with the normal enzyme.
Electrophoresis, Polyacrylamide Gel↗
Is the HLA-linked haemochromatosis allele implicated in idiopathic refractory sideroblastic anaemia?
In order to assess the previously reported association of HLA-linked idiopathic haemochromatosis with idiopathic refractory sideroblastic anaemia (IRSA), the prevalence of HLA-A3 antigen in a group of 22 patients with IRSA was compared to that observed in healthy controls and in patients with homozygous idiopathic haemochromatosis and to that calculated for a population heterozygous for idiopathic haemochromatosis. The prevalence of A3 in patients with IRSA (0.23) was quite similar to that observed in controls (0.29) and significantly different from that observed in homozygous (0.73; P less than 10(-5] and heterozygous (0.57; P less than 10(-3] haemochromatosis. Serum iron, transferrin saturation, serum ferritin and liver iron concentration showed no difference in IRSA patients with or without A3. It is concluded that there is neither systematic association between the haemochromatosis allele and IRSA nor systematic implication of such an allele in the development of iron overload observed in IRSA.
Aged↗
Effects of succinylacetone on dimethylsulfoxide-mediated induction of heme pathway enzymes in mouse friend virus-transformed erythroleukemia cells.
Heme has been reported to exert a control over its own biosynthesis and to affect the erythroid differentiation process at different sites. In this study, succinylacetone, a powerful inhibitor of delta-aminolevulinic acid dehydrase was used to block heme synthesis and to study the effects of heme depletion on the dimethylsulfoxide (DMSO)-mediated induction of the heme pathway enzymes in Friend virus-transformed erythroleukemia cells. The presence of succinylacetone in the medium during the DMSO treatment (1) potentiates the induction of delta-aminolevulinic acid synthetase (the first enzyme of the pathway) and this effect is reversed by the addition of exogenous hemin; (2) does not affect the induction of delta-aminolevulinic acid dehydrase (the second enzyme); (3) prevents the induction of porphobilinogen deaminase (the third enzyme), since no increase could be detected in either the enzyme activity or the immunoreactive protein and this effect could not be reversed by the addition of exogenous hemin; (4) does not affect the induction of ferrochelatase. The possible role of heme or of intermediate metabolites of the pathway on the induction of these enzymes during the erythroid differentiation process is discussed.
5-Aminolevulinate Synthetase↗