Human methylenetetrahydrofolate reductase: isolation of cDNA mapping and mutation identification.
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Amino Acid Sequence↗
Biomedical subjects
R Rozen
Publications and source records attributed to R Rozen.
Assignment of the gene for the core protein II (UQCRC2) subunit of the mitochondrial cytochrome bc1 complex to human chromosome 16p12.
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Chromosome Mapping↗
Translational control of ornithine-delta-aminotransferase (OAT) by estrogen.
Ornithine-delta-aminotransferase (OAT) catalyzes the reversible transamination of ornithine to glutamate semialdehyde. OAT is abundant in liver, kidney and retina; hereditary deficiency of the enzyme leads to chorioretinal degeneration. Studies of OAT regulation in retinoblastomas have revealed an alternatively spliced OAT mRNA, which contains an additional exon (exon 2) in the 5' untranslated region. Estrogen and thyroid hormone were previously shown to increase OAT mRNA levels approximately 3-fold and 5-fold, respectively, in these cells. To determine the mechanism of hormonal action in retinoblastomas, we performed nuclear transcription assays and analyzed the distribution of OAT mRNAs in individual fractions of a polysome gradient. Thyroid hormone increased the rate of transcription of the OAT mRNA in these cells. Estrogen did not stimulate transcription; it was associated with increased translation, since it resulted in a shift of the major (spliced) OAT mRNA species into denser fractions of the polysome gradient. Cycloheximide treatment suggested that the latter effect was due to increased initiation of translation. The unspliced OAT mRNA, which is inefficiently compared to the spliced mRNA, was insensitive to estrogen in these experiments.
Alternative Splicing↗
Tritium dilution space measurement is not modified by a doubling in fluid intake.
The purpose of this study was to examine the effects of a doubling in water intake on the total body water measured by the tritium dilution technique. Overdrinking was obtained by presenting tap water and sweet water to the rats. Total body water was measured twice, tritium dilution vs. dessication. Body water volumes differed between the two methods but not between the two groups. Thus the isotopic dilution technique can be used to measure total body water regardless of the flux of fluid through the subject.
Adipose Tissue↗
Cystic fibrosis mutations in French Canadians: three CFTR mutations are relatively frequent in a Quebec population with an elevated incidence of cystic fibrosis.
The French-Canadian population in the Saguenay-Lac St. Jean region of northeastern Quebec has an elevated frequency of cystic fibrosis (CF). The average incidence of cystic fibrosis was 1 in 891 births and the prevalence of CF carriers was estimated to be 1 in 15. We tested for 10 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in 133 French-Canadian CF families from Quebec. Ninety-one families were from the Saguenay-Lac St. Jean region and 42 families were referred from other regions of Quebec. We detected the CFTR mutation in 93 and 92% of the CF chromosomes in the Saguenay-Lac St. Jean and the major-urban Quebec families, respectively. The two groups of French-Canadian families were significantly different for the proportions of CFTR mutations. The three most common mutations in the Saguenay-Lac St. Jean families were delta F508 (58%), 621 + 1G----T (23%), and A455E (8%); and in the major-urban Quebec families were delta F508 (71%), 711 + 1G----T (9%), and 621 + 1G----T (5%). These results provide evidence for the role of founder effect in the elevated incidence of cystic fibrosis in the Saguenay-Lac St. Jean population.
Cystic Fibrosis↗
Five mutations at the PAH locus account for almost 90% of PKU mutations in French-Canadians from eastern Quebec.
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Base Sequence↗
In vitro and in vivo correlations for I65T and M1V mutations at the phenylalanine hydroxylase locus.
Mutations at the phenylalanine hydroxylase (PAH) locus are the major cause of hyperphenylalaninemia. We have previously described four mutations (M1V, IVS12nt1, R408W, and S349P) at the PAH locus in French Canadians with ancestry in eastern Quebec. Here we report (1) identification of another mutation, on a haplotype 9 chromosome, which converts codon 65 from isoleucine (ATT) to threonine (ACT), (2) expression analysis of the I65T mutation in COS cells demonstrating 75% loss of both immunoreactive protein and enzyme activity, and (3) expression analysis of the most prevalent PKU allele (M1V) in eastern Quebec, showing nondetectable levels of PAH protein and activity, a finding compatible with a mutation in the translation initiation codon. Homozygosity for M1V and codominant inheritance of I65T/R408W were both associated with classical phenylketonuria.
Amino Acid Metabolism, Inborn Errors↗
Newborn screening for sickle cell and other hemoglobinopathies: a Canadian pilot study.
We estimated incidence of HbS disease in Quebec. It is approximately 9 cases per 100,000 births (equivalent to the incidence of the hyperphenylalanemias). Accordingly, we performed a voluntary pilot study in 9 self-identified ethnic groups; 3528 families were counselled about the relevance of newborn screening for hemoglobinopathies; and 2779 cord blood samples were collected (participation rate, 78.7%) and analyzed for Hemoglobin S and other hemoglobin variants by cellulose acetate electrophoresis. There were 95 (3.42%) positive tests on the initial (cord blood) samples, of which only 40 could be confirmed because of low participation in follow-up. We identified 8 false-positive tests; 7 had been classified initially as alpha-thalassemia trait and one as HbC heterozygosity on the first test. The relative frequency of hemoglobinopathy genes (confirmed) was: 52.5% HbS; 22.5% alpha-thalassemia; 22.5% other mutation; all but one patient with sickle cell disease were heterozygotes; the majority (71%) of HbS genes were accounted for by the 7% of screened newborns who were Black; a further 24% of the HbS genes were accounted for by 7% with Central American ancestry. Record linkage of the findings in heterozygotes for use later in life is an unsolved problem. Seventy five first-degree relatives of the 48 probands were screened in follow-up studies (64% of parents participated); 5 couples at risk for having a future child with a hemoglobinopathy were identified. Attitudes toward follow-up varied among the ethnic groups. The single family with an affected newborn (sickle cell anemia) was counselled effectively; the infant received penicillin prophylaxis.(ABSTRACT TRUNCATED AT 250 WORDS)
Anemia, Sickle Cell↗
Translational control of ornithine aminotransferase. Modulation by initiation factor eIF-4E.
Ornithine aminotransferase (OAT) is a mitochondrial enzyme expressed at high levels in liver, kidney, and retina. To characterize OAT regulation in retinal lines, we have been studying OAT synthesis in retinoblastomas RB355 and Y79. Our previous data (Fagan, R.J., Sheffield, W.P., and Rozen, R. (1989) J. Biol. Chem. 264, 20513-20517) indicated similar OAT mRNA levels in the two strains with 3-fold greater immunoreactive OAT protein and enzyme activity in Y79. To examine the regulatory mechanisms in these cell lines, we performed nuclear runoff experiments and characterized polysome-associated OAT mRNAs. The nuclear runoff data did not reveal any differences in transcription between the two strains. However, OAT mRNA of the RB355 strain was present in the lighter polysome fractions as compared with Y79. Treatment with cycloheximide, which slows the rate of elongation, indicated that initiation was decreased in RB355. Eukaryotic initiation factor eIF-4E mRNA and protein were reduced in RB355, suggesting that eIF-4E might be rate-limiting for OAT translation. Overexpression of a wild-type eIF-4E in RB355 shifted the OAT mRNA into denser fractions of the gradient and increased the amount of OAT protein to the level observed in Y79; overexpression of a mutant eIF-4E had no such effect. We previously identified an alternatively spliced OAT mRNA (containing exon 2) in these cells. This mRNA appeared in the lightest fractions of the gradient in both strains and was not affected by eIF-4E overexpression.
Cycloheximide↗
A rapid procedure for extracting genomic DNA from leukocytes.
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Blotting, Southern↗
Deficient synthesis of MTHFD, a trifunctional folate-dependent enzyme, in the CHO Ade E mutant.
MTHFD is a folate-dependent trifunctional protein comprised of three activities: N5,N10-methylenetetrahydrofolate dehydrogenase, N5,N10-methenyltetrahydrofolate cyclohydrolase, and N10-formyltetrahydrofolate synthetase. The enzymes catalyze sequential interconversion of tetrahydrofolate derivatives required for purine, methionine, and thymidylate synthesis. A Chinese hamster ovary cell line (Ade-E), reported to have reduced cyclohydrolase activity, was studied to characterize the nature of the mutation. Enzymatic assays showed reduced activities of all three enzymes of the polypeptide. Immunoblotting and immunoprecipitation of radiolabeled cell extracts indicated that MTHFD protein was greatly reduced or absent in the mutant. Northern analysis of a clonal derivative of Ade-E revealed normal levels of MTHFD mRNA. These results suggest that the mutation affects a posttranscriptional process in the synthesis of the trifunctional enzyme.
Amidophosphoribosyltransferase↗
A pseudogene on the X chromosome for the human trifunctional enzyme MTHFD (methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase).
The human trifunctional folate-dependent protein MTHFD has been mapped to chromosome 14q24 and to the X chromosome was identified by screening an X-chromosome-specific library. Amplification by the polymerase chain reaction (PCR) and sequencing of PCR products indicate that the sequence is an intronless pseudogene.
Aminohydrolases↗
Energy-metabolism adaptation in obese adults on a very-low-calorie diet.
In this study, six obese women received a very-low-calorie diet (VLCD) for 3 wk. At day 0, body composition was assessed with a bioelectric impedance analyzer. The evolution in lean body mass (LBM) during the VLCD was estimated from nitrogen balance, measuring urine and fecal losses and taking into account skin, nitrate, and menstrual losses to avoid underestimation bias that could explain the decreased ratio of resting metabolic rate (RMR) to LBM previously reported. RMR was measured at days 0, 3, 5, and 21. The RMR-LBM ratio declined significantly during the VLCD period and decreased faster during the first week; the day 3, day 5, and day 21 ratio values were 94%, 91%, and 82%, respectively, of the original. The RMR-LBM ratio decrease after 21 d of a VLCD was near that found in chronic undernutrition. Results of previous studies that did not find any drop in the RMR-LBM ratio in obese adults on VLCDs might be explained by their LBM-assessment methods.
Adaptation, Physiological↗
Deficiency of complex III of the mitochondrial respiratory chain in a patient with facioscapulohumeral disease.
Facioscapulohumeral disease (FSHD), an inherited neuromuscular disorder, is characterized by progressive wasting of specific muscle groups, particularly the proximal musculature of the upper limbs; the primary defect in this disorder is unknown. We studied a patient with FSHD to determine whether the mitochondrial respiratory chain was functionally abnormal. Muscle biopsy revealed fiber atrophy with patchy staining for oxidative enzymes. Electron microscopy of a liver section showed many enlarged mitochondria with paracrystalline inclusions. Decreased oxidation of the respiratory substrates-alanine and succinate-in skin fibroblasts suggested a deficiency of complex III of the electron-transport chain; cytochrome c oxidase activity (complex IV) was in the normal range. Biochemical analysis of liver supported the fibroblast data, since succinate oxidase activity (electron-transport activity through complexes II-IV) was reduced, whereas complex IV activity was normal. Furthermore, analysis of the cytochrome spectrum in liver revealed typical peaks for cytochromes cc1 and aa3, whereas cytochrome b (a component of complex III) was undetectable. Southern blot analysis of fibroblast mtDNA revealed no major deletions or rearrangements. Our study provides the first documentation of a specific enzyme-complex deficiency associated with FSHD.
Adult↗
Congenital deficiency of a 20-kDa subunit of mitochondrial complex I in fibroblasts.
The first component of the mitochondrial electron-transport chain is especially complex, consisting of 19 nuclear and seven mitochondrion-encoded subunits. Accordingly, a wide range of clinical manifestations are produced by the various mutations occurring in human populations. In this study, we analyze the subunit structure of complex I in fibroblasts from two patients who have distinct clinical phenotypes associated with complex I deficiency. The first patient died in the second week of life from overwhelming lactic acidosis. Severe complex I deficiency was evident in her fibroblasts, since alanine oxidation was markedly reduced whereas succinate oxidation was normal. Absence of a 20-kDa subunit was demonstrable when newly synthesized proteins were immunoprecipitated from pulse-labeled fibroblasts by anti-complex I antibody. Disordered assembly or decreased stability of the complex was suggested by deficiency of multiple subunits on Western immunoblots. The second patient exhibited a milder clinical phenotype, characterized by moderate lactic acidosis and developmental delay in childhood and by onset of seizures at 8 years of age. Oxidation studies demonstrated expression of the complex I deficiency in fibroblasts, but no subunit abnormalities were detected by immunoprecipitation or Western immunoblotting. This report demonstrates the utility of cultured fibroblasts in studying mutations affecting synthesis and assembly of complex I.
Alanine↗
Negative allesthesia and decreased endogenous opiate system activity in anorexia nervosa.
The combined effects of an intragastric load of glucose compared to water and of naltrexone compared to placebo were tested on preference for sucrose in six anorectic patients. While in normal subjects, glucose-induced negative allesthesia is known to disappear upon loss of weight, it persisted in anorexia nervosa (AN) despite a major weight loss; furthermore, in contrast with its effects in normoponderal subjects, naltrexone at the dose of 25 mg did not decrease the preference for sucrose nor did it enhance glucose-induced allesthesia. Basal plasma beta endorphin level determined by radioimmunoassay was higher in AN than in normal subjects (75 +/- 6.1 pmoles/l vs. 13 +/- 3.8 pmoles/l) (p less than 0.001). It is suggested that a decrease in endogenous system opiate activity might be associated with food refusal and body weight loss in anorexia nervosa.
Adolescent↗
Purification and characterization of methylenetetrahydrofolate reductase from human cadaver liver.
Methylenetetrahydrofolate reductase from human cadaver liver was purified to homogeneity. The purified enzyme had a molecular mass of 150 kDa. On SDS-polyacrylamide gel electrophoresis it was dissociated into a single fragment with a molecular mass of 39 kDa. In contrast, fresh lymphocyte enzyme extract showed a major band with a molecular mass of 75 kDa and a minor band of 39 kDa. Fresh liver enzyme was inhibited by S-adenosylmethionine while the purified enzyme from human cadaver liver was not inhibited. These observations suggest that human methylenetetrahydrofolate reductase is composed of two identical subunits of 75 kDa each but is cleaved into a major single band due to autolysis in cadaver liver. The purified cadaver enzyme was a FAD-specific protein. The pH optimum was 6.6 for methylenetetrahydrofolate-NADPH oxidoreductase, 6.5 for methyltetrahydrofolate-menadione oxidoreductase, and 7.2 for NADP-menadione oxidoreductase. The Km values of human liver methylenetetrahydrofolate reductase were 17 microns for NADPH and 38 microns for methyltetrahydrofolate in the reduction of menadione, and 12 microns for NADPH in the reduction of methylenetetrahydrofolate.
Cadaver↗
Recurrent mutation, gene conversion, or recombination at the human phenylalanine hydroxylase locus: evidence in French-Canadians and a catalog of mutations.
The codon 408 mutation (CGG----TGG, Arg----Trp) in exon 12 of the phenylalanine hydroxylase (PAH) gene occurs on haplotype 1 in French-Canadians; elsewhere this mutation (R408W) occurs on haplotype 2. A CpG dinucleotide is involved. The finding is compatible with a recurrent mutation, gene conversion, or a single recombination between haplotypes 2 and 1. A tabulation of 20 known mutations at the PAH locus reveals three instances of putative recurrent mutation.
Alleles↗