Biomedical subjects

M A Magnuson

Publications and source records attributed to M A Magnuson.

85 records · Page 5Linked to original sources

PubMed1989

Rat glucokinase gene: structure and regulation by insulin.

The glucokinase gene is 15.5-kilobases long, appears to be present as a single copy, and contains 10 exons that range in size from 96 to 977 base pairs. The transcription start site was located 127 nucleotides upstream from the translation initiation codon. The 5' flanking DNA contains several regions similar to defined promoter elements. These include a probable "TATA box," an Sp1 binding site, and several elements related to liver-specific gene expression. In addition, we determined that transcription of the glucokinase gene increased at least 20-fold when diabetic rats were treated with insulin for 2 hr.

Animals↗

PubMed1989

Structural characterization of the rat malic enzyme gene.

We have identified and characterized lambda bacteriophage clones containing genomic DNA encoding rat malic enzyme [(S)-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating); EC 1.1.1.40]. The malic enzyme gene is unexpectedly large, spanning at least 95 kilobases. It is divided into 14 exons that range in size from 76 to 1513 base pairs. The sizes and boundaries of the exons were determined by Southern blotting and DNA sequencing. The sequences at the 5' and 3' ends of each intron conformed to the consensus sequence for mammalian introns. S1 nuclease and primer-extension assays showed that transcription of the malic enzyme gene initiates at multiple sites, the strongest one at position -31 relative to the ATG. "TATA and CCAAT box" homologies are not present in the proximal promoter region. Analysis of the 3' end of the gene showed that the utilization of alternate polyadenylylation signals in exon 14 results in two mRNAs with 3' untranslated regions of 345 and 1345 nucleotides, respectively.

Amino Acid Sequence↗

PubMed1989

Coding nucleotide sequence of rat malic enzyme mRNA and tissue specific regulation by thyroid hormone.

A nearly full length ME cDNA has been obtained and sequenced. The identity has been established by comparison of the translated nucleotide sequence with the amino acid sequence of 7 tryptic peptides from purified ME. Northern analysis with this cDNA shows that ME mRNA consists of two different messages of about 27S and 21S. The size difference between two ME mRNAs (approximately equal to 27S and 21S) is attributed to the differences in the 3' noncoding regions. The relative ratios of the two ME mRNAs differ in various tissues examined (liver, heart, kidney, brain, lung, spleen, and testis). Their regulation by T3 is tissue specific with coordinate stimulation of both mRNAs in liver, heart and kidney, suggesting a single promoter for both mRNAs and no stimulation of either in the other tissues. T3 regulates ME mRNA synthesis via a dual-tissue specific mechanism by increasing the rate of transcription in liver and heart and stabilizing nuclear ME RNA sequences only in liver.

Amino Acid Sequence↗

PubMed1988

Location and characterization of two widely separated glucocorticoid response elements in the phosphoenolpyruvate carboxykinase gene.

Chimeric genes were constructed by fusion of various regions of the 5'-flanking sequence from the phosphoenolpyruvate carboxykinase (GTP) (PEPCK) gene to the chloramphenicol acetyltransferase-coding sequence and to simian virus 40 splice and polyadenylation sequences. These were used to demonstrate that two glucocorticoid regulatory elements (GREs) combine to confer glucocorticoid responsiveness upon the PEPCK gene in H4IIE hepatoma cells. Both elements, a distal one whose 5' boundary is located between -1264 and -1111 base pairs and a proximal one located between -468 and -420 base pairs relative to the transcription initiation site, act independently, in various positions and orientations, and upon the heterologous thymidine kinase promoter. Each element accounts for half of the maximal response of the chimeric genes. Therefore, two widely separated enhancerlike elements contribute equally to the response of the PEPCK gene to glucocorticoid hormones. Neither of the PEPCK GREs contains the TGTTCT consensus sequence associated with most other GREs.

Acetyltransferases↗

PubMed1988

Identification of basal and cyclic AMP regulatory elements in the promoter of the phosphoenolpyruvate carboxykinase gene.

Promoter elements important for basal and cyclic AMP (cAMP)-regulated expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene have been identified by analysis of a series of PEPCK promoter mutations in transfection experiments. Fusion genes containing wild-type and mutated PEPCK promoter sequences from -600 to +69 base pairs (bp) fused to the coding sequence for chloramphenicol acetyltransferase were studied. Internal deletion mutations that replaced specific bases with a 10-bp linker within the region from -129 bp to -18 bp of the PEPCK promoter were examined. In addition, wild-type and mutated DNA templates were used as probes in DNase I protection experiments to determine sites of protein-DNA interaction. The PEPCK promoter contains a binding site for nuclear factor 1-CAAT. Deletion of the 5' end of this binding site reduced the size of the DNase I footprint in this region but had no effect on promoter activity. In contrast, deletion or disruption of the 3' end of this binding site completely eliminated protein binding and reduced promoter activity by 50%. Deletion of core sequences of the cAMP regulatory element (CRE) resulted in loss of cAMP responsiveness and an 85% decrease in basal promoter activity, indicating that the CRE also functions as a basal stimulatory element. Mutation of the core sequence of the CRE resulted in loss of the DNase I footprint over the CRE. Internal deletions flanking the CRE showed no loss of induction by cAMP but did have reduced promoter activity. This delimits the CRE to an 18-bp region between nucleotides -100 and -82. Analysis of mutations that disrupted bases between the CRE and the initiation site identified a basal inhibitory element adjacent to a basal stimulatory element, both located just 3' of the CRE, as well as a basal stimulatory element coincident with the TATA consensus sequence centered at -27. These data demonstrate that several cis-acting elements are located within 130 nucleotides of the initiation site of the PEPCK gene and that the CRE is essential for both basal promoter activity and cAMP-regulated expression of this gene.

Animals↗

PubMed1987

Multihormonal regulation of phosphoenolpyruvate carboxykinase-chloramphenicol acetyltransferase fusion genes. Insulin's effects oppose those of cAMP and dexamethasone.

The multihormonal regulation of phosphoenolpyruvate carboxykinase (PEPCK) was studied using chimeric genes composed of various regions of the PEPCK gene promoter region fused to the coding sequence of the chloramphenicol acetyltransferase (CAT) gene. These constructions, transfected into H4IIE hepatoma cells, are regulated like the endogenous PEPCK gene: dexamethasone and cAMP both stimulate PEPCK-CAT gene expression and their effects are additive; insulin inhibits the individual or combined effects of these stimulatory agents; and insulin inhibits dexamethasone-stimulated PEPCK-CAT fusion gene expression in a concentration-dependent fashion that is half-maximal at 10(-11) M. The induction by dexamethasone and the inhibition by insulin is specific for the DNA sequences that flank the 5' end of the PEPCK gene because similar effects were not observed for a plasmid in which the promoter and enhancer sequences of simian virus 40 (SV40) are fused to CAT. These results imply that the DNA adjacent to the transcription start site of the PEPCK gene contains the cis-acting hormone response elements responsible for the multihormonal regulation of this gene, including the insulin response.

Acetyltransferases↗

PubMed1986

Transcriptional regulation by thyroid hormone of an mRNA homologous to a protease inhibitor.

We have previously cloned a cDNA of a rat liver mRNA, designated 4-12B, markedly induced by triiodothyronine (T3) at a pretranslational level [Magnuson, M.A., Dozin, B., & Nikodem, V.M. (1985) J. Biol. Chem. 260, 5906-5912]. Here we show that this hormonal effect is due in part to an increase of the rate of transcription of the 4-12B gene. In addition, the nucleotide sequence of 4-12B cDNA has been determined, revealing significant similarity with the sequences of the superfamily of serine protease inhibitors and a very high homology with contrapsin, a mouse serum trypsin inhibitor, at the level of nucleotide and amino acid sequence (77.9 and 66.8%, respectively). The optimized alignment of the putative reactive center region of 4-12B with four related members of this superfamily revealed that lysine-serine residues are located at the reactive site or adjacent to it, thus suggesting that the triiodothyronine-regulated rat 4-12B mRNA might code for a protease inhibitor with trypsin-like specificity. Although not enough data are presently available to assign definitively antitryptic activity to this protein, the high degree of similarity with members of the superfamily of serine protease inhibitors leaves no doubt that 4-12B is a member of this superfamily.

Amino Acid Sequence↗

PubMed1986

Thyroid hormone regulation of malic enzyme synthesis. Dual tissue-specific control.

The regulatory mechanism(s) involved in the tissue-specific induction of cytosolic malic enzyme (EC 1.1.1.40) by triiodothyronine (T3) have been investigated in rat liver and heart. In these two tissues, cellular malic enzyme mRNA accumulates to different extents in response to hormonal stimulation (11-16- and 3-4-fold above the respective basal levels) (Dozin, B., Magnuson, M. A., and Nikodem, V. M. (1985) Biochemistry 24, 5581-5586). To gain further insight into this pretranslational control, nuclear in vitro run-off transcription assays were performed and correlated with the levels of malic enzyme RNA sequences in cytoplasm. The data demonstrate that the rate of transcription of the malic enzyme gene is stimulated by T3 to similar extents in liver and heart (3-4-fold above the basal activity). In liver, T3 also promotes an additional increase in cellular malic enzyme mRNA. This additional effect could be due to a tissue-specific change in the rate of degradation of cytoplasmic mRNA or to an effect on malic enzyme mRNA in the nucleus.

Animals↗

PubMed1986

Coding nucleotide sequence of rat liver malic enzyme mRNA.

The nucleotide sequence of the mRNA for malic enzyme ((S)-malate NADP+ oxidoreductase (oxaloacetate-decarboxylating, EC 1.1.1.40) from rat liver was determined from three overlapping cDNA clones. Together, these clones contain 2078 nucleotides complementary to rat liver malic enzyme mRNA. The single open reading frame of 1761 nucleotides codes for a 585-amino acid polypeptide with a calculated molecular mass of about 65,460 daltons. The cloned cDNAs contain the complete 3'-noncoding region of 301 nucleotides for the major mRNA species of rat liver and 16 nucleotides of the 5'-noncoding region. Amino acid sequences of seven tryptic peptides (67 amino acids) from the purified protein are distributed through the single open reading frame and show excellent correspondence with the translated nucleotide sequence. The putative NADP-binding site for malic enzyme was identified by amino acid sequence homology with the NADP-binding site of the enoyl reductase domain of fatty acid synthetase.

Amino Acid Sequence↗

PubMed1985

Tissue-specific regulation of two functional malic enzyme mRNAs by triiodothyronine.

Rat liver malic enzyme (ME) synthesis is known to be regulated by 3,5,3'-triiodo-L-thyronine (T3). Hybridization of 32P-labeled ME cDNA with RNA extracted from normal and T3-induced livers (15 or 50 micrograms/100 g body weight for 10 days) showed an increase in the ME mRNA concentration by approximately 11-fold in T3-treated rats. ME activity and ME mass were stimulated to the same degree as ME mRNA. Northern blot analysis of either total or poly(A+) RNA revealed two distinct ME mRNAs (21 and 27 S) which were equally induced by T3 treatment. Both mRNAs were shown by in vitro translation assay to program the synthesis of the same immunoprecipitable protein corresponding to full-sized ME. From all the above, we concluded that both messages code for active enzyme. ME activity and ME mRNA were also detected in nonhepatic tissues for which different responses to T3 induction were observed without direct correlation with their respective content of T3 nuclear receptor. Increases in ME activity and level of hybridizable ME mRNA were seen 48 h after a single administration of T3 (200 micrograms/100 g body weight) in liver, kidney, and heart (10.3- and 15.5-, 1.7- and 2.6-, and 1.72- and 3.4-fold above basal values, respectively). Lower levels of induction could already be detected after 24 h, liver being the most stimulated tissue. ME was not affected in brain, lung, testis, and spleen. Northern blot analysis showed that both ME mRNAs are present in all tissues tested, although in different relative proportions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1985

Regulation of specific rat liver messenger ribonucleic acids by triiodothyronine.

A plasmid cDNA library was constructed using poly(A+) RNA isolated from the livers of rats treated with 3,5,3'-triiodothyronine (T3) and fed a high carbohydrate diet. This library was screened by differential colony hybridization with [32P]cDNA probes made from hypothyroid and hyperthyroid rat liver poly(A+) RNA to obtain clones representing T3-inducible mRNAs. Using plasmid cDNAs to 4 different T3-inducible mRNAs, we have studied by hybridization assay the responses of these mRNAs to different thyroidal steady states and to a high carbohydrate diet. The fold of induction (hypothyroid to hyperthyroid) varied from about 4.0 (mRNA 5-8D) to 13.2 (mRNA 4-12B). The linearity of response with regard to nuclear receptor occupancy was estimated by assessing the relative mRNA levels in a euthyroid state. Three of the mRNAs demonstrated nonlinear responses with the largest portion of the induction occurring in the euthyroid to hyperthyroid transition. An induction by the high carbohydrate diet was clearly seen for only one mRNA (5-8D) suggesting that these two pathways of induction are independent. In a study of the response kinetics of each mRNA to a nuclear receptor saturating dose of T3 in hypothyroid animals, an increase was seen within 4 h (the earliest time point examined) for one of the mRNAs. The other 3 mRNAs did not increase significantly until 8 h after the T3 dose. Northern analysis showed a single mRNA corresponding to each of these 4 clones with sizes ranging from about 1375 to 7600 bases. Two mRNAs (5-9E and 4-12B) were shown by hybrid-selected translation to code for proteins of molecular mass of about 27 and 46 kDa, respectively. The availability of several different cDNA probes to T3 responsive liver mRNAs should facilitate future studies on the mechanism of action of this hormone.

Animals↗

PubMed1983

Molecular cloning of a cDNA sequence for rat malic enzyme. Direct evidence for induction in vivo of rat liver malic enzyme mRNA by thyroid hormone.

Malic enzyme (EC 1.1.1.40) mRNA was partially purified to 12% of total mRNA activity (greater than 150-fold enrichment) from 3-5-3'-triiodo-L-thyronine-carbohydrate-stimulated rat liver by polysome immunopurification followed by oligo(dT)-cellulose chromatography. This preparation was used as the template for synthesis of cDNA on a pBR322-SV40 hybrid plasmid DNA primer which was then circularized with linker DNA and used to transform Escherichia coli. The resulting transformants were screened by in situ differential hybridization using 32P-labeled poly(A+)RNA prepared from uninduced and 3-5-3'-triiodo-L-thyronine-carbohydrate-induced rat livers. Of 750 transformants screened, 6 were found to hybridize differentially; 1 of these, prME, contained an insert of about 1250 base pairs and hybrid-selected an mRNA which directed synthesis of malic enzyme in a cell-free translation system. Using this cDNA as a probe, we demonstrated that the level of malic enzyme mRNA after thyroid hormone treatment was markedly increased and the size of the major malic enzyme mRNA was shown by Northern analysis to be about 2700 nucleotides in length.

Animals↗

PubMed1975

Evaluation of ristocetin-Willebrand factor assay and ristocetin-induced platelet aggregation.

Normal subjects, patients with various bleeding disorders, and patients with von Willebrand's disease were studied. All patients with von Willebrand's disease had decreased levels of ristocetin-Willebrand factor (range, 0 to 41%) as compared with all other subjects (range, 79 to 202%). Ristocetin-induced platelet aggregation of platelet-rich plasma was abnormal in all patients with von Willebrand's disease tested, and it was possible to correct this abnormal response by addition of normal platelet-poor plasma. Abnormal ristocetin-induced platelet aggregation was seen in patients with intrinsic platelet disorders or, on some occasions, in normal patients who had ingested aspirin. Ristocetin-induced platelet aggregation is not diagnostic, but it may be useful as a simple screening test for patients with possible von Willebrand's disease. In conjunction with other tests, the assay for ristocetin-Willebrand factor will be useful in diagnosis and evaluation of these patients.

Anticoagulants↗