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Stoney S Simons - One of the best experts on this subject based on the ideXlab platform.
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a negative Tyrosine Aminotransferase gene element that blocks glucocorticoid modulatory element regulated modulation of glucocorticoid induced gene expression
Molecular Endocrinology, 1996Co-Authors: Clayton D Collier, Hisaji Oshima, Stoney S SimonsAbstract:Tyrosine Aminotransferase (TAT) is the prototypic steroid-inducible gene. Recently, we have found that the modulation of TAT induction properties is reproduced by a novel cis-acting TAT gene element, the glucocorticoid modulatory element (GME). This GME lies about 1 kb upstream of the glucocorticoid response elements (GREs) of the TAT gene and binds a heterooligomer of two recently defined proteins. We now report the existence of an additional TAT gene element between the GME and the GREs that blocks the action of the GME and thus prevents the left shift in the glucocorticoid dose-response curve caused by the GME. This negative element has the properties of a silencer because its activity is relatively position- and orientation-independent. The interaction appears to be stoichiometric in that the effects of a single negative element can be overcome by a second GME. This negative element also has an intrinsic inhibitory activity in the absence of the GME. The majority of the negative element activity could...
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the factor binding to the glucocorticoid modulatory element of the Tyrosine Aminotransferase gene is a novel and ubiquitous heteromeric complex
Journal of Biological Chemistry, 1995Co-Authors: Hisaji Oshima, Daniele Szapary, Stoney S SimonsAbstract:Glucocorticoid induction of the Tyrosine Aminotransferase gene deviates from that of many glucocorticoid-responsive genes by having a lower EC50 and displaying more agonist activity with a given antiglucocorticoid. A cis-acting element, located 3646 base pairs upstream of the start of Tyrosine Aminotransferase gene transcription, has been found to be sufficient to reproduce these variations with heterologous genes and promoters (Oshima, H., and Simons, S.S., Jr. (1992) Mol. Endocrinol. 6, 416-428). This element has been called a glucocorticoid modulatory element, or GME. Others have called this sequence a cyclic AMP-responsive element (CRE) due to the binding of the cyclic AMP response element binding protein (CREB). We now report the partial purification and characterization of two new proteins (GMEB1 and -2) of 88 and 67 kDa that bind to the GME/CRE as a heteromeric complex. This purification was followed by the formation of a previously characterized, biologically relevant band in gel shift assays. By several biochemical criteria, the GMEBs differed from many of the previously described CREB/CREM/ATF family members. Partial peptide sequencing revealed that the sequences of these two proteins have not yet been described. Size exclusion chromatography and molecular weight measurements of the gel-shifted band demonstrated that the GMEBs bound to the GME as a macromolecular complex of about 550 kDa that could be dissociated by deoxycholate. Similar experiments showed that CREB bound to the GME as heteromeric complexes of about 310 and 360 kDa. As determined from gel shift assays, GMEB1 and -2 are not restricted to rat liver cells but appear to be ubiquitous. Thus, these novel GMEBs may participate in a similar modulation of other glucocorticoid-inducible genes in a variety of cells.
Hisaji Oshima - One of the best experts on this subject based on the ideXlab platform.
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a negative Tyrosine Aminotransferase gene element that blocks glucocorticoid modulatory element regulated modulation of glucocorticoid induced gene expression
Molecular Endocrinology, 1996Co-Authors: Clayton D Collier, Hisaji Oshima, Stoney S SimonsAbstract:Tyrosine Aminotransferase (TAT) is the prototypic steroid-inducible gene. Recently, we have found that the modulation of TAT induction properties is reproduced by a novel cis-acting TAT gene element, the glucocorticoid modulatory element (GME). This GME lies about 1 kb upstream of the glucocorticoid response elements (GREs) of the TAT gene and binds a heterooligomer of two recently defined proteins. We now report the existence of an additional TAT gene element between the GME and the GREs that blocks the action of the GME and thus prevents the left shift in the glucocorticoid dose-response curve caused by the GME. This negative element has the properties of a silencer because its activity is relatively position- and orientation-independent. The interaction appears to be stoichiometric in that the effects of a single negative element can be overcome by a second GME. This negative element also has an intrinsic inhibitory activity in the absence of the GME. The majority of the negative element activity could...
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the factor binding to the glucocorticoid modulatory element of the Tyrosine Aminotransferase gene is a novel and ubiquitous heteromeric complex
Journal of Biological Chemistry, 1995Co-Authors: Hisaji Oshima, Daniele Szapary, Stoney S SimonsAbstract:Glucocorticoid induction of the Tyrosine Aminotransferase gene deviates from that of many glucocorticoid-responsive genes by having a lower EC50 and displaying more agonist activity with a given antiglucocorticoid. A cis-acting element, located 3646 base pairs upstream of the start of Tyrosine Aminotransferase gene transcription, has been found to be sufficient to reproduce these variations with heterologous genes and promoters (Oshima, H., and Simons, S.S., Jr. (1992) Mol. Endocrinol. 6, 416-428). This element has been called a glucocorticoid modulatory element, or GME. Others have called this sequence a cyclic AMP-responsive element (CRE) due to the binding of the cyclic AMP response element binding protein (CREB). We now report the partial purification and characterization of two new proteins (GMEB1 and -2) of 88 and 67 kDa that bind to the GME/CRE as a heteromeric complex. This purification was followed by the formation of a previously characterized, biologically relevant band in gel shift assays. By several biochemical criteria, the GMEBs differed from many of the previously described CREB/CREM/ATF family members. Partial peptide sequencing revealed that the sequences of these two proteins have not yet been described. Size exclusion chromatography and molecular weight measurements of the gel-shifted band demonstrated that the GMEBs bound to the GME as a macromolecular complex of about 550 kDa that could be dissociated by deoxycholate. Similar experiments showed that CREB bound to the GME as heteromeric complexes of about 310 and 360 kDa. As determined from gel shift assays, GMEB1 and -2 are not restricted to rat liver cells but appear to be ubiquitous. Thus, these novel GMEBs may participate in a similar modulation of other glucocorticoid-inducible genes in a variety of cells.
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Modulation of glucocorticoid induction of Tyrosine Aminotransferase gene expression by variations in cell density.
Endocrinology, 1992Co-Authors: Hisaji Oshima, S. Stoney SimonsAbstract:Glucocorticoids induce Tyrosine Aminotransferase (TAT) in hepatoma cells. We have previously shown that both the concentration of the agonist dexamethasone (Dex) required for half-maximal induction (EC50) and the amount of agonist activity produced by the antagonist dexamethasone 21-mesylate (Dex-Mes), expressed as a percentage of maximum induction achieved by Dex, are different in Fu5-5 and HTC cells. Furthermore, both activities vary over several weeks in each cell line in an apparently random manner, but, nevertheless, are correlated by a linear semilog plot. We now find that this long term and previously unpredictable variation in both the Dex EC50 and the amount of Dex-Mes agonist activity for the induction of TAT enzyme activity can be made to occur reproducibly in 40 h or less by changing the cell density and/or amount of medium in the tissue culture plates. Thus, a higher cell density and/or a lower volume of medium produced both higher amounts of Dex-Mes agonist activity and lower EC50 values for...
Gunther Schutz - One of the best experts on this subject based on the ideXlab platform.
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the distal enhancer implicated in the developmental regulation of the Tyrosine Aminotransferase gene is bound by liver specific and ubiquitous factors
Molecular and Cellular Biology, 1993Co-Authors: Doris Nitsch, Gunther SchutzAbstract:Tyrosine Aminotransferase gene expression is confined to parenchymal cells of the liver, is inducible by glucocorticoids and glucagon, and is repressed by insulin. Three enhancers control this tissue-specific and hormone-dependent activity, one of which, located at -11 kb, is implicated in establishing an active expression domain. We have studied in detail this important regulatory element and have identified a 221-bp fragment containing critical enhancer sequences which stimulated the heterologous thymidine kinase promoter more than 100-fold in hepatoma cells. Within this region, we have characterized two essential liver-specific enhancer domains, one of which was bound by proteins of the hepatocyte nuclear factor 3 (HNF3) family. Analyses with the dedifferentiated hepatoma cell line HTC suggested that HNF3 alpha and/or -gamma, but not HNF3 beta, are involved in activating the Tyrosine Aminotransferase gene via the -11-kb enhancer. Genomic footprinting and in vitro protein-DNA binding studies documented cell-type-specific binding of ubiquitous factors to the second essential enhancer domain, which by itself stimulated the thymidine kinase promoter preferentially in hepatoma cells. These results will allow further characterization of the role of these enhancer sequences in developmental activation of the Tyrosine Aminotransferase gene.
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perinatal activation of a Tyrosine Aminotransferase fusion gene does not occur in albino lethal mice
Mechanisms of Development, 1993Co-Authors: Friedrich Beermann, Erika Schmid, E Hummler, Gunther SchutzAbstract:To investigate developmental expression of the rat Tyrosine Aminotransferase (TAT) gene in normal and in albino lethal mice we generated transgenic mice carrying a fusion gene of TAT 5'-sequences (11 kb) and the bacterial chloramphenicol acetyltransferase (CAT) gene. In four lines, CAT activity was found only in liver. RNA analyses on a high-expressing line showed that transgenic expression follows expression of mouse TAT mRNA: it is inducible by glucocorticoids and activated perinatally. This perinatal activation of transgene expression does not occur in lethal albino mice (c14CoS/c14CoS) which are characterized by reduced mRNA levels of several liver-specific enzymes involved in gluconeogenesis. In conclusion, the data show that the 5'-flanking region of the rat TAT gene contains elements specifying regulated expression and establish that the 5'-flanking region of the TAT gene is responsive to the enzyme deficiency characteristic of the albino lethal mice.
Cristina Nowicki - One of the best experts on this subject based on the ideXlab platform.
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A recombinant Tyrosine Aminotransferase from Trypanosoma cruzi has both Tyrosine Aminotransferase and alanine Aminotransferase activities
FEMS microbiology letters, 1995Co-Authors: Marisa Montemartini, Jacqueline Búa, Esteban J. Bontempi, Cecilia Zelada, Andrés M. Ruiz, Joséa. Santomé, Juan Josécazzulo, Cristina NowickiAbstract:Tyrosine Aminotransferase purified from epimastigotes of Trypanosoma cruzi displays an additional activity of alanine Aminotransferase, absent in all other Tyrosine Aminotransferases characterized so far. Since the parasite's genome contains a high number of copies of the Tyrosine Aminotransferase gene, we could not rule out the possibility that two very similar proteins, with changed specificity due to a few amino acid substitutions, might be responsible for the two activities. We have now expressed in Escherichia coli a recombinant Tyrosine Aminotransferase as a fusion protein with glutathione S-trans-ferase. The purified fusion protein, intact or after thrombin cleavage, displays Tyrosine Aminotransferase and alanine Aminotransferase activities with apparent Km values similar to those for the natural enzyme, thus proving that they belong to the same protein.
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purification and partial structural and kinetic characterization of Tyrosine Aminotransferase from epimastigotes of trypanosoma cruzi
Biochemical Journal, 1993Co-Authors: Marisa Montemartini, Joséa. Santomé, Juan Jose Cazzulo, Cristina NowickiAbstract:Tyrosine Aminotransferase was purified to homogeneity from epimastigotes of Trypanosoma cruzi by a method involving chromatography on DEAE-cellulose, gel filtration on Sephacryl S-200 and chromatography on Mono Q in an f.p.l.c. system. The purified enzyme showed a single band in SDS/PAGE, with an apparent molecular mass of 45 kDa. Since the apparent molecular mass of the native enzyme, determined by gel filtration, is 91 kDa, the native enzyme is a dimer of similar subunits. The amino-acid composition was determined, as well as the sequences of three internal peptides obtained by CNBr cleavage at Met residues. Both criteria suggest considerable similarity with the Tyrosine Aminotransferases from rat and from human liver. The enzyme contains nine 1/2 Cys residues, three free and the others forming three disulphide bridges. The enzyme is not N-glycosylated. The isoelectric point is 4.6-4.8. The optimal pH for the reaction of the enzyme with Tyrosine as a substrate is 7.0. The apparent Km values for Tyrosine, phenylalanine and tryptophan, with pyruvate as a co-substrate, were 6.8, 17.9 and 21.4 mM, respectively, whereas those for pyruvate, alpha-oxoglutarate and oxaloacetate, with Tyrosine as a substrate, were 0.5, 38 and 16 mM respectively. The purified Tyrosine Aminotransferase acts as an alanine Aminotransferase as well and the activity seems to reside in the same enzyme molecule. The results suggest that the enzyme is a general aromatic-amino-acid transaminase, with high sequence similarity to Tyrosine Aminotransferases from rat and human liver.
David J Prieur - One of the best experts on this subject based on the ideXlab platform.
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comparison of the Tyrosine Aminotransferase cdna and genomic dna sequences of normal mink and mink affected with Tyrosinemia type ii
Journal of Heredity, 2005Co-Authors: Steven R Leib, Travis C Mcguire, David J PrieurAbstract:Type II Tyrosinemia, designated Richner-Hanhart syndrome in humans, is a hereditary metabolic disorder with autosomal recessive inheritance characterized by a deficiency of Tyrosine Aminotransferase activity. Mutations occur in the human Tyrosine Aminotransferase gene, resulting in high levels of Tyrosine and disease. Type II Tyrosinemia occurs in mink, and our hypothesis was that it would also be associated with mutation(s) in the Tyrosine Aminotransferase gene. Therefore, the transcribed cDNA and the genomic Tyrosine Aminotransferase gene were sequenced from normal and affected mink. The gene extended over 11.9 kb and had 12 exons coding for a predicted 454-amino-acid protein with 93% homology with human Tyrosine Aminotransferase. FISH analysis mapped the gene to chromosome 8 using the Mandahl and Fredga (1975) nomenclature and chromosome 5 using the Christensen et al. (1996) nomenclature. The hypothesis was rejected because sequence analysis disclosed no mutations in either cDNA or introns that were associated with affected mink. This suggests that an unlinked gene regulatory mutation may be the cause of Tyrosinemia in mink.
