Isolation and characterization of the human tyrosine aminotransferase gene.
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Structure and sequence of the human gene for tyrosine aminotransferase (TAT) was determined by analysis of cDNA and genomic clones. The gene extends over 10.9 kbl and consists of 12 exons giving rise to a 2,754 nucleotide long mRNA (excluding the poly(A)tail). The human TAT gene is predicted to code for a 454 amino acid protein of molecular weight 50,399 dalton. The overall sequence identity within the coding region of the human and the previously characterized rat TAT genes is 87% at the nucleotide and 92% at the protein level. A minor human TAT mRNA results from the use of an alternative polyadenylation signal in the 3' exon which is present but not used at the corresponding position in the rat TAT gene. The non-coding region of the 3' exon contains a complete Alu element which is absent in the rat TAT gene but present in apes and old world monkeys. Two functional glucocorticoid response elements (GREs) reside 2.5 kb upstream of the rat TAT gene. The DNA sequence of the corresponding region of the human TAT gene shows the distal GRE mutated and the proximal GRE replaced by Alu elements. (+info)
Protein-protein interactions facilitate DNA binding by the glucocorticoid receptor DNA-binding domain.
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We have studied the interaction of the DNA-binding domain of the glucocorticoid receptor with a glucocorticoid response element from the tyrosine aminotransferase gene. This response element consists of two binding sites (half-sites) for the glucocorticoid receptor DNA-binding domain. The sequences of these two half-sites are not identical, and we have previously shown that binding occurs preferentially to one of the half-sites (Tsai, S.-Y., Carlstedt-Duke, J., Weigel, N. L., Dahlman, K., Gustafsson, J.-A., Tsai, M.-J., and O'Malley, B. W. (1988) Cell 55, 361-369). We show here that binding to the low affinity half-site is dependent on previous occupancy of the high affinity half-site. This facilitated binding is dependent on the distance between the two half-sites and their relative orientation but is not dependent on the integrity of the DNA backbone. This is consistent with a model where DNA binding is not only dependent on interactions between the protein and its DNA target sequence but is also influenced by interactions between the protein molecules bound. (+info)
mRNA levels and methylation patterns of the tyrosine aminotransferase gene in aging inbred rats.
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We have examined the mRNA levels and methylation patterns of the liver-specific tyrosine aminotransferase (TAT) gene in inbred female rats aged 6, 24 and 36 months. Northern hybridization analysis of total RNA showed a 65% decrease in the steady state transcript level of TAT in the liver of 24- and 36-month old rats as compared to 6-month old animals. The TAT gene as studied by Southern hybridization analysis using the isoschizomers Hpa II and Msp I was found to be hypomethylated in the liver as compared to spleen and brain at six CpG sites within the gene. Methylation at these sites remained unchanged during aging. (+info)
In vivo monitoring of a cAMP-stimulated DNA-binding activity.
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The transcriptional activity of the tyrosine aminotransferase (TAT) gene is influenced by two major signal transduction pathways, by glucocorticoids and by glucagon acting via cAMP. We analyzed the effect of cAMP on protein-DNA interactions in vivo and on the transcription rate of the TAT gene. We demonstrate that a cAMP-responsive element (CRE) is located in a tissue-specific DNase I-hypersensitive region, 3.6 kb upstream of the start site of transcription. By using the genomic footprinting technique, we show that this sequence is occupied by protein in uninduced cells and that the in vivo footprint is transiently increased upon cAMP induction. Protein binding at the TAT-CRE correlates with the rate of transcription of the TAT gene. Cycloheximide treatment reveals that the genomic footprint is subject to rapid turnover; however, subsequent cAMP induction in the continued presence of cycloheximide restores the footprint partially. We conclude that as a part of the signal transduction pathway, a cAMP-dependent, post-translational modification increases the DNA-binding activity of a protein to the TAT-CRE and thereby stimulates the transcription rate of the TAT gene. (+info)
Glucocorticoids locally disrupt an array of positioned nucleosomes on the rat tyrosine aminotransferase promoter in hepatoma cells.
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Transcriptional activation by steroid hormones is often associated with the appearance of a DNase I hypersensitive site resulting from a local alteration of the nucleoprotein structure of the promoter. For the mouse mammary tumor virus long terminal repeat, a viral promoter under glucocorticoid control, a model has been proposed: the appearance of the hormonodependent DNase I hypersensitive site reflects the displacement of a single precisely positioned nucleosome associated with the glucocorticoid responsive elements. To determine if such a mechanism is of general relevance in transcriptional activation by steroid hormones, we have investigated the nucleosomal organization of the rat tyrosine aminotransferase promoter over a 1-kilobase region that contains the glucocorticoid regulatory target. This region displays a hormonodependent DNase I hypersensitive site. In the absence of hormone, micrococcal nuclease digestion of nuclei demonstrates the presence of positioned nucleosomes, with cutting sites centered around positions -3080, -2900, -2700, -2800, -2255, and -2040. Treatment of the cells with dexamethasone induces a disruption of the chromatin structure over a relatively short stretch of DNA (approximately positions -2400 to -2650) that overlaps two nucleosomes. These observations suggest a strong similarity in the role of chromatin structure in glucocorticoid-dependent transcriptional activation of mouse mammary tumor virus and tyrosine aminotransferase promoters. (+info)
Abrogation of glucocorticoid receptor dimerization correlates with dissociated glucocorticoid behavior of compound a.
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Glucocorticoid receptors in Morris hepatomas and host liver and the correlation of biological activity with receptor levels.
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Glucocorticoid-binding macromolecules were examined in Morris hepatomas 7787, 5123tc, 3683F, 7800, and 3683 and the Reuber hepatoma H-35 with the use of the synthetic glucocorticoid, triamcinolone acetonide. The physical properties of the triamcinolone acetonide-binding macromolecules of the hepatomas indicate that they are specific glucocorticoid receptors. The equilibrium association constants (Ka), sedimentation coefficients, and sensitivity to sulfhydryl-blocking reagents were found to be similar when hepatoma receptors were compared with the known properties of the liver receptor. Probably the most convincing criterion that the triamcinolone acetonide-binding macromolecules from the hepatomas are specific receptors is that 50 to 90% of the receptor can be depleted from hepatoma cytosol by treating rats with cortisol. In adrenalectomized tumor-bearing rats, the receptor levels in hepatomas 7787, 7800, 5123tc, and H-35 are comparable to or greater than receptor levels of host liver. However, tryptophan oxygenase was not responsive to glucocorticoids in hepatoma 7800 although receptor levels were quite high, and there were no indications that the receptor molecules were altered. Hepatomas 3683 and 3686F have low levels of receptor which may be related to resistance of these tumors to glucocorticoid treatment. (+info)
Early effects of hypervitaminosis A on gluconeogenic activity and amino acid metabolizing enzymes of rat liver.
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In an earlier report from this laboratory, one of the early manifestations of hypervitaminosis A was shown to be a marked stimulation of hepatic gluconeogenesis. In the present study, effects of feeding 30,000 IU of retinyl palmitate to young rats (80-100 g), once daily, for 2 days on the incorporation of 14C-labeled precursors into glucose and glycogen by liver slices, levels of amino acids in blood and tissues, and activities of some important amino acid catabolizing enzymes in the liver were investigated. A stimulation of hepatic gluconeogenesis in hypervitaminosis A was indicated by the increased incorporation of 14C-labeled alanine and bicarbonate into glucose and glycogen by liver slices. Excessive intake of retinol caused a marked increase in the activities of hepatic alanine aminotransferase and ornithine aminotransferase and a decrease in that of tryptophan pyrrolase, without affecting those of tyrosine aminotransferase and serine dehydratase. The ratio of NADH:NAD in the livers of rats fed excess retinol was significantly increased. It is suggested that enhancement of glucoeogenesis in hypervitaminosis A is caused by a stimulation of gluconeogenic activity of the liver. (+info)