Nucleotide sequence of rat liver tyrosine aminotransferase gene fragment

The sequence of a 3677 nucleotide EcoRI fragment was determined that codes for part of the rat liver tyrosine aminotransferase gene. The sequence was compared with the previously determined cDNA sequence and the intron and exon boundaries were deduced.     

Nucleotide sequence of a reiterated rat DNA fragment

A reiterated component of rat DNA was isolated by restriction with HindIII endonuclease and polyacrylamide gel electrophoresis. Sequence analysis revealed that the fragment was 179 nucleotides long. Unlike the known 370N reiterated rat DNA fragment which contained a high m5C content (2.7 mole%), this repetitive element contained a rather low m5C content (0.5 mole%). The present rat repetitive sequence appeared to be of alpha-type as shown by its significant homologies with alpha DNA sequences of African green monkey and human. The possibility of sequence heterogeneity is discussed.     

Decreased glucocorticoid receptor activity following glucocorticoid receptor antisense RNA gene fragment transfection.

Depression is often characterized by increased cortisol secretion caused by hyperactivity of the hypothalamic-pituitary-adrenal axis and by nonsuppression of cortisol secretion following dexamethasone administration. This hyperactivity of the hypothalamic-pituitary-adrenal axis could result from a reduced glucocorticoid receptor (GR) activity in neurons involved in its control. To investigate the effect of reduced neuronal GR levels, we have blocked cellular GR mRNA processing and/or translation by introduction of a complementary GR antisense RNA strand. Two cell lines were transfected with a reporter plasmid carrying the chloramphenicol acetyltransferase (CAT) gene under control of the mouse mammary tumor virus long terminal repeat (a glucocorticoid-inducible promoter). This gene construction permitted assay of the sensitivity of the cells to glucocorticoid hormones. Cells were also cotransfected with a plasmid containing 1,815 bp of GR cDNA inserted in the reverse orientation downstream from either a neurofilament gene promoter element or the Rous sarcoma virus promoter element. Northern (RNA) blot analysis demonstrated formation of GR antisense RNA strands. Measurement of the sensitivity of CAT activity to exogeneous dexamethasone showed that although dexamethasone increased CAT activity by as much as 13-fold in control incubations, expression of GR antisense RNA caused a 2- to 4-fold decrease in the CAT response to dexamethasone. Stable transfectants bearing the GR antisense gene fragment construction demonstrated a 50 to 70% decrease of functional GR levels compared with normal cells, as evidenced by a ligand-binding assay with the type II glucocorticoid receptor-specific ligand [3H]RU 28362. These results validate the use of antisense RNA to GR to decrease cellular response to glucocorticoids.     

Comparative analysis of tryptophan oxygenase activity and glucocorticoid receptor under the influence of insulin

This investigation addresses the interaction of insulin (INS) and glucocorticoid (GC) signaling in the hepatic regulation of tryptophan oxygenase (TO) enzyme activity in the rat. Male Wistar rats (200-250 g b.w) received an injection of the different doses of INS (10, 25, 50, 70 and 100 microg/200 g b.w., i.p.) and were used for experiments 3 h and 18 h after INS administration. This study shows that maximum of TO activity was found at dose of 50 microg of INS with peak increases observed at 3 h and 18 h after injection of INS, while INS had no effect on TO activity in adrenalectomized rats. The analysis of INS effects on glucocorticoid receptor-complex (GC/GR complex) stability shows that complexes from INS-treated rats are less stable than those from control animals. In addition, INS-stimulated stability of glucocorticoid receptor (GR) protein was significantly increased from the controls. Furthermore, the results show that GC/GR complexes from INS-treated rats could be activated and accumulated at higher rate in cell nuclei of control animals. These data support the involvement of INS in modulation of GC signaling pathway which mediates, in part, the activity of TO.     

Nucleotide sequence of a cloned fragment of rat mitochondrial DNA containing the replication origin

The nucleotide sequence was determined for the 717 bp HapII subfragment (HapEcoA5) of the EcoRI-A fragment of rat mitochondrial DNA, which contains the heavy-strand replication origin. Analysis of the heavy-strand initiation segments released from the D-loop molecules has revealed that some 5'-ends of these initiation segments are linked to ribonucleotide(s) and are heterogeneous. Sequence analysis of the 5'-end portion of the initiation segment indicated that one of the start points of the deoxyribonucleotide polymerization corresponds to the 425th bp on the HapEcoA5. Two-fold rotational symmetry and palindrome structures, and a G-cluster sequence around the start point have been discussed in connection with unidirectional replication.