Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We previously identified a DNA binding element termed the upstream regulatory element (URE) that contains the consensus initiator sequence (Inr) in the upstream promoter of the rat prodynorphin gene. The URE displays specific binding to the upstream regulatory element binding protein (UREB1), a novel transcription regulator. Here, we report that the URE functions as a suppressor element. A series of chloramphenicol acetyltransferase reporters (pCAT) were constructed by subcloning either wild-type or mutated URE sequences into a pCAT reporter plasmid 5' of bases -135 to +58 of the prodynorphin gene. The basal -135 to +58 dynorphin promoter (pCAT 0.2) has robust transcriptional activity in Chinese hamster ovary (CHO) cells but not in rat pheochromocytoma PC12 cells. This robust transcriptional activity was completely inhibited in the presence of wildtype URE, whereas the mutations of the URE had no effect. Gel mobility shift assays showed that the complex formed by the URE and nuclear protein extracts can be competed by addition of wild-type URE oligonucleotide but not by specific mutations of the URE, defining particular bases required for protein interaction with the URE. The identical URE sequence is also found upstream in the promoter of human macrophage inflammatory protein 1 beta (hMIP 1 beta). The suppressive activity of the rat dynorphin URE can be replaced by the hMIP 1 beta URE. These data suggest that the URE may serve as a suppressor element in the regulation of dynorphin and hMIP 1 beta gene transcription.
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PMID:URE, an initiator (Inr)-like site, suppresses the promoter of the rat dynorphin gene. 907 Feb 43

To investigate the involvement of the cAMP/protein kinase A (PKA) second messenger system in mediating the hormonal regulation of prodynorphin expression, a normal, homologous cell transfection model was used. Ovarian granulosa cells, which express high levels of prodynorphin mRNA in response to hormonal stimulation, were obtained from the ovaries of gonadotropin-primed 26-day-old rats, pooled, and cultured under serum-free conditions. Cells were transfected with a plasmid construct (proDYN-CAT) containing approximately 2 kb of the rat prodynorphin gene (-1858 to +133 by relative to the transcription start site) fused to the chloramphenicol acetyltransferase (CAT) reporter gene. Cultures were cotransfected with either a control plasmid (pUC13) or the Mt-REV expression plasmid; Mt-REV overexpresses a mutated form of the RIalpha regulatory subunit of PKA which binds to and thereby inhibits the activity of the catalytic subunit. When granulosa cells were cotransfected with proDYN-CAT and a control plasmid, CAT activity was stimulated by human FSH (20 ng/ml), human chorionic gonadotropin (10 ng/ml), and 8-(4-chlorphenyl thio)-cAMP (0.5 mM) (10-, 11-, and 6-fold, respectively; P < 0.005). However, when cells were cotransfected with proDYN-CAT and Mt-REV there was a complete abolition of hormone and cAMP stimulation of CAT activity (P > 0.4 vs controls). The effect of Mt-REV cotransfection was specific since cotransfection with Mt-REVwt, a plasmid which overexpresses the normal, wild-type regulatory subunit, had no effect on hormonal responsiveness. Furthermore, cotransfection with Mt-CalphaEV or Mt-CbetaEV (which overexpress the alpha or beta catalytic subunits of PKA and thus mimic the effects of PKA activation) significantly stimulated prodynorphin promoter activity by 14-fold (P < 0.05). These results support the hypothesis that gonadotropin stimulation of granulosa cells increases prodynorphin promoter activity via a process which requires a functional PKA second messenger system. Thus, PKA may be a generalized mechanism by which stimulatory input increases cellular prodynorphin expression.
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PMID:Stimulation of prodynorphin gene expression requires a functional protein kinase A. 1991 70