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

In order to locate the promoter region of the human alpha 2A adrenergic receptor gene we used RNase protection analysis and antisense RNA probes to map the cap site of the alpha 2 transcripts. Prior sequence analysis has shown two potential TATA box motifs in the human alpha 2A adrenergic receptor gene, TATATAT and TATAAAA, located 427 and 1037 base pairs (bp), respectively, upstream of the protein coding region. RNase protection experiments and primer extension show that transcription starts downstream of the distal TATAAAA, indicating that the 5'-untranslated region is approximately 1 kilobase in length. We have used the chloramphenicol acetyltransferase reporter gene and transient transfection into HT29, a human adenocarcinoma cell line that expresses the alpha 2A receptor, to show that as little as 150 bp upstream of the cap site can direct transcription. Sequence analysis shows that although this region contains the TATA box motif it lacks a CCAAT box motif. DNase I footprint analysis of a fragment from -17 to -193 (where +1 is the transcription initiation site), using nuclear extracts from HT29, showed hypersensitive sites (-68/-69) and two protected regions: -70 to -87, which includes a 10-bp palindrome, and -92 to -105, which includes a GC box, a common motif for Sp1 nuclear factor binding. Gel mobility shift assays indicate that Sp1 or a related factor may bind to this GC box. Deletion of the GC box and the palindrome from chloramphenicol acetyltransferase constructs abolishes transcription. We propose that these cis sequences may function in lieu of a CCAAT box to regulate transcription of the human alpha 2A adrenergic receptor gene.
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PMID:Promoter region of the human alpha 2A adrenergic receptor gene. 138 31

Since cAMP has a number of important effects regulating activity of adrenergic receptor pathways, we wondered if expression of the alpha 2A adrenergic receptor gene is regulated by this second messenger. We have examined the effects of a cAMP analog (Bt2cAMP) on the expression of alpha 2A adrenergic receptors in HT-29 cells. Bt2cAMP induced a 5.3 +/- 0.8-fold increase in alpha 2A receptor mRNA abundance as did forskolin and vasoactive intestinal polypeptide which both increase cAMP accumulation in these cells. Bt2cAMP increased alpha 2A receptor number up to 2.4 +/- 0.3-fold. The rate of alpha 2A receptor gene transcription increased 7.8 +/- 3.2-fold in cells treated with Bt2cAMP for 2 h; after 24 h, the transcription rate was 3.7 +/- 1.7-fold higher than in controls. The increased rate of transcription occurred in the presence of the protein synthesis inhibitor cycloheximide. The half life of the alpha 2A receptor mRNA in cells incubated with Bt2cAMP for 2 h increased by 1.5-fold but returned to the original value after exposure to Bt2cAMP for 24 h. The increased expression of alpha 2A receptors was associated with an increased efficacy of inhibition of cAMP accumulation mediated by the alpha 2 adrenergic agonist UK14304. Using a chloramphenicol acetyltransferase (CAT) reporter plasmid containing 5'-flanking sequences of the alpha 2A receptor gene, we found that co-transfection of JEG-3 cells with expression vectors containing cAMP-dependent protein kinase regulatory subunit cDNA with mutations at both cAMP binding sites inhibited basal and Bt2cAMP-stimulated expression of CAT activity. These results demonstrate that an alpha 2A adrenergic receptor gene is regulated by the second messenger cAMP via cAMP-dependent protein kinase, mainly by controlling the rate of transcription, which leads to an increased expression of these receptors.
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PMID:cAMP regulates transcription of the alpha 2A adrenergic receptor gene in HT-29 cells. 184 58