Abstract
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.
Highlights
In order tolocate the promoter region of the human only apreceptor in human platelets andthe aZBreceptor was a z A adrenergic receptor gene we used RNase protec- the only azreceptor in neonatal rat lung, whereas other tissues tion analysis and antisensReNA probes to map the cap showed a mixtureof the two subtypes (Bylund, 1985).Recent site of the a2 transcripts
As in the case of many other genes of that encode for Gprotein-linked receptors, the protein coding region of the human azA adrenergic receptor gene is encoded in a single exon (Kobilka et al, 1987; Fraser et al, 1989)
Northern blot analysis of RNA from human spleen and HT29cells shows that the transcripdt erived from can direct transcription.Sequence analysis shows that the azA adrenergic receptor gene is 3.8 kb in size (Flordellis this region contains the TATA box motif it et al, 1990), indicating that the azA transcript contains long lacks a CCAAT box motif
Summary
Location of the Start Site of Transcription-In order to locate the promoter region of the human aZA adrenergic receptor gene, we first identified the 5' end of the a2transcript. Antiharvested, lysed by freeze-thawing, and assayed for @-galactosidase activity and CAT enzyme activity (Herbomel et al, 1984; Gorman et al, 1982).In order to correct for transfection efficiencies,CAT assays sense RNA probes and theRNase protection assay were used in order to map the transcription initiation site used in two were performed on cell extracts that contained equivalent amounts cell lines, HT29andHEL, that express the human a2A of @-galactosidaseactivity (Herbomel et al, 1984). Functional promoter, an antisense RNA probe of 259 nt (Fig. 1,probe a ) was used that overlaps this TATA box. Cells were pelleted at 1400 rpm for 5 min at 4 "C,resuspended in phosphate-buffered saline, repelleted and resuspended in 1packed cell volume of 10 mM HEPES, pH 7.9, promoter, transcription will start further upstream and the entire 259-nt region will be protected by the transcript.
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