Affinity labelling and cloning of steroid and thyroid hormone receptors / edited by H. Gronemeyer.

Date:
[1988]
Catalogue details

Licence: Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)

Credit: Affinity labelling and cloning of steroid and thyroid hormone receptors / edited by H. Gronemeyer. Source: Wellcome Collection.

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    302 Analysis of the human glucocorticoid receptor gene promoter [Ch. 16 CTAG 9876 54321 z Primer extention and SI nuclease ^ Protection of human GR gene Fig. 3 — Mapping of the human glucocorticoid receptor mRNA cap site. The Smal-Smal fragment of the HGR gene (Fig. 2) was subcloned into M13mpl8 and BSM13. The sequencing of the M13mpl8 clone with oligonucleotide 5'-TCCGCAGTTCCCGCCGCG-3' '338' as primer is shown on the left side. The BS plasmid containing the insert in the 5' to 3' orientation with regard to T3-RNA promoter was linearized at the Ncol site and a uniformly [^^P]CTP labeled anti-mRNA probe was synthesized using T7-RNA polymerase. 2 /ig of each polyA^ RNA from LNCaP (lane 3) and MCF-7 (lane 4) were hybridized with the anti-mRNA probe and digested with SI nuclease, and the protected fragments were separated on 7 M urea-6% Polyacrylamide sequencing gel. Lane 1 contained the labeled probe alone; lane 2 contained the probe hybridized to tRNA and SI digested. Primer extension mapping was performed using [^^P]-end-labeled primer '338', reverse transcriptase and polyA^ RNA from LNCaP (lanes 5 and 6) and MCF-7 (lanes 7 and 8) cells. The primer was extended in the presence and absence (lane 9) of RNA without treatment with SI nuclease. The results obtained are presented schematically on the right. probe was hybridized with 2 ßg each of LNCaP and MCF-7 polyA^ RNA and digested with nuclease SI, muhiple protected fragments were detected. The major fragments had lengths of 88, 81 and 80 bases (Fig. 3). The largest of these protected fragments co-migrated with the major primer extension product demonstrating that the 5' end of the primer is at the 3' end of the exon. The primer extension, nuclease SI mapping and sequencing data (Figs. 2 and 3) together suggest that the human glucocorticoid receptor cDNAs isolated from an MCF-7 cDNA library (Govindan et al., 1985, and unpublished results) extends to the