Isolation of a genomic sublibrary enriched for glucocorticoid-regulated genes.
Glucocorticoids regulate gene expression by causing the glucocorticoid receptor to bind to an enhancer-like DNA element termed the glucocorticoid regulatory element (GRE). The resultant effect on transcription of specific genes causes a cascade of intracellular events that determines the growth or differentiated function of the target tissue. Although virtually all animal tissues respond to glucocorticoids, it has proven difficult to elucidate the molecular events which underlie physiologically important glucocorticoid effects such as lymphocyte death or poor wound healing. In this paper, a tryptic fragment of the glucocorticoid receptor (17K-GR) is shown to bind selectively to DNA containing a GRE. When a mixture of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) region and plasmid vector DNA was extracted using the intact glucocorticoid receptor or the 17K-GR, the 17K-GR retained a greater proportion of LTR vs. plasmid DNA. The 17K-GR-LTR complex was also more resistant to salt extraction. Extraction of Bam HI-digested mouse genomic DNA resulted in enrichment of the pro-opiomelanocortin (POMC) gene 5' fragment (which contains a GRE) vs. the 3' fragment which does not. A mouse genomic phage library was enriched for GRE-containing sequences by extraction using the 17K-GR. The frequency of POMC-positive plaques was determined to gauge enrichment of down-regulated genes, and the frequency of phosphoenolpyruvate carboxy-kinase-positive plaques was determined to gauge enrichment of up-regulated genes. The frequencies obtained (1.2 x 10(-3) and 3.5 x 10(-3), respectively) indicated that a family of glucocorticoid-regulated genes totaling approximately 300 had been isolated in a genomic sublibrary.
Harrison
R W
RW
Division of Endocrinology/Metabolism, University of Arkansas for Medical Sciences, Little Rock 72205.
Lippman
S S
SS
Hendry
W J
WJ
Chien
M C
MC
D000818:Animals; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000302:isolation & purification*; D004268:DNA-Binding Proteins / Q000502:physiology*; D015698:Genomic Library; D005938:Glucocorticoids / Q000502:physiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D011965:Receptors, Glucocorticoid / Q000502:physiology*; D012045:Regulatory Sequences, Nucleic Acid
D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D005938:Glucocorticoids; D011965:Receptors, Glucocorticoid