Hence, only the coding region is actually translated into protein. Of the alternate exon 1 sequences identified, four correspond to exon 1 sequences previously identified in mouse, exons 11,15, 19, and 110 84,85 Most selleckchem alternative exons are located in a 3-kb CpG island upstream of exon 2 that exhibits substantial promoter selleck screening library activity in transfected cells (Figure 2). Ribonuclease protection assays demonstrate significant levels of six alternative exon 1 sequences in vivo in the
rat, with differential expression in the liver, hippocampus, and thymus presumably reflecting tissue-specific differences in promoter activity. The different promoters respond to different signals, Inhibitors,research,lifescience,medical which Inhibitors,research,lifescience,medical forms the basis for tissue-specific laterations in gene expression. Simply put, it is the process by which environmental or hormonal signals can alter GR expression in one region of the body, without affecting expression in another. Hippocampal RNA contains significant levels of the exon 17-containing GR mRNA variants expressed at undetectable levels in liver and thymus. These studies thus identify a brain-specific GR Inhibitors,research,lifescience,medical promoter, the exon 17 sequence. Figure 2.
Map of the noncoding exon 1 region of the glucocorticoid receptor (GR) gene cloned from rat hippocampus.83 The sequence of the critical exon 17 region is provided below, highlighting the NGFIA (nerve growth factor-induced clone A) consensus sequence. … In transient transfection experiments, a construct
encoding the entire regulatory region of the GR gene, including eight of the alternate exon 1 sequences and the splice acceptor site within the intron 5′ of exon 2, Inhibitors,research,lifescience,medical was fused to a lucif erase reporter gene. The lucif Inhibitors,research,lifescience,medical erase gene is activated by the coupled promoters and its activity thus reflects the ability of the regulatory sites to activate gene transcription – hence the term reporter gene. Fusion to the socalled reporter gene permits a measure of the degree to which individual sequences can potentially influence gene expression. This alteration in activity results from various sequences originating at any point within the regulatory region and, we Drug_discovery presume, represents the sum of the activity of individual promoters on the genomic DNA fragment. In subsequent studies examining the potency of the individual promoters, we found that the relative activity of the individual exon 1 sequences is similar, with one notable exception, the exon 17 promoter sequence. The fused exon 17 has the highest transcriptional activity of any single promoter construct. More recent studies confirm the transactivational effect of NGFIA at the exon 17sequence. We used a cotransfection model with human embryonic kidney (HEK) cells (intentionally aiming as far from the neural target as possible) with an NGFIA expression vector and an exon 17-luciferase construct.