Characterization of the 5' flanking region of rat glucokinase gene

The rat glucokinase (GK) gene containing the first exon was isolated and its 5' flanking region was characterized by the bacterial chloramphenicol acetyltransferase (CAT) assay. A transient expression assay with a series of 5' deletion constructs (-5.5 k to -48) of GK-CAT fusion genes indicated that the 5' flanking sequence up to nucleotide -87 was sufficient for promoter activity in adult rat hepatocytes, but its activity was much weaker than that of the SV40 enhancer/promoter. Similar promoter activity was also detected in dRLh-84 hepatoma cells, which do not express glucokinase. Insulin treatment caused no change in the CAT activity of hepatocytes transfected with the fusion genes. These results suggest that the 5' flanking region of the glucokinase gene up to -5.5 k does not contain enhancer elements responsible for tissue-specific expression or insulin regulation.     

Major cis-regulatory elements for rice bidirectional promoter activity reside in the 5′-untranslated regions

Bidirectional promoters are defined as those that regulate adjacent genes organized in a divergent fashion (head to head orientation) and separated by < 1 kb. In order to dissect bidirectional promoter activity in a model plant, deletion analysis was performed for seven rice promoters using promoter-reporter gene constructs, which identified three promoters to be bidirectional. Regulatory elements located in or close to the 5′-untranslated regions (UTR) of one of the genes (divergent gene pair) were found to be responsible for their bidirectional activity. DNA footprinting analysis identified unique protein binding sites in these promoters. Deletion/alteration of these motifs resulted in significant loss of expression of the reporter genes on either side of the promoter. Changes in the motifs at both the positions resulted in a remarkable decrease in bidirectional activity of the reporter genes flanking the promoter. Based on our results, we propose a novel mechanism for the bidirectionality of rice bidirectional promoters.