1. The use of reporter genes to define DNA regulatory elements
The use of reporter genes to define DNA regulatory elements. A DNA fragment bearing regulatory cis-elements (triangles, square, circles in diagram) from the gene in question—in this example, approximately 2 kb of 5′-flanking DNA and cognate promoter—is ligated into a plasmid vector that contains a suitable reporter gene—in this case, the enzyme firefly luciferase, abbreviated LUC. As noted in Figure 38–10 in such experiments, the reporter cannot be present endogenously in the cells transfected. Consequently, any detection of these activities in a cell extract means that the cell was successfully transfected by the plasmid. Not shown here, but typically one cotransfects an additional reporter such as Renilla luciferase to serve as a transfection efficiency control. Assay conditions for the firefly and Renilla luciferases are different, hence the two activities can be sequentially assayed using the same cell extract. An increase of firefly luciferase activity over the basal level, for example, after addition of one or more hormones, means that the region of DNA inserted into the reporter gene plasmid contains functional hormone response elements (HRE). Progressively shorter pieces of DNA, regions with internal deletions, or regions with point mutations can be constructed and inserted upstream of the reporter gene to pinpoint the response element (see Figure 38–13).
Source: Structure, Function, & Replication of Informational Macromolecules, Harper's Illustrated Biochemistry, 30e
Citation: Rodwell VW, Bender DA, Botham KM, Kennelly PJ, Weil P. Harper's Illustrated Biochemistry, 30e; 2015 Available at: Accessed: October 22, 2017
Copyright © 2017 McGraw-Hill Education. All rights reserved