Promoter deletion assay

The methods used for the evaluation of regulation of gene expression are too numerous to be described in detail here. They include Northern analysis to determine levels of a particular mRNA, nuclear run on to determine whether an increase in mRNA is due to an increase in the rate of transcription, and promoter deletion analysis to identify specific elements in the promoter region responsible for the control of expression. Of much current interest is the use of microarrays that permit the study of the expression of hundreds to thousands of genes at the same time. Reverse transcriptase-polymerase chain reaction and RNase protection assay techniques are used to amplify and quantitate mRNAs, while the electrophoretic mobility shift assay is used to measure binding of a transcription factor to its specific DNA consensus sequence. 

Promoter Deletion Analysis/Reporter Gene Assays... 

Figure 2.9. Luciferase reporter gene assay/promoter deletion analysis. Cells are transfected with the CYP1A1 promoter reporter (luciferase) constructs in which the promoter region is increasing deleted. The cells are then treated with benzo[a]pyrene (an inducer of CYP1A1), and cells are harvested 24-48hr later and luciferase activity is measured. Promoter deletion analysis revealed enhancer element between -1140 and -1029. Contained within this region is xenobiotic response element (XRE) to which the aryl hydrocarbon receptor (AHR) binds.

A combination of promoter deletion analysis, linker scanning, site-directed mutagenesis, gain-of-function analysis, gel mobility shift assays, DNA methylation interference and DNase 1 footprinting have been used to search for AuxREs within auxin-responsive promoters. Two types of AuxREs have been identified using these experimental approaches. One of these is the ocs or as-1 element and the other is theTGTCNC element (discussed in Section 4.3.). 

The promoters of ecCopA and YacK are apparently regulated by CueR. Both copper and silver are inducers, but not zinc or mercury. The loss of copper activation at both promoters in the cueR deletion strain can be rescued by complementing it with a plasmid carrying the gene. Interaction between CueR and the ecCopA promoter has been shown with a DNase I protection assay. It showed that CueR binds in vitro to a sequence with dyad symmetry within a 19-spacer sequence in the promoter (Stoyanov et al., 2001). CueR is thus the primary copper-responsive activator of the ecCopA copper efflux system of Es. coli. 

A detailed functional analysis of the immunoglobulin promoter elements is not available. However, comparison of sequences from a number of immunoglobulin promoters revealed the presence of a well-conserved octanucleotide consensus (ATGCAAAT) in all VH promoters and its complement (ATTTGCAT) in all VL promoters as well as in the heavy chain enhancer [54,55], This octanucleotide has been shown to be essential for transcription in both VH and VK promoters, as deletion of it abolishes promoter activity [46,55,56]. Furthermore, in the presence of the IgH enhancer, the octanucleotide is a sufficient VH upstream promoter element as measured in transfection assays [57],... 

So far we have only described oncogenes that behave in a dominant manner when mutated. For example, the success of the transformation assay depended on the fact that the "activated" Ras protein is oncogenic in cells that have normal Ras. In contrast, there is another class of oncogenes that result from "loss-of-function" mutations and they have been called tumor-suppressor genes. Mutations or deletions of tumor-suppressor genes promote cancer... 

To analyzes the functional elements in the 2.8 kb fragment, several deletions were made in the 5 and the 3 region in a plasmid containing TK promoter and the sequence encoding luciferase. Transfection assays were performed with these various deleted constructs in HeLa cells. Preliminary transfection results seem to suggest that this localized PPRE element is not the only one in controlling thiolase B expression by PPARa. 

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