CAT Assays Measure Transcriptional Activity

One way to assess promoter activity is to engineer and clone a DNA sequence containing a reporter gene attached to the promoter that is being assessed. When this sequence, or construct, is introduced into eukaryotic cells, transcription will be initiated from the promoter if it is active, and the reporter gene will be transcribed and its protein product synthesized. Measuring the amount of this protein produced is thus a way to determine the activity of the promoter.

Most reporter genes are chosen because they encode proteins that can be easily measured, such as the enzyme chloramphenicol acetyltransferase (CAT), which transfers the acetyl group from acetyl-CoA to the antibiotic chloramphenicol (Figure 23-13). The more active the promoter, the more CAT will be produced within the transfected cell. By introducing mutations into promoter sequences and then assaying for promoter activity with the corresponding reporter gene, conserved sequence motifs have been identified within promoters. Another reporter gene, the firefly luciferase gene, is also convenient and easy to use. Luciferase activity is analyzed by the emission of light, which is detected by a luminometer.

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