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In Vitro Transcription and Translation Protocols pp 409–421Cite as

Transcriptional activation analysis by the Chloramphenicol Acetyl Transferase (CAT) enzyme assay

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 37))

Abstract

Gene expression is controlled by cis-regulatory elements. Generally, the most important elements that are required for transcription are contained in the promoter sequences located upstream of a gene (1). Eukaryotic RNA polymerase requires several accessory factors, such as TFIID, TFIIA, TFIIB, and TFIIF, as well as transcription factors that augment or regulate developmental expression of various genes (2, 3). Tissue-specific factors have been shown to bind DNA in a sequence-specific manner and interact with other transcription factors in order to regulate gene expression (3, 4). The sequences that many transcription factors bind to are located in both promoter and enhancer elements (5, 6). To study regulation of cloned promoter sequences, one needs to introduce these promoter sequences into specific cells, and by linking them to an appropriate reporter gene, one can estimate promoter activity by an increase in reporter gene activity either by enzymatic assay or mRNA expression. Various reporter genes can be used to measure transcription activity, such as chloramphenicol acetyl transferase (CAT), luciferase, β-galactosidase, and human growth hormone (7). To examine the transcription enhancement activity of a putative promoter/enhancer sequence, the cloned DNA fragment being evaluated is inserted upstream of a particular reporter gene-containing vector. The recombinant promoter-reporter gene construct is then introduced into an appropriate cell type, either by CaPO4 transfection or electroporation (8–10). Transcription activity can then be measured directly by estimating enzymatic activity or mRNA expression (11, 12). Generally, a control plasmid is also introduced and used to normalize transfection efficiency (13). After making corrections for this transfection efficiency and averaging replicate experiments, the expression of the reporter genes appear to be directly proportional to the transcriptional activity of the promoter sequences employed for such studies.

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Author information

Authors and Affiliations

  1. Laboratory of Molecular Oncology, National Cancer Institute, Frederick, MD

    Delores M Thompson, Alexandra Panayiotakis & Arun Seth

Authors
  1. David R. Hodge
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  2. Delores M Thompson
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  3. Alexandra Panayiotakis
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  4. Arun Seth
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Editor information

Editors and Affiliations

  1. Monash University Clayton, Victoria, Australia

    Martin J. Tymms

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© 1995 Humana Press Inc.

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Hodge, D.R., Thompson, D.M., Panayiotakis, A., Seth, A. (1995). Transcriptional activation analysis by the Chloramphenicol Acetyl Transferase (CAT) enzyme assay. In: Tymms, M.J. (eds) In Vitro Transcription and Translation Protocols. Methods in Molecular Biology, vol 37. Humana Press. https://doi.org/10.1385/0-89603-288-4:409

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  • DOI: https://doi.org/10.1385/0-89603-288-4:409

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-288-0

  • Online ISBN: 978-1-59259-524-2

  • eBook Packages: Springer Protocols

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