Advertisement

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

  • David R. Hodge
  • Delores M Thompson
  • Alexandra Panayiotakis
  • Arun Seth
Part of the Methods in Molecular Biology book series (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, 9, 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.

Keywords

Transfection Efficiency Human Growth Hormone Chloramphenicol Acetyl Transferase Chloramphenicol Acetyl Transferase Activation Normalize Transfection Efficiency 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Seth, A. and Papas, T. S. (1993) Principles of molecular cell biology of cancer: general aspects of gene regulation, in Cancer: Principles and Practice of Oncology (DeVita, V. T., Jr., Hellman, S., and Rosenberg, S. A., eds.), J. B. Lippincott, Philadelphia, pp. 23–34.Google Scholar
  2. 2.
    Klausner, R. D. and Hartford, J. B. (1989) Cis-trans models for post-transcriptional gene regulation. Science 246, 870–872.PubMedCrossRefGoogle Scholar
  3. 3.
    Shenk, T (1981) Transcriptional control regions: nucleotide sequences requirements for initiation by RNA polymerase I and II. Curr. Etop. Microbiol. Immunol. 93, 25–40.Google Scholar
  4. 4.
    Zwartkruis, F., Hoeijmakers, T., Deschamps, J., and Meijlink, F. (1991) Characterization of the murine Hox-2.3 promoter: involvement of the transcription factor USF (MLTF). Genes Dev. 33(3), 179–190.Google Scholar
  5. 5.
    Haberstroh, L., Galindo, J., and Firtel, R. A. (1991) Developmental and spatial regulation of a Dictyostelium prespore gene: cis-acting elements and a cAMP-induced, developmentally regulated DNA binding activity. Development 113, 947–958.PubMedGoogle Scholar
  6. 6.
    Serfling, E., Jasin, M., and Schaffner, W. (1985) Enhancers and eukaryotic gene transcription. Trends Genet. 1, 224–230.CrossRefGoogle Scholar
  7. 7.
    Alam, J. and Cook, J. L. (1990) Reporter genes: application to the study of mammalian gene transcription. Anal. Biochem. 188, 245.PubMedCrossRefGoogle Scholar
  8. 8.
    Pahl, H. L., Burn, T. C, and Tenen, D. G. (1991) Optimization of transient transfection into human myeloid cell lines using a luciferase reporter gene. Exp. Hematol. 10, 1038–1041.Google Scholar
  9. 9.
    Ray, J. and Gaga, F. H. (1992) Gene transfer into established and primary fibroblast cell lines: comparison of transfection methods and promoters. Biotechniques 13, 598–603.PubMedGoogle Scholar
  10. 10.
    Nickoloff, J. A. and Reynolds, R. J. (1992) Electroporation-mediated gene transfer efficiency is reduced by linear plasmid carrier DNAs. Anal. Biochem. 205(2), 237–243.PubMedCrossRefGoogle Scholar
  11. 11.
    Martin, J. D. (1990) Application of the two-phase assay for chloramphenicol acety 1 transferase (CAT) to transfections with simian virus 40-CAT plasmids. Anal. Biochem. 191, 242–246.PubMedCrossRefGoogle Scholar
  12. 12.
    Sankaran, L. A. (1992) Simple quantitative assay for chloramphenicol acetyltransferase by direct extraction of the labeled product into scintillation cocktail. Anal. Biochem. 200, 180–186.PubMedCrossRefGoogle Scholar
  13. 13.
    Miller, J. H. (1972) in Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 352.Google Scholar
  14. 14.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  15. 15.
    Rosenthal, N. (1987) Identification of regulatory elements of cloned genes with functional assays. Methods Enzymol. 152, 704–720.PubMedCrossRefGoogle Scholar
  16. 16.
    Salier, J.-P. and Kurachi, K. (1989) A CAT expression vector with virtually no background: pUMSVO-CAT. Biotechniques 7, 30,31.PubMedCrossRefGoogle Scholar
  17. 17.
    McGeady, M. L., Wood, T. G., Maizel, J. V., and Vande Woude, G. F. (1986) Sequences upstream from the mouse c-mos oncogene may function as a transcriptional terminal signal. DNA 5, 289–298.PubMedGoogle Scholar
  18. 18.
    Gendloff, E. H., Bowen, B., and Buchholz, W. G. (1990) Quantitation of chloramphenicol acetyl transferase in transgenic tobacco plants by ELISA and correlation with gene copy number. Plant Mol. Biol. 14, 575–583.PubMedCrossRefGoogle Scholar
  19. 19.
    Ow, D. W., Wood, K. V., DeLuca, M., Dewet, J. R., Helinski, D. R., and Howell, S. H. (1986) Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants. Science 234, 856–859.PubMedCrossRefGoogle Scholar
  20. 20.
    deMartin, R., Strasswimmer, J., and Philipson, L. (1993) A new luciferase promoter insertion vector for the analysis of weak transcriptional activities. Gene 124, 137,138.CrossRefGoogle Scholar
  21. 21.
    Caricasole, A. and Ward, A. (1993) A luciferase-reporter vector with blue-white selection for rapid subcloning and mutational analysis of eukaryotic promoters. Gene 124, 139,140.PubMedCrossRefGoogle Scholar
  22. 22.
    Hall, C. V., Jacob, P. E, Ringold, G. M, and Lee, F. (1983) Expression and regulation of Escherichia coli lac Z gene fusions in mammalian cells. J. Mol. Appl. Gen. 2, 101.Google Scholar
  23. 23.
    Ribela, M. T., Murata, Y., Morganti, L., Toniolo, D., and Bartolini, P. (1993) The use of recombinant human growth hormone for radioiodination and standard preparation in radioimmunoassay. J. Immunol. Methods 159, 269–274.PubMedCrossRefGoogle Scholar
  24. 24.
    Wan Nazaimoon, W. M., Satgunasingam, N., and Khalid, B. (1990) Development of an in-house radioimmunoassay for human growth hormone. Malays. J. Pathol. 12, 13–20.Google Scholar
  25. 25.
    Dinesen, B. (1991) Immunochemical aspects of growth hormone assays. Horm. Res. 36, 11–16.PubMedCrossRefGoogle Scholar
  26. 26.
    Gervasi, G., Samy, M., and Scholler, R. (1990) Comparison of four human growth hormone (hGH) immunoassay kits and analysis of recognition of circulating forms. Pathol. Biol. 38, 912–919.PubMedGoogle Scholar
  27. 27.
    Seth, A., Robinson, L., Thompson, D. M., Panayiotakis, A., Smyth, F. E., Watson, D. K., and Papas, T. S. (1993) Transactivation of GATA-1 promoter with ETS1, ETS2 and ERGB/Hu-FLI-1 proteins: stabilization of the ETS1 protein binding on GATA-1 promoter sequences by monoclonal antibody. Oncogene 8, 1783–1790.PubMedGoogle Scholar
  28. 28.
    Seth, A., Hodge, D. R., Thompson, D. M., Robinson, L., Panayiotakis, A., Watson, D. K., and Papas, T. S. (1993) ETS family proteins activate transcription from HIV-1 LTR. AIDS Res. Hum. Retroviruses 9, 1017–1023.PubMedCrossRefGoogle Scholar
  29. 29.
    Seth, A., Ascione, R., Fisher, R. J., Mavrothalassitis, G. J., Bhat, N. K., and Papas, T. S. (1992) The ets gene family. Cell Growth Differ. 3, 327–334.PubMedGoogle Scholar
  30. 30.
    Seth, A., Robinson, L., Panayiotakis, A., Thompson, D. M., Hodge, D., Zhang, X. K., Watson, D. K., Ozato, K., and Papas, T. S. (1994) The EndoA enhancer contains multiple ETS binding site repeats and is regulated by ETS proteins. Oncogene 9, 469–477.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 1995

Authors and Affiliations

  • David R. Hodge
  • Delores M Thompson
    • 1
  • Alexandra Panayiotakis
    • 1
  • Arun Seth
    • 1
  1. 1.Laboratory of Molecular OncologyNational Cancer InstituteFrederick

Personalised recommendations