Abstract
Nuclear extracts prepared by appropriate methods contain all of the components for transcription (1). Although nuclear extracts are not defined like transcription systems utilizing purified transcription components (2–4) the extracts can be used to study aspects of promoter regulation. If suitable nuclear extracts can be prepared that contain the necessary transcription factors in an active state, and suitable DNA templates containing regulator sequences are available, processes such as differential gene expression (5,6) and hormonal simulation of transcription can be reconstituted (7,8).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1, 1475–1489
Tyree, C. M., George, C. P., Lira-DeVito, L. M., Wampler, S. L., Dahmus, M. E., Zawel, L., and Kadonaga, J. T. (1993) Identification of a minimal set of proteins that is sufficient for accurate initiation of transcription by RNA polymerase II. Genes Dev. 7, 1254–1265.
Goodrich, J. A. and Tjian, R. (1994) Transcription factors IIE and IIH and ATP hydrolysis direct promoter clearance by RNA polymerase II. Cell 77, 145–156.
Hansen, S. K., Takada, S., Jacobson, R. H., Lis, J. T., and Tjian, R. (1997) Transcription properties of a cell type-specific TATA-binding protein, TRF. Cell 91, 71–83.
Flanagan, W. M. and Crabtree, G.R. (1992) In vitro transcription faithfully reflecting T-cell activation requirements. J. Biol. Chem. 267, 399–406.
Gorski, K, Carneiro, M., and Schibler U. (1986) Tissue-specific in vitro transcription from mouse albumin promoter. Cell 47, 767–776
Bagchi, M. K., Tsai, S. Y., and Tsai, M.-J. (1995) In vitro reconstitution of progesterone-dependent RNA transcription in nuclear extracts of human breast carcinoma cells, in In Vitro Transcription and Translation Protocols (Tymms, M. J., ed.), Humana, Totowa, NJ, pp. 107–120.
Fondell, J. D., Brunel, F., Hisatake, K., and Roeder, R. G. (1996) Unliganded thyroid hormone receptor alpha can target TATA-binding protein for transcriptional repression. Mol. Cell Biol. 16, 281–287.
Wallrath, L. L., Lu, Q., Granok, H., and Elgin, S. C. R. (1994) Architectural variations of inducible eukaryotic promoters:preset and remodeling chromatin structures. Bioessays 16, 165–170.
Razin, A. (1998) CpG methylation, chromatin structure and gene silencing-a three-way connection. EMBO J. 17, 4905–4908.
Kadonaga, J. T. (1998) Eukaryotic transcription: an interlaced network of transcription factors and chromatin-modifying machines. Cell 92, 307–313.
Archer, T. K., Cordingley, M. G., Wolford, R. G., and Hager, G. L. (1991) Transcription factor access is mediated by accurately positioned nucleosomes on the mouse mammary tumor virus promoter. Mol. Cell Biol. 11, 688–698.
Chang, L. A., Smith, T., Pognonec, P., Roeder, R. G., and Murialdo, H. (1992) Identification of USF as the ubiquitous murine factor that binds to and stimulates transcription from the immunoglobulin lambda 2-chain promoter. Nucleic. Acids. Res. 20, 287–293.
Abate, C., Luk, D., and Curran, T. (1991) Transcriptional regulation by Fos and Jun In Vitro: interaction among multiple activator and regulatory domains. Mol. Cell. Biol. 11, 3624–3632.
Goldberg, Y., Treier, M., Ghysdael, J., and Bohmann, D. (1994) Repression of AP-1-stimulated transcription by c-Ets-1. J. Biol. Chem. 269, 16,566–16,576.
Song, C. Z., Tierney, C. J., Loewenstein, P. M., Pusztai, R., Symington, J. S., Tang, Q. Q., Toth, K., Nishikawa, A., Bayley, S. T., and Green, M. (1995) Transcriptional repression by human adenovirus E1A N terminus/conserved domain 1 polypeptides in vivo and in vitro in the absence of protein synthesis. J. Biol. Chem. 270, 23,263–23,267.
Yu, M., Yang, X. Y., Schmidt, T., Chinenov, Y., Wang, R., and Martin, M. E. (1997) GA-binding protein-dependent transcription initiator elements. Effect of helical spacing between polyomavirus enhancer a factor 3(PEA3)/Ets-binding sites on initiator activity. J. Biol. Chem. 272, 29,060–29,067.
Tymms, M. J. (1995) Quantitative measurement of mRNA using the RNase protection assay, in In Vitro Transcription and Translation Protocols (Tymms, M. J., ed.) Humana, Totowa, NJ, pp. 31–46.
Viville, S. and Mantovani, R. (1994) S1 mapping using single-stranded DNA probes, in Protocols for Gene Analysis (Harwood, A. J., ed.) Humana, Totowa, NJ, pp. 299–305.
Simpson, C. G. and Brown, J. W. (1995) Primer extension assay, in Plant Gene Transfer and Expression Protocols (Jones, H., ed.) Humana, Totowa, NJ, pp. 249–256.
Pugh, B. F. (1995) Preparation of HeLa nuclear extracts, in In Vitro Transcription and Translation Protocols (Tymms, M. J., ed.) Humana, Totowa, NJ, pp. 349–357.
Sawadogo, M. and Roeder, R. G. (1985) Factors involved in specific transcription by human RNA polymerase II: analysis by a rapid and quantitative in vitro assay. Proc. Natl. Acad. Sci. USA 82, 4394–4398.
Klein-Hitpass, L., Tsai, S. Y., Weigel, N. L., Allan, G. F., Riley, D., Rodriguez, R., Schrader, W. T., Tsai, M. J., and O’Malley, B. W. (1990) The progesterone receptor stimulates cell-free transcription by enhancing the formation of a stable preinitiation complex. Cell 60, 247–257.
Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2000 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Tymms, M.J. (2000). In Vitro Promoter Analysis Using Nuclear Extracts and G-Free Cassette Vectors. In: Tymms, M.J. (eds) Transcription Factor Protocols. Methods in Molecular Biology™, vol 130. Humana Press. https://doi.org/10.1385/1-59259-686-X:247
Download citation
DOI: https://doi.org/10.1385/1-59259-686-X:247
Publisher Name: Humana Press
Print ISBN: 978-0-89603-573-7
Online ISBN: 978-1-59259-686-7
eBook Packages: Springer Protocols