Abstract
The method described in this chapter has been used in the molecular cloning of transcription factors and other factors with DNA-binding activity toward specific double-stranded DNA sequences. The protocol is based on the method of Singh et al. (1) and shares feature with the immunological approach to screening cDNA expression libraries (see Chapter 13). The principle is to probe a cDNA expression library (usually a λ-phage expression library) with a labeled double-stranded DNA probe containing the sequence recognized by the factor in question. Recombinants expressing a protein capable of binding the probe sequence in the presence of nonspecific competitor DNA are thus identified and can be isolated.
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References
Singh, H., LeBowitz, J. H., Baldwin, A. S., and Sharp, P. A. (1988) Molecular cloning of an enhancer binding protein: isolation by screening of an expression library with a recognition site DNA. Cell 52, 415–423.
Huynh, T. V., Young, R. W., and Davis, R. W. (1988) Construction and screening of cDNA libraries in lambda gt11 and lambda gt10, in Cloning: A Practical Approach, vol. 1 (Glover, D. M., ed.), IRL, Oxford, UK, pp. 49–78.
Maekawa, T., Sakura, H., Kanei Ishii, C., Sudo, T., Yoshimura, T., Fujisawa, J., Yoshida, M., and Ishii, S. (1989) Leucine zipper structure of the protein CRE-BP1 binding to the cyclic AMP response element in brain. EMBO J. 8, 2023–2028.
Hai, T., Liu, F., Coukos, W. J., and Green, M. R. (1989) Transcription factor ATF cDNA clones: an extensive family of leucine zipper proteins able to selectively form DNA-binding heterodimers. Genes Dev. 3, 2083–2090.
Poll, V., Mancini, F. P., and Cortese, R. (1990) IL-6DBP, a nuclear protein involved in interleukin-6 signal transduction, defines a new family of leucine zipper proteins related to C/EBP. Cell 63, 643–653.
Cowell, I. G., Skinner, A., and Hurst, H. C. (1992) Transcriptional repression by a novel member of the bzip family of transcription factors. Mol. Cell Biol. 12, 3070–3077.
Kageyama, R., and Pastan, I. (1989) Molecular cloning and characterisation of a human DNA binding factor that represses transcription. Cell 59, 815–825.
Williams, T. M., Moolten, D., Burlein, J., Romano, J., Bhaerman, R., Godillot, A., Mellon, M., Rauscher, F. J., III, and Kant, J. A. (1991) Identification of a zinc finger protein that inhibits IL-2 expression. Science 254, 1791–1793.
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© 1997 Humana Press Inc., Totowa, NJ
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Cowell, I.G. (1997). Cloning Sequence-Specific DNA-Binding Factors from cDNA Expression Libraries Using Oligonucleotide Binding Site Probes. In: Cowell, I.G., Austin, C.A. (eds) cDNA Library Protocols. Methods in Molecular Biology™, vol 69. Humana Press. https://doi.org/10.1385/0-89603-383-X:161
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DOI: https://doi.org/10.1385/0-89603-383-X:161
Publisher Name: Humana Press
Print ISBN: 978-0-89603-383-2
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