Abstract
Central to the regulation of transcription of eukaryotic genes is the interaction of specific DNA binding proteins with promoter and enhancer elements. The molecular cloning of such DNA binding factors is an important step toward understanding this process and a number of strategies have been devised to meet this aim. Oligonucleotide probes derived from peptide sequence data may be used to screen cDNA libraries by hybridization or, alternatively, cDNA expression libraries may be screened immunologically, using antibodies raised against the factor of interest. The protocol described here, which is based on a method described by Singh et al. (1), is analogous to immunological screening but uses a labeled DNA binding site oligonucleotide probe to screen a cDNA expression library; DNA binding factors are thus cloned by virtue of their binding activity.
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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.
Maekawa, T., Sakura, H., Kanei-Ishii, C, Sudo, T., Yoshimura, T., Fujisawa, J.-I., Yoshida, M., and Ishii, S. (1989) Leucine zipper structure of the protein CRE-BP1 binding to the cAMP 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 Devel. 3, 2083–2090.
Poli, 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.
Klemsz, M. J., McKercher, S. R., Celada, A., Van Beveren, C., and Maki, R. A. (1990) The macrophage and B cell specific transcription factor PU. 1 is related to the ets oncogene. Cell 61, 113–124.
Xiao, J. H., Davidson, I., Matthes, H., Gamier, J.-M., and Chambon, P. (1991) Cloning, expression and transcriptional properties of the human enhancer factor TEF-1. Cell 65, 551–568.
Huynn, T. V., Young, R. A., and Davis, R. W. (1985) Construction and screening of cDNA libraries in Xgtl 1 and gtlO, in DNA Cloning-A Practical Approach, vol. 1, (Glover, D. M., ed.) IRL, Oxford, pp. 49–78.
Vinson, C. R., LaMarco, K. L., Johnson, P. F., Landshulz, W. H., and McKnight, S. L. (1988) In situ detection of sequence-specific DNA binding activity speci-fied by a recombinant bacteriophage. Genes Dev. 2, 801–806.
Lum, L. S. Y., Sultman, L. A., Kaufman, R, J., Linzer, D. I., and Wu, B. J. (1990) A cloned human CCAAT-box-binding factor stimulates transcription from the human hsp70 promoter. Mol. Cell. Biol. 10, 6709–6717.
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© 1994 Humana Press Inc., Totowa, NJ
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Coivell, I.G., Hurst, H.C. (1994). Cloning DNA Binding Proteins from cDNA Expression Libraries Using Oligonucleotide Binding Site Probes. In: Harwood, A.J. (eds) Protocols for Gene Analysis. Methods in Molecular Biology, vol 31. Humana Press. https://doi.org/10.1385/0-89603-258-2:363
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DOI: https://doi.org/10.1385/0-89603-258-2:363
Publisher Name: Humana Press
Print ISBN: 978-0-89603-258-3
Online ISBN: 978-1-59259-518-1
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