Gene Isolation by Screening λgt11 Expression Libraries with DNA Binding-Site Probes

  • Bill Kalionis
Part of the Methods in Molecular Biology book series (MIMB, volume 37)

Abstract

A fundamental goal of gene analysis is to determine what factors regulate the temporal and spatial expression of the gene of interest. This type of analysis requires the identification of the promoter region of the gene, characterization of the specific DNA sequences that regulate the expression of the gene, and the determination of the DNA-binding factors that recognize these sequences. The first two requirements can be met by routine in vitro molecular biological techniques, such as mobility shift DNA-binding assays (1) and DNase footprinting (2), which allow the DNA-binding sequence to be defined to a short DNA sequence, but isolating the specific DNA-binding factors that regulate the transcription of the gene, has proven to be problematic. One common strategy is to isolate the DNA-binding factor from complex mixtures of proteins and then to use a “reverse genetics” approach to clone the gene that encodes the factor. In many cases, access to large amounts of the required tissue or cell type required for biochemical purification is impracticable. A more direct approach is to screen an expression library with a short radioactively labeled DNA sequence defined in vitro as a binding site for the factor. The vector most commonly used for library construction is λgtl1 (3), where cDNA inserts are cloned into a unique EcoRI site near the C-terminal end of the coding region of the lacZ gene. The β-galactosidase fusion protein can be induced to high levels in the presence of the inducer IPTG.

Keywords

Agar MgCl2 Boiling HEPES MgSO4 

References

  1. 1.
    Garner, M. and Revzin, A. (1981) A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: applications to components of the E. coli lactose operon regulator system. Nucleic Acids Res. 9, 3047–3060.PubMedCrossRefGoogle Scholar
  2. 2.
    Galas, D. and Schmitz, A. (1978) DNase footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res. 5, 3157–3170.PubMedCrossRefGoogle Scholar
  3. 3.
    Huynh, T. V., Young, R. A., and Davis, R. W. (1985) Construction and screening cDNA libraries in λgt10 and λgtl1, in DNA Cloning, vol. 1. A Practical Approach (Glover, D M., ed.), IRL, Oxford, pp. 49–78.Google Scholar
  4. 4.
    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.PubMedCrossRefGoogle Scholar
  5. 5.
    Snyder, M., Elledge, S., Sweetser, D., Young, R. A., and Davis, R. W. (1987) λgtll: gene isolation with antibody probes and other applications. Methods Enzymol. 154, 107–129.PubMedCrossRefGoogle Scholar
  6. 6.
    Mierendorf, R. C, Percy, C, and Young, R. A. (1987) Gene isolation by screening λgtll libraries with antibodies. Methods Enzymol. 152, 458–469.PubMedCrossRefGoogle Scholar
  7. 7.
    Clerc, R. G., Corcoran, L. M., LeBowitz, J. H., Baltimore, D., and Sharp, P. A. (1988) The B-cell specific Oct-2 protein contains Pou box and homeo box-type domains. Genes Dev. 2, 1570–1581.PubMedCrossRefGoogle Scholar
  8. 8.
    Staudt, L. M., Clerc, R. G., Singh, H., LeBowitz, J. H., Sharp, P. A., and Baltimore, D. (1988) Cloning of a lymphoid specific cDNA encoding a protein binding the regulatory octamer DNA motif. Science 241, 577–580.PubMedCrossRefGoogle Scholar
  9. 9.
    Vinson, C. R., LaMarco, K. L., Johnson, P. F., Landschulz, W. H., and McKnight, S. L. (1988) In situ detection of sequence-specific DNA-binding activity specified by a recombinant bacteriophage. Genes Dev. 2, 801–806.PubMedCrossRefGoogle Scholar
  10. 10.
    Kalionis, B. and O’Farrell, P. H. (1993) A universal target sequence is bound in vitro by diverse homeodomains. Mech. Dev. in press.Google Scholar
  11. 11.
    Tan, T.-H. (1991) A nonradioactive screening method for cloning genes encoding sequence-specific DNA-binding proteins. Anal. Biochem. 192, 17–22.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 1995

Authors and Affiliations

  • Bill Kalionis
    • 1
  1. 1.Department of Obstetrics and Gynaecology, School of MedicineThe Flinders University of South AustraliaAdelaideAustralia

Personalised recommendations