Abstract
The yeast two-hybrid assay has proven useful for detecting protein-protein interactions. A variation on the theme can be used for finding proteins that interact with a particular DNA sequence. The one-hybrid assay, so far carried out only in Saccharomyces cerevisiae, in its simplest form (Fig. 1) consists of a DNA sequence of interest placed upstream of a reporter gene. The reporter gene can be either on a plasmid (1) or integrated into the chromosome (2). The protein or library being tested is cloned into a vector that expresses that protein fused to a transcription activation domain (TAD), the equivalent of the prey protein in a two-hybrid assay. This hybrid protein is expressed in the strain carrying the reporter gene. If the protein is able to interact with the sequence of interest, by either binding directly to the DNA or indirectly via interaction with a DNA-binding protein, transcription of the reporter gene is activated.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Wang, M. M. and Reed, R. R. (1993) Molecular cloning of the olfactory neuronal transcription factor Olf-1 by genetic selection in yeast. Nature 364, 121–126.
Li, J. J. and Herskowitz, I. (1993) Isolation of ORC6, a component of the yeast origin recognition complex by a one-hybrid system. Science 262, 1870–1874.
Bourns, B. D., Alexander, M. K., Smith, A. M., and Zakian, V. A. (1998) Sir proteins, Rif proteins and Cdc13p bind Saccharomyces telomeres in vivo. Mol. Cell. Biol. 18, 5600–5608.
Ortiz, J., Stemmann, O., Rank, S., and Lechner, J. (1999) A putative protein complex consisting of Ctf19, Mcm21, and Okp1 represents a missing link in the budding yeast kinetochore. Genes Dev. 13, 1140–1155.
Spencer, J. A., Baron, M. H., and Olson, E. N. (1999) Cooperative transcriptional activation by serum response factor and the high mobility group protein SSRP1. J. Biol. Chem. 274, 15,686–15,693.
Yu, Y., Yussa, M., Song, J., Hirsch, J., and Pick, L. (1999) A double interaction screen identifies positive and negative ftz gene regulators and ftz-interacting proteins. Mech. Dev. 83, 95–105.
Luo, Y., Vijaychander, S., Stile, J., and Zhu, L. (1996) Cloning and analysis of DNA-binding proteins by yeast one-hybrid and one-two-hybrid systems. Biotechniques 20, 564–568.
Wilson, T. E., Fahrner, T. J., Johnston, M., and Milbrandt, J. (1991) Identification of the DNA binding site for NGFI-B by genetic selection in yeast. Science 252, 1296–1300.
Liu, J., Wilson, T., Milbrandt, J., and Johnston, M. (1993) Identifying DNA-binding sites and analyzing DNA-binding domains using a yeast selection system. Methods 5, 125–137.
Celniker, S. E., Sweder, K., Srienc, F., Bailey, J. E., and Campbell, J. L. (1984) Deletion mutations affecting autonomously replicating sequence ARS1 of Saccharomyces cerevisiae. Mol. Cell. Biol. 4, 2455–2466.
Mannhaupt, G., Schnall, R., Karpov, V., Vetter, I., and Feldmann, H. (1999) Rpn4p acts as a transcription factor by binding to PACE, a nonamer box found upstream of 26S proteasomal and other genes in yeast. FEBS Lett. 450, 7–34.
Thatcher, J. D., Haun, C., and Okkema, P. G. (1999) The DAF-3 Smad binds DNA and represses gene expression in the Caenorhabditis elegans pharynx. Development 126, 97–107.
Wei, Z., Angerer, R. C., and Angerer, L. M. (1999) Identification of a new sea urchin ets protein, SpEts4, by yeast one-hybrid screening with the hatching enzyme promoter. Mol. Cell. Biol. 19, 1271–1278.
Liu, Q., Kasuga, M., Sakuma, Y., Abe, H., Miura, S., Yamaguchi-Shinozaki, K., and Shinozaki, K. (1998) Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought-and low-temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell 10, 1391–1406.
Oda, E., Shirasuna, K., Suzuki, M., Nakano, K., Nakajima, T., and Oda, K. (1998) Cloning and characterization of a GC-box binding protein, G10BP-1, responsible for repression of the rat fibronectin gene. Mol. Cell. Biol. 18, 4772–4782.
Cooper, J. P., Nimmo, E. R., Allshire, R. C., and Cech, T. R. (1997) Regulation of telomere length and function by a Myb-domain protein in fission yeast. Nature 385, 744–747.
De Felice, B., Pontecorvo, G., and Carfagna, M. (1999) Identification of a new gene encoding pericentromeric dodeca-satellite binding protein in Drosophila melanogaster. FEBS Lett. 455, 31–35.
Sandell, L. L. and Zakian, V. A. (1993) Loss of a yeast telomere: arrest, recovery and chromosome loss. Cell 75, 729–739.
Conrad, M. N., Wright, J. H., Wolf, A. J., and Zakian, V. A. (1990) RAP1 protein interacts with yeast telomeres in vivo: overproduction alters telomere structure and decreases chromosome stability. Cell 63, 739–750.
Gyuris, J., Golemis, E., Chertkov, H., and Brent, R. (1993) Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2. Cell 75, 791–803.
Kennedy, B. K., Austriaco, N. R., Zhang, J., and Guarente, L. (1995) Mutation in the silencing gene SIR4 can delay aging in S. cerevisiae. Cell 80, 485–496.
Chien, C. T., Bartel, P. L., Sternglanz, R., and Fields, S. (1991) The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc. Natl. Acad. Sci. USA 88, 9578–9582.
Mastick, G. S., McKay, R., Oligino, T., Donovan, K., and Lopez, A. J. (1995) Identification of target genes regulated by homeotic proteins in Drosophila melanogaster through genetic selection of Ultrabithorax protein-binding sites in yeast. Genetics 139, 349–363.
Wotton, D. and Shore, D. (1997) A novel Rap1p-interacting factor, Rif2p, cooperates with Rif1p to regulate telomere length in Saccharomyces cerevisiae. Genes Dev. 11, 748–760.
Conrad, M. N., Dominguez, A. M., and Dresser, M. E. (1997) Ndj1p, a meiotic telomere protein required for normal chromosome synapsis and segregation in yeast. Science 276, 1252–1255.
Chua, P. R. and Roeder, G. S. (1997) Tam1, a telomere-associated meiotic protein, functions in chromosome synapsis and crossover interference. Genes Dev. 11, 1786–1800.
Holmes, S. G., Rose, A. B., Steuerle, K., Saez, E., Sayegh, S., Lee, Y. M., and Broach, J. R. (1997) Hyperactivation of the silencing proteins, Sir2p and Sir3p, causes chromosome loss. Genetics 145, 605–614.
Struhl, K. and Davis, R. W. (1977) Production of a functional eukaryotic enzyme in Escherichia coli: cloning and expression of the yeast structural gene for imidazole-glycerolphosphate dehydratase (his3). Proc. Natl. Acad. Sci. USA 74, 5255–5259.
Gottschling, D. E., Aparicio, O. M., Billington, B. L., and Zakian, V. A. (1990) Position effect at S. cerevisiae telomeres: reversible repression of Pol II transcription. Cell 63, 751–762.
Wright, J. H., Gottschling, D. E., and Zakian, V. A. (1992) Saccharomyces telomeres assume a non-nucleosomal chromatin structure. Genes Dev. 6, 197–210.
Runge, K. W. and Zakian, V. A. (1990) Properties of the transcriptional enhancer in Saccharomyces cerevisiae telomeres. Nucleic Acids Res. 18, 1783–1787.
Shore, D. and Nasmyth, K. (1987) Purification and cloning of a DNA binding protein from yeast that binds to both silencer and activator elements. Cell 51, 721–732.
Donze, D., Adams, C. R., Rine, J., and Kamakaka, R. T. (1999) The boundaries of the silenced HMR domain in Saccharomyces cerevisiae. Genes Dev. 13, 698–708.
Bi, X. and Broach, J. R. (1999) UASrpg can function as a heterochromatin boundary element in yeast. Genes Dev. 13, 1089–1101.
Nimmo, E. R., Cranston, G., and Allshire, R. C. (1994) Telomere-associated chromosome breakage in fission yeast results in variegated expression of adjacent genes. EMBO J. 13, 3801–3811.
Farr, C., Fantes, J., Goodfellow, P., and Cooke, H. (1991) Functional reintroduction of human telomeres into mammalian cells. Proc. Natl. Acad. Sci. USA 88, 7006–7010.
Hanish, J. P., Yanowitz, J. L., and De Lange, T. (1994) Stringent sequence requirements for the formation of human telomeres. Proc. Natl. Acad. Sci. USA 91, 8861–8865.
Nogi, Y., Shimada, H., Matsuzaki, Y., Hashimoto, H., and Fukasawa, T. (1984) Regulation of expression of the galactose gene cluster in Saccharomyces cerevisiae. II. The isolation and dosage effect of the regulatory gene GAL80. Mol. Gen. Genet. 195, 29–34.
Wolf, S., Roder, K., and Schweizer, M. (1996) Construction of a reporter plasmid that allows expression libraries to be exploited for the one-hybrid system. Biotechniques 20, 568–574.
Zakian, V. A. and Scott, J. F. (1982) Construction, replication and chromatin structure of TRP1 RI circle, a multiple-copy synthetic plasmid derived from Saccharomyces cerevisiae chromosomal DNA. Molec. Cell. Biol. 2, 221–232.
Breeden, L. and Nasmyth, K. (1985) Regulation of the yeast HO gene. Cold Spring Harbor Symp. Quant. Biol. 50, 643–650.
Guarente, L. (1983) Yeast promoters and lac Z fusions designed to study expression of cloned genes in yeast. Methods Enzymol. 101C, 181–191.
Gietz, D., St. Jean, A., Woods, R. A., and Schiestl, R. H. (1992) Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 20, 1425.
Kimmerly, W. J. and Rine, J. (1987) Replication and segregation of plasmids containing cis-acting regulatory sites of silent mating-type genes in Saccharomyces cerevisiae are controlled by the SIR genes. Mol. Cell. Biol. 7, 4225–4237.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2001 Humana Press Inc.
About this protocol
Cite this protocol
Alexander, M.K., Bourns, B.D., Zakian, V.A. (2001). One-Hybrid Systems f hor Detecting Protein-DNA Interactions. In: MacDonald, P.N. (eds) Two-Hybrid Systems. Methods in Molecular Biology, vol 177. Humana Press. https://doi.org/10.1385/1-59259-210-4:241
Download citation
DOI: https://doi.org/10.1385/1-59259-210-4:241
Publisher Name: Humana Press
Print ISBN: 978-0-89603-832-5
Online ISBN: 978-1-59259-210-4
eBook Packages: Springer Protocols