title: Design of a single plasmid-based modified yeast one-hybrid system for investigation of in vivo protein-protein and protein-DNA interactions.
abstract: We have developed a modified yeast one-hybrid system (MY1H) useful for in vivo investigation of protein-protein and protein-DNA interactions. Our single-plasmid expression system is capable of differential protein expression levels; in addition to a GAL4 activation domain (AD) fusion protein, a second protein can be coexpressed at either comparable or higher transcriptional levels from expression vectors pCETT or pCETF, respectively. This second protein can play a structural, modifying, or inhibitory role that restores or blocks reporter gene expression. Our MY1H was validated by use of the well-characterized DNA-binding protein p53 and its inhibitory partners, large T antigen (LTAg) and 53BP2. By coexpressing LTAg or 53BP2 at comparable or higher levels than the GAL4AD-p53 fusion in the MY1H, we show that DNA binding of p53 decreases by different, measurable extents dependent on the expression level of inhibitory partner. As with the traditional Y1H, our system could also be used to investigate proteins that provide coactivational or bridging functions and to identify novel protein- or DNA-binding partners through library screening. Our MY1H provides a system for investigation of simultaneous protein-protein and protein-DNA interactions, and thus is a useful addition to current methods for in vivo investigation of such interactions.
authors.affiliation: Department of Chemistry, University of Toronto, Mississauga, Ontario, Canada.
authors.forename: Gang
authors.initials: G
authors.lastname: Chen
authors.forename: Lisa
authors.initials: L
authors.lastname: DenBoer
authors.forename: Jumi
authors.initials: J
authors.lastname: Shin
mesh_terms: D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism; D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D037102:Lectins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: D000952:Antigens, Polyomavirus Transforming; D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; D037102:Lectins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C090468:TP53BP2 protein, human; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; C027112:fucose-binding lectin; D004247:DNA
references.citation: Mech Dev. 1999 May;83(1-2):95-105
references.pmid: 10381570
references.citation: Cell. 1992 Feb 7;68(3):597-612
references.pmid: 1339307
references.citation: Mol Cell Biol. 1995 Apr;15(4):2157-65
references.pmid: 7891710
references.citation: Proc Natl Acad Sci U S A. 1997 May 13;94(10):5172-6
references.pmid: 9144210
references.citation: Gene. 1990 May 31;90(1):79-85
references.pmid: 2199331
references.citation: Nature. 1989 Jul 20;340(6230):245-6
references.pmid: 2547163
references.citation: Genes Dev. 2006 Sep 1;20(17):2373-82
references.pmid: 16951253
references.citation: Annu Rev Cell Biol. 1990;6:539-57
references.pmid: 2125830
references.citation: Proc Natl Acad Sci U S A. 1989 Apr;86(7):2172-5
references.pmid: 2648393
references.citation: Nature. 1992 Jul 2;358(6381):83-6
references.pmid: 1614538
references.citation: Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):6098-102
references.pmid: 8016121
references.citation: Curr Opin Genet Dev. 1998 Oct;8(5):552-9
references.pmid: 9794820
references.citation: Methods Enzymol. 1983;101:192-201
references.pmid: 6310322
references.citation: Nucleic Acids Res. 2006 Feb 07;34(3):955-67
references.pmid: 16464826
references.citation: Nature. 1993 Jul 8;364(6433):121-6
references.pmid: 8321284
references.citation: J Steroid Biochem Mol Biol. 2003 Dec;87(4-5):223-31
references.pmid: 14698202
references.citation: Gene. 1986;44(2-3):177-83
references.pmid: 3023181
references.citation: Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):13671-6
references.pmid: 8942992
references.citation: J Biol Chem. 2001 Apr 13;276(15):12120-7
references.pmid: 11152481
references.citation: Genes Dev. 1992 Oct;6(10):1886-98
references.pmid: 1398068
references.citation: Nature. 1997 Apr 10;386(6625):569-77
references.pmid: 9121580
references.citation: Oncogene. 1993 Mar;8(3):543-8
references.pmid: 8382354
references.citation: Proc Natl Acad Sci U S A. 1981 Oct;78(10):6354-8
references.pmid: 6273866
references.citation: Science. 1996 Nov 8;274(5289):1001-5
references.pmid: 8875926
references.citation: Mol Cell Biol. 1995 Oct;15(10):5820-9
references.pmid: 7565735
references.citation: Mol Cell Biol. 1994 Dec;14(12):8315-21
references.pmid: 7969167
references.citation: Biotechniques. 1990 Sep;9(3):298, 300
references.pmid: 2223068
references.citation: Proc Natl Acad Sci U S A. 1997 May 13;94(10):4919-24
references.pmid: 9144165
references.citation: Biotechniques. 1996 Apr;20(4):564-8
references.pmid: 8800670
references.citation: Genes Dev. 1995 Apr 15;9(8):1009-19
references.pmid: 7774807