Genetics, Vol 144, 1425-1436, Copyright © 1996

INVESTIGATIONS

Genomic Libraries and a Host Strain Designed for Highly Efficient Two-Hybrid Selection in Yeast

P. James, J. Halladay and E. A. Craig
Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706

The two-hybrid system is a powerful technique for detecting protein-protein interactions that utilizes the well-developed molecular genetics of the yeast Saccharomyces cerevisiae. However, the full potential of this technique has not been realized due to limitations imposed by the components available for use in the system. These limitations include unwieldy plasmid vectors, incomplete or poorly designed two-hybrid libraries, and host strains that result in the selection of large numbers of false positives. We have used a novel multienzyme approach to generate a set of highly representative genomic libraries from S. cerevisiae. In addition, a unique host strain was created that contains three easily assayed reporter genes, each under the control of a different inducible promoter. This host strain is extremely sensitive to weak interactions and eliminates nearly all false positives using simple plate assays. Improved vectors were also constructed that simplify the construction of the gene fusions necessary for the two-hybrid system. Our analysis indicates that the libraries and host strain provide significant improvements in both the number of interacting clones identified and the efficiency of two-hybrid selections.

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				M. Kohli, S. Buck, and H.-P. Schmitz The function of two closely related Rho proteins is determined by an atypical switch I region J. Cell Sci., April 1, 2008; 121(7): 1065 - 1075. [Abstract] [Full Text] [PDF]

				T. A. Hennen-Bierwagen, F. Liu, R. S. Marsh, S. Kim, Q. Gan, I. J. Tetlow, M. J. Emes, M. G. James, and A. M. Myers Starch Biosynthetic Enzymes from Developing Maize Endosperm Associate in Multisubunit Complexes Plant Physiology, April 1, 2008; 146(4): 1892 - 1908. [Abstract] [Full Text] [PDF]

				A. Formstone, R. Carballido-Lopez, P. Noirot, J. Errington, and D.-J. Scheffers Localization and Interactions of Teichoic Acid Synthetic Enzymes in Bacillus subtilis J. Bacteriol., March 1, 2008; 190(5): 1812 - 1821. [Abstract] [Full Text] [PDF]

				H. A. van den Burg, D. I. Tsitsigiannis, O. Rowland, J. Lo, G. Rallapalli, D. MacLean, F. L.W. Takken, and J. D.G. Jones The F-Box Protein ACRE189/ACIF1 Regulates Cell Death and Defense Responses Activated during Pathogen Recognition in Tobacco and Tomato PLANT CELL, March 1, 2008; 20(3): 697 - 719. [Abstract] [Full Text] [PDF]

				R. Yamamura, N. Nishimura, H. Nakatsuji, S. Arase, and T. Sasaki The Interaction of JRAB/MICAL-L2 with Rab8 and Rab13 Coordinates the Assembly of Tight Junctions and Adherens Junctions Mol. Biol. Cell, March 1, 2008; 19(3): 971 - 983. [Abstract] [Full Text] [PDF]

				S. Van der Ent, B. W.M. Verhagen, R. Van Doorn, D. Bakker, M. G. Verlaan, M. J.C. Pel, R. G. Joosten, M. C.G. Proveniers, L.C. Van Loon, J. Ton, et al. MYB72 Is Required in Early Signaling Steps of Rhizobacteria-Induced Systemic Resistance in Arabidopsis Plant Physiology, March 1, 2008; 146(3): 1293 - 1304. [Abstract] [Full Text] [PDF]

				T. L. Brady, P. G. Fuerst, R. A. Dick, C. Schmidt, and D. F. Voytas Retrotransposon Target Site Selection by Imitation of a Cellular Protein Mol. Cell. Biol., February 15, 2008; 28(4): 1230 - 1239. [Abstract] [Full Text] [PDF]

				G. R. Pilkington and R. Parker Pat1 Contains Distinct Functional Domains That Promote P-Body Assembly and Activation of Decapping Mol. Cell. Biol., February 15, 2008; 28(4): 1298 - 1312. [Abstract] [Full Text] [PDF]

				R. P. Darst, S. N. Garcia, M. R. Koch, and L. Pillus Slx5 Promotes Transcriptional Silencing and Is Required for Robust Growth in the Absence of Sir2 Mol. Cell. Biol., February 15, 2008; 28(4): 1361 - 1372. [Abstract] [Full Text] [PDF]