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Development of an optimized interaction-mating protocol for large-scale yeast two-hybrid analyses.

Soellick TR, Uhrig JF - Genome Biol. (2001)

Bottom Line: This significantly reduces the effort and cost of cDNA library screening and allows multiple parallel approaches.The improved yeast two-hybrid interaction-mating protocol presented here allows the multiple parallel screening of cDNA libraries.It can be carried out without specialized equipment and has the potential to be standardized and automated.

View Article: PubMed Central - HTML - PubMed

Affiliation: Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg, D-50829 Köln, Germany. juhrig@mpiz-koeln.mpg.de

ABSTRACT

Background: Protein-protein interactions have decisive roles in almost all aspects of the structural and functional organization of cells. But in spite of the increasing amount of complete genome sequence data, the ability to predict protein function from sequences alone is limited. Therefore comprehensive analysis of protein-protein interactions, as derived from the yeast two-hybrid mating system, will yield valuable information for functional biology on a proteomic scale.

Results: We have developed an optimized interaction mating protocol for the yeast two-hybrid system, which gives increased mating efficiencies. This significantly reduces the effort and cost of cDNA library screening and allows multiple parallel approaches. Improved preincubation conditions before mating, and optimal cell densities and cell ratios enable almost quantitative mating of the yeast cells carrying the cDNA library. We have proved the applicability of this technology using 20 bait proteins to screen an Arabidopsis thaliana cDNA library, in spite of bait-dependent variations in mating efficiency.

Conclusions: The improved yeast two-hybrid interaction-mating protocol presented here allows the multiple parallel screening of cDNA libraries. It can be carried out without specialized equipment and has the potential to be standardized and automated.

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Related in: MedlinePlus

Successive transformation of yeast strain PJ69-4A. Yeast strain PJ69-4A was transformed with vector pTS195.1, raised in liquid selective medium lacking uracil (SD-U) and then successively transformed with plasmid pCL1. Numbers of doubly transformed cells were monitored by propagating aliquots on selective medium lacking leucine and uracil (SD-LU) and efficiency in terms of doubly transformed cells versus all cells was calculated.
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Figure 1: Successive transformation of yeast strain PJ69-4A. Yeast strain PJ69-4A was transformed with vector pTS195.1, raised in liquid selective medium lacking uracil (SD-U) and then successively transformed with plasmid pCL1. Numbers of doubly transformed cells were monitored by propagating aliquots on selective medium lacking leucine and uracil (SD-LU) and efficiency in terms of doubly transformed cells versus all cells was calculated.

Mentions: Yeast strain PJ69-4A propagating plasmid pTS195.1 was successively transformed with different amounts of vector pCL1 using a standard transformation procedure [22]. Transformation efficiencies ranged between 0.01% (0.1 μg DNA per 108 cells) and 0.1% (5 μg DNA per 108 cells, Figure 1). Extrapolation of the data revealed saturation at approximately 0.11%.


Development of an optimized interaction-mating protocol for large-scale yeast two-hybrid analyses.

Soellick TR, Uhrig JF - Genome Biol. (2001)

Successive transformation of yeast strain PJ69-4A. Yeast strain PJ69-4A was transformed with vector pTS195.1, raised in liquid selective medium lacking uracil (SD-U) and then successively transformed with plasmid pCL1. Numbers of doubly transformed cells were monitored by propagating aliquots on selective medium lacking leucine and uracil (SD-LU) and efficiency in terms of doubly transformed cells versus all cells was calculated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC64837&req=5

Figure 1: Successive transformation of yeast strain PJ69-4A. Yeast strain PJ69-4A was transformed with vector pTS195.1, raised in liquid selective medium lacking uracil (SD-U) and then successively transformed with plasmid pCL1. Numbers of doubly transformed cells were monitored by propagating aliquots on selective medium lacking leucine and uracil (SD-LU) and efficiency in terms of doubly transformed cells versus all cells was calculated.
Mentions: Yeast strain PJ69-4A propagating plasmid pTS195.1 was successively transformed with different amounts of vector pCL1 using a standard transformation procedure [22]. Transformation efficiencies ranged between 0.01% (0.1 μg DNA per 108 cells) and 0.1% (5 μg DNA per 108 cells, Figure 1). Extrapolation of the data revealed saturation at approximately 0.11%.

Bottom Line: This significantly reduces the effort and cost of cDNA library screening and allows multiple parallel approaches.The improved yeast two-hybrid interaction-mating protocol presented here allows the multiple parallel screening of cDNA libraries.It can be carried out without specialized equipment and has the potential to be standardized and automated.

View Article: PubMed Central - HTML - PubMed

Affiliation: Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg, D-50829 Köln, Germany. juhrig@mpiz-koeln.mpg.de

ABSTRACT

Background: Protein-protein interactions have decisive roles in almost all aspects of the structural and functional organization of cells. But in spite of the increasing amount of complete genome sequence data, the ability to predict protein function from sequences alone is limited. Therefore comprehensive analysis of protein-protein interactions, as derived from the yeast two-hybrid mating system, will yield valuable information for functional biology on a proteomic scale.

Results: We have developed an optimized interaction mating protocol for the yeast two-hybrid system, which gives increased mating efficiencies. This significantly reduces the effort and cost of cDNA library screening and allows multiple parallel approaches. Improved preincubation conditions before mating, and optimal cell densities and cell ratios enable almost quantitative mating of the yeast cells carrying the cDNA library. We have proved the applicability of this technology using 20 bait proteins to screen an Arabidopsis thaliana cDNA library, in spite of bait-dependent variations in mating efficiency.

Conclusions: The improved yeast two-hybrid interaction-mating protocol presented here allows the multiple parallel screening of cDNA libraries. It can be carried out without specialized equipment and has the potential to be standardized and automated.

Show MeSH
Related in: MedlinePlus