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Yeast two-hybrid interaction partner screening through in vivo Cre-mediated Binary Interaction Tag generation.

Hastie AR, Pruitt SC - Nucleic Acids Res. (2007)

Bottom Line: Nonetheless, the logistics of pair-wise screening has resulted in a cumbersome and incomplete application of this method to complex genomes.Once linked, DNA from complex pools of clones can be processed without losing the identity of the interacting partners.The technology described here sufficiently improves the throughput of the Y2H approach to make feasible the generation of near comprehensive interaction maps for complex organisms.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Biophysics and Biochemistry, Roswell Park Cancer Institute, Buffalo, NY, USA.

ABSTRACT
Yeast two-hybrid (Y2H) has been successfully used for genome-wide screens to identify protein-protein interactions for several model organisms. Nonetheless, the logistics of pair-wise screening has resulted in a cumbersome and incomplete application of this method to complex genomes. Here, we develop a modification of Y2H that eliminates the requirement for pair-wise screening. This is accomplished by incorporating lox sequences into Y2H vectors such that cDNAs encoding interacting partners become physically linked in the presence of Cre recombinase in vivo. Once linked, DNA from complex pools of clones can be processed without losing the identity of the interacting partners. Short linked sequence tags from each pair of interacting partner (binary interaction Tags or BI-Tags) are then recovered and sequenced. To validate the approach, comparisons between interactions found using traditional Y2H and the BI-Tag method were made, which demonstrate that the BI-Tag technology accurately represents the complexity of the interaction partners found in the screens. The technology described here sufficiently improves the throughput of the Y2H approach to make feasible the generation of near comprehensive interaction maps for complex organisms.

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BI-tag Y2H vectors. The prey vector (panel A) pGADt7lox71 includes an Adc1 promoter, GAL4 AD cDNA, a hemagglutinin epitope, a gap-repair cloning sequence that includes the lox71 sequence, the 2 μ ori, an ampicillin resistance gene for bacterial selection and the LEU2 gene for yeast selection. The figure also shows a cDNA molecule flanked by vector homology, an MmeI RE binding site, and a lox71 site. The bait vector (panel B) pCD.2lox66HoxA1 includes an Adc1 promoter, GAL4 DBD cDNA, a hemagglutinin epitope, the HoxA1 sequence, an ampicillin resistance gene for bacterial selection, a CEN sequence for low copy number replication, and a TRP1 gene for yeast selection. A bait vector pGADt7lox71 for library creation (panel C) includes an ADH1 promoter, GAL4 DBD cDNA, a cMyc epitope, a gap-repair cloning sequence that includes the lox66 sequence, the 2 μ ori, a kanamycin resistance gene for bacterial selection and the TRP1 gene for yeast selection.
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Figure 1: BI-tag Y2H vectors. The prey vector (panel A) pGADt7lox71 includes an Adc1 promoter, GAL4 AD cDNA, a hemagglutinin epitope, a gap-repair cloning sequence that includes the lox71 sequence, the 2 μ ori, an ampicillin resistance gene for bacterial selection and the LEU2 gene for yeast selection. The figure also shows a cDNA molecule flanked by vector homology, an MmeI RE binding site, and a lox71 site. The bait vector (panel B) pCD.2lox66HoxA1 includes an Adc1 promoter, GAL4 DBD cDNA, a hemagglutinin epitope, the HoxA1 sequence, an ampicillin resistance gene for bacterial selection, a CEN sequence for low copy number replication, and a TRP1 gene for yeast selection. A bait vector pGADt7lox71 for library creation (panel C) includes an ADH1 promoter, GAL4 DBD cDNA, a cMyc epitope, a gap-repair cloning sequence that includes the lox66 sequence, the 2 μ ori, a kanamycin resistance gene for bacterial selection and the TRP1 gene for yeast selection.

Mentions: To produce Y2H libraries configured for use in the BI-Tag Y2H method, Y2H vectors were modified to contain mutant lox sequences (15) adjacent to the 3′ end of the cDNA insertion site (Figure 1). Lox sites are recognized by the site-specific recombinase, Cre (16). The lox66 and lox71 sites can recombine with each other to form a wild-type loxP site and double mutant lox66/71 site for which Cre has a very low affinity, suppressing recombination at the double mutant site (15). In the present application, the lox66/71 site is between the linked cDNAs.Figure 1.


Yeast two-hybrid interaction partner screening through in vivo Cre-mediated Binary Interaction Tag generation.

Hastie AR, Pruitt SC - Nucleic Acids Res. (2007)

BI-tag Y2H vectors. The prey vector (panel A) pGADt7lox71 includes an Adc1 promoter, GAL4 AD cDNA, a hemagglutinin epitope, a gap-repair cloning sequence that includes the lox71 sequence, the 2 μ ori, an ampicillin resistance gene for bacterial selection and the LEU2 gene for yeast selection. The figure also shows a cDNA molecule flanked by vector homology, an MmeI RE binding site, and a lox71 site. The bait vector (panel B) pCD.2lox66HoxA1 includes an Adc1 promoter, GAL4 DBD cDNA, a hemagglutinin epitope, the HoxA1 sequence, an ampicillin resistance gene for bacterial selection, a CEN sequence for low copy number replication, and a TRP1 gene for yeast selection. A bait vector pGADt7lox71 for library creation (panel C) includes an ADH1 promoter, GAL4 DBD cDNA, a cMyc epitope, a gap-repair cloning sequence that includes the lox66 sequence, the 2 μ ori, a kanamycin resistance gene for bacterial selection and the TRP1 gene for yeast selection.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2189736&req=5

Figure 1: BI-tag Y2H vectors. The prey vector (panel A) pGADt7lox71 includes an Adc1 promoter, GAL4 AD cDNA, a hemagglutinin epitope, a gap-repair cloning sequence that includes the lox71 sequence, the 2 μ ori, an ampicillin resistance gene for bacterial selection and the LEU2 gene for yeast selection. The figure also shows a cDNA molecule flanked by vector homology, an MmeI RE binding site, and a lox71 site. The bait vector (panel B) pCD.2lox66HoxA1 includes an Adc1 promoter, GAL4 DBD cDNA, a hemagglutinin epitope, the HoxA1 sequence, an ampicillin resistance gene for bacterial selection, a CEN sequence for low copy number replication, and a TRP1 gene for yeast selection. A bait vector pGADt7lox71 for library creation (panel C) includes an ADH1 promoter, GAL4 DBD cDNA, a cMyc epitope, a gap-repair cloning sequence that includes the lox66 sequence, the 2 μ ori, a kanamycin resistance gene for bacterial selection and the TRP1 gene for yeast selection.
Mentions: To produce Y2H libraries configured for use in the BI-Tag Y2H method, Y2H vectors were modified to contain mutant lox sequences (15) adjacent to the 3′ end of the cDNA insertion site (Figure 1). Lox sites are recognized by the site-specific recombinase, Cre (16). The lox66 and lox71 sites can recombine with each other to form a wild-type loxP site and double mutant lox66/71 site for which Cre has a very low affinity, suppressing recombination at the double mutant site (15). In the present application, the lox66/71 site is between the linked cDNAs.Figure 1.

Bottom Line: Nonetheless, the logistics of pair-wise screening has resulted in a cumbersome and incomplete application of this method to complex genomes.Once linked, DNA from complex pools of clones can be processed without losing the identity of the interacting partners.The technology described here sufficiently improves the throughput of the Y2H approach to make feasible the generation of near comprehensive interaction maps for complex organisms.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Biophysics and Biochemistry, Roswell Park Cancer Institute, Buffalo, NY, USA.

ABSTRACT
Yeast two-hybrid (Y2H) has been successfully used for genome-wide screens to identify protein-protein interactions for several model organisms. Nonetheless, the logistics of pair-wise screening has resulted in a cumbersome and incomplete application of this method to complex genomes. Here, we develop a modification of Y2H that eliminates the requirement for pair-wise screening. This is accomplished by incorporating lox sequences into Y2H vectors such that cDNAs encoding interacting partners become physically linked in the presence of Cre recombinase in vivo. Once linked, DNA from complex pools of clones can be processed without losing the identity of the interacting partners. Short linked sequence tags from each pair of interacting partner (binary interaction Tags or BI-Tags) are then recovered and sequenced. To validate the approach, comparisons between interactions found using traditional Y2H and the BI-Tag method were made, which demonstrate that the BI-Tag technology accurately represents the complexity of the interaction partners found in the screens. The technology described here sufficiently improves the throughput of the Y2H approach to make feasible the generation of near comprehensive interaction maps for complex organisms.

Show MeSH
Related in: MedlinePlus