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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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Cre-mediated recombination in vivo. A Southern blot of HindIII and PstI-digested yeast DNA probed with a GAL4 AD fragment is shown. Lane 1: Bait and prey vectors in yeast strain AH109 in the absence of Cre expression vector (AH109-pCDlox66, pGADt7lox71). Lane 2: Bait and prey vectors in yeast strain AH109 in the presence of Cre expression vector (AH109-pCDlox66, pGADt7lox71, pFA6a2 μ-Adc1Cre).
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Figure 2: Cre-mediated recombination in vivo. A Southern blot of HindIII and PstI-digested yeast DNA probed with a GAL4 AD fragment is shown. Lane 1: Bait and prey vectors in yeast strain AH109 in the absence of Cre expression vector (AH109-pCDlox66, pGADt7lox71). Lane 2: Bait and prey vectors in yeast strain AH109 in the presence of Cre expression vector (AH109-pCDlox66, pGADt7lox71, pFA6a2 μ-Adc1Cre).

Mentions: Y2H vectors were transformed into yeast in the presence or absence of the Cre expression vector, pFA6a2 μ-Adc1Cre, and assessed for recombination (Figure 2). Recombination occurs between lox71 AD vectors and lox66 DBD vectors in the presence of Cre, while no recombination is detected in its absence. It also should be noted that recombination between lox66 and lox71 creates a loxP site in addition to the lox66/71 site and that the loxP site will recombine with other lox sites forming higher order plasmids. These molecules are likely not stable and were not assayed for in the Southern blot or in downstream applications (e.g. BI-Tag purification).Figure 2.


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

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

Cre-mediated recombination in vivo. A Southern blot of HindIII and PstI-digested yeast DNA probed with a GAL4 AD fragment is shown. Lane 1: Bait and prey vectors in yeast strain AH109 in the absence of Cre expression vector (AH109-pCDlox66, pGADt7lox71). Lane 2: Bait and prey vectors in yeast strain AH109 in the presence of Cre expression vector (AH109-pCDlox66, pGADt7lox71, pFA6a2 μ-Adc1Cre).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Cre-mediated recombination in vivo. A Southern blot of HindIII and PstI-digested yeast DNA probed with a GAL4 AD fragment is shown. Lane 1: Bait and prey vectors in yeast strain AH109 in the absence of Cre expression vector (AH109-pCDlox66, pGADt7lox71). Lane 2: Bait and prey vectors in yeast strain AH109 in the presence of Cre expression vector (AH109-pCDlox66, pGADt7lox71, pFA6a2 μ-Adc1Cre).
Mentions: Y2H vectors were transformed into yeast in the presence or absence of the Cre expression vector, pFA6a2 μ-Adc1Cre, and assessed for recombination (Figure 2). Recombination occurs between lox71 AD vectors and lox66 DBD vectors in the presence of Cre, while no recombination is detected in its absence. It also should be noted that recombination between lox66 and lox71 creates a loxP site in addition to the lox66/71 site and that the loxP site will recombine with other lox sites forming higher order plasmids. These molecules are likely not stable and were not assayed for in the Southern blot or in downstream applications (e.g. BI-Tag purification).Figure 2.

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