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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 method. (A) PCR amplification (with primers that anneal to GAL4 AD and DBD cDNAs) across linked cDNAs and lox sequence of the HoxA1 Y2H positive colony DNA (bar). (B) MmeI digestion of the PCR product to produce the 86 bp BI-Tag (arrow). (C) Left lane: 160 bp PCR product that includes the BI-Tag and 40 bp linkers (arrow), Middle lane: 94 bp BI-Tag (arrow) generated by NotI digestion with NotI compatible overhangs for concatenation. (D) Amplicons of BI-Tag concatamer inserts in a cloning vector.
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Figure 4: BI-Tag method. (A) PCR amplification (with primers that anneal to GAL4 AD and DBD cDNAs) across linked cDNAs and lox sequence of the HoxA1 Y2H positive colony DNA (bar). (B) MmeI digestion of the PCR product to produce the 86 bp BI-Tag (arrow). (C) Left lane: 160 bp PCR product that includes the BI-Tag and 40 bp linkers (arrow), Middle lane: 94 bp BI-Tag (arrow) generated by NotI digestion with NotI compatible overhangs for concatenation. (D) Amplicons of BI-Tag concatamer inserts in a cloning vector.

Mentions: The BI-Tag method was then used (diagrammed in Figure 3). First, DNA, which includes recombined plasmid DNA (Figure 3A and B), was isolated from a pool of the ∼1000-colony HoxA1 interaction library. Amplicons across interacting cDNAs were generated using PCR with GAL4 DBD- and GAL4 AD-specific primers (Figure 3C). This reaction resulted in DNA fragments ranging from 1500 bp and larger in length when assessed on a 1% agarose gel (Figure 4A). Each amplicon includes the HoxA1-DBD fusion cDNA, an MmeI site, the lox66/71 double mutant recombination product, a second MmeI site, and the interacting AD-cDNA fusion. MmeI digestion was then used to excise an ∼86 bp fragment from each amplicon (Figure 3C). These fragments are visible on a PAGE gel (Figure 4B). These DNA fragments contain lox66/71 flanked by MmeI sites and the 19–21 bp BI-Tags used to identify the two interaction partners. Linkers with NotI cleavage sites were ligated to each end and used as primer binding sites in PCR amplification (Figures 3D and 4C, left lane). NotI digestion, which results in ∼94 bp fragments with complementary overhangs for concatenation, are gel purified by 6% PAGE (Figure 4C, lane center lane). Purified DNA was ligated (Figure 3E), and the resulting concatamers >500 bp were purified and cloned into a NotI-digested cloning vector. Figure 4D shows amplicons across concatenated BI-Tags with size distributions between ∼300 and 700 bp. DNAs recovered from the clones were sequenced.Figure 3.


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 method. (A) PCR amplification (with primers that anneal to GAL4 AD and DBD cDNAs) across linked cDNAs and lox sequence of the HoxA1 Y2H positive colony DNA (bar). (B) MmeI digestion of the PCR product to produce the 86 bp BI-Tag (arrow). (C) Left lane: 160 bp PCR product that includes the BI-Tag and 40 bp linkers (arrow), Middle lane: 94 bp BI-Tag (arrow) generated by NotI digestion with NotI compatible overhangs for concatenation. (D) Amplicons of BI-Tag concatamer inserts in a cloning vector.
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Related In: Results  -  Collection

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Figure 4: BI-Tag method. (A) PCR amplification (with primers that anneal to GAL4 AD and DBD cDNAs) across linked cDNAs and lox sequence of the HoxA1 Y2H positive colony DNA (bar). (B) MmeI digestion of the PCR product to produce the 86 bp BI-Tag (arrow). (C) Left lane: 160 bp PCR product that includes the BI-Tag and 40 bp linkers (arrow), Middle lane: 94 bp BI-Tag (arrow) generated by NotI digestion with NotI compatible overhangs for concatenation. (D) Amplicons of BI-Tag concatamer inserts in a cloning vector.
Mentions: The BI-Tag method was then used (diagrammed in Figure 3). First, DNA, which includes recombined plasmid DNA (Figure 3A and B), was isolated from a pool of the ∼1000-colony HoxA1 interaction library. Amplicons across interacting cDNAs were generated using PCR with GAL4 DBD- and GAL4 AD-specific primers (Figure 3C). This reaction resulted in DNA fragments ranging from 1500 bp and larger in length when assessed on a 1% agarose gel (Figure 4A). Each amplicon includes the HoxA1-DBD fusion cDNA, an MmeI site, the lox66/71 double mutant recombination product, a second MmeI site, and the interacting AD-cDNA fusion. MmeI digestion was then used to excise an ∼86 bp fragment from each amplicon (Figure 3C). These fragments are visible on a PAGE gel (Figure 4B). These DNA fragments contain lox66/71 flanked by MmeI sites and the 19–21 bp BI-Tags used to identify the two interaction partners. Linkers with NotI cleavage sites were ligated to each end and used as primer binding sites in PCR amplification (Figures 3D and 4C, left lane). NotI digestion, which results in ∼94 bp fragments with complementary overhangs for concatenation, are gel purified by 6% PAGE (Figure 4C, lane center lane). Purified DNA was ligated (Figure 3E), and the resulting concatamers >500 bp were purified and cloned into a NotI-digested cloning vector. Figure 4D shows amplicons across concatenated BI-Tags with size distributions between ∼300 and 700 bp. DNAs recovered from the clones were sequenced.Figure 3.

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