TY - JOUR
T1 - Chromosomal integration of a binding domain:bait gene into yeast enhances detection in the two-hybrid system
AU - Miyashita, Shuhei
AU - Shirako, Yukio
N1 - Funding Information:
This research was partially supported by the Ministry of Education, Science, Sports and Culture, Grant-in-Aid for Scientific Research (B), 2005–2007, 16380034.
PY - 2008/5
Y1 - 2008/5
N2 - We have developed a modified yeast two-hybrid system using the GAL4 transcription activator by integrating a BD:bait gene (GAL4 binding domain:bait gene) into the host chromosome. Locus-specific integration by homologous recombination and use of a strong transcription promoter enabled uniform expression of the integrated BD:bait gene in all host cells. Moreover, after introduction of an AD:prey (GAL4 activation domain:prey) plasmid, false positives were nearly undetectable and false negatives were decreased significantly. Using this improved method a cDNA library could be efficiently screened using roughly 1/10 of the materials required for conventional sequential two-hybrid screen.
AB - We have developed a modified yeast two-hybrid system using the GAL4 transcription activator by integrating a BD:bait gene (GAL4 binding domain:bait gene) into the host chromosome. Locus-specific integration by homologous recombination and use of a strong transcription promoter enabled uniform expression of the integrated BD:bait gene in all host cells. Moreover, after introduction of an AD:prey (GAL4 activation domain:prey) plasmid, false positives were nearly undetectable and false negatives were decreased significantly. Using this improved method a cDNA library could be efficiently screened using roughly 1/10 of the materials required for conventional sequential two-hybrid screen.
KW - Aureobasidine A
KW - cDNA library screening
KW - Chromosomal integration
KW - GAL4 transcription activator
KW - Protein-protein interactions
KW - Two-hybrid system
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U2 - 10.1016/j.mimet.2008.02.006
DO - 10.1016/j.mimet.2008.02.006
M3 - Article
C2 - 18374435
AN - SCOPUS:42249103436
SN - 0167-7012
VL - 73
SP - 179
EP - 184
JO - Journal of Microbiological Methods
JF - Journal of Microbiological Methods
IS - 2
ER -