2.10 Yeast two-hybrid analysis and transcription activation
activity
A yeast two-hybrid assay was performed using a HybriZAP-2.1 two-hybrid
vector system (Stratagene, https://www.agilent.com/) as described
previously (Matsuoka, Yasufuku, Furuya, & Manmori, 2015). The
full-length coding regions of CRCT , GF14A and GF14Bwere amplified by RT-PCR using gene-specific primers (Table S1). The PCR
product of CRCT was cloned into the pAD-GAL4-2.1 vector for the
expression of GAL4 transcriptional activation domain fusion proteins.
The PCR products of GF14A and GF14B were inserted into the
pBD-GAL4 Cam vector for the expression of GAL4 DNA-binding domain fusion
proteins. Each pair of bait and prey vectors was cotransformed into
yeast (strain YRG-2). The transformation was confirmed by growth on the
synthetic media plate lacking Leu and Trp (SD2-). The
protein interaction was monitored by growth on the synthetic media plate
lacking Leu, Trp and His (SD3-).
Transcriptional activation activity was analyzed using the two-hybrid
vector system without pAD-GAL4-2.1 vector. Eight different CRCTfragments were amplified by PCR using primers listed in Table S1 and
cloned into the pBD-GAL4 Cam vector. The vectors were transformed into
the yeast. The transformation and transcription activation activity were
monitored by growth of yeast on the synthetic media plate lacking Trp
(SD1-) and Trp and His (SD2-),
respectively.