Skip to main content
. 2006 Sep 7;25(18):4326–4337. doi: 10.1038/sj.emboj.7601313

Figure 3.

Figure 3

Characterisation of the AtBARD1 and AtBRCA1 interaction by yeast two-hybrid assay and by BiFC in transiently transfected mustard seedlings. (A) Yeast two-hybrid assay. Different constructs of AtBRCA1 or AtBARD1 were used either as bait (DNA-BD) or prey (DNA-AD) and tested for their ability to activate the lacZ reporter gene (X-gal) and the nutritional marker gene leucine (Leu). The full-length protein AtBARD1 was, when fused to a DNA-binding domain (DNA-BD), able to activate the lacZ reporter gene as well as the leucine reporter gene. The use of AtBRCA1 as bait led to inconsistent results. A truncated version of AtBRCA1, containing the first 59 N-terminal aa (representing the RING domain) interacted with the complete AtBARD1 protein fused to the activation domain and resulted in the activation of the lacZ reporter gene and the leucine reporter gene. No interaction could be demonstrated between the AtBRCA1 RING domain and a C-terminal part of AtBARD1 (AtBARD1 C-T). Furthermore, none of the single used constructs was able to activate transcription. Blue staining of the yeast colonies appeared within 30 min for the AtBARD1 protein, to up to 2 h for the AtBRCA1 AtBARD1 interaction, whereas the growth of yeast colonies on LEU lacking medium was determined after 2 days. (B–I) (B′–I′) and (B″–I″) BiFC analysis in transiently transfected mustard seedlings. The pictures B–I show an YFP signal in case of a protein interaction, in the nucleus of a representative cell, owing to the restoration of the YFP complex. The pictures B′–I′ show a CFP signal from a cotransfected nuclear marker. The pictures B″–I″ show the same cells as in (B–I) and (B′–I′), respectively, by bright field microscopy. Bars=20 μm. B, B′ and B″ AtBRCA1 (YC) and AtBARD1 (YN); C, C′ and C″ AtBRCA1 (YC) and empty vector pMAV-GW-YN; D, D′ and D″ AtBRCA1 RING (YC) and AtBARD1 (YN); E, E′ and E″ AtBRCA1 RING (YC) and empty vector pMAV-GW-YN; F, F′ and F″ AtBRCA1 C-terminus (YC) and AtBARD1 (YN); G, G′ and G″ AtBARD1 (YN) and empty vector pMAV-GW-YC; H, H′ and H″ ASK1 (YN) and EID1 (YC), positive control; I, I′ and I″ ASK1 (YN) and EID1ΔF (YC), negative control.