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. 1997 Feb 4;94(3):820–825. doi: 10.1073/pnas.94.3.820

Figure 2.

Figure 2

The yeast BUR6 gene encodes the homologue of the DRAP1 corepressor. (A) Sequence alignment of the human (DRAP1) and yeast (Bur6) proteins. Identical residues are denoted by |; similar residues are denoted by : or .. (B) BUR6 is essential for cell viability. Two fragments of the BUR6 gene (open rectangles), corresponding to the indicated 5′ and 3′ regions of BUR6, were ligated into the TRP1 integrating vector pRS304. The resulting plasmid was used to disrupt a single copy of BUR6 in a trp1/trp1 BUR6/BUR6 diploid strain. Sporulation and dissection yielded 2 viable spores for each of 16 tetrads, all of which were phenotypically Trp−. (C) Expression of human DRAP1 complements a bur6::TRP1 null mutation in a human Dr1-dependent manner. Strain YMH218 (YDR1+ bur6::TRP1 leu2 [BUR6-URA3]) was transformed with the MET25/hDRAP1-LEU2 plasmid, selecting for Leu+ transformants. When cured of the BUR6-URA3 plasmid under conditions that induce expression from the MET25 promoter (5-FOA/-Met) no growth was observed. However, strain YMH234 (ydr1::HIS3 bur6::TRP1 ura3 leu2 ade2 [MET25/hDr1-LEU2] [BUR6-URA3]), which expresses human Dr1 rather than yeast YDR1, is viable when cured of the BUR6-URA3 plasmid under conditions that induce expression of hDRAP1 (5-FOA/-Met). This effect is hDRAP1-dependent since the control strain carrying the ADE2 vector alone failed to grow on the same medium. (D) The interaction between Dr1 and DRAP1 (BUR6) is species-specific. Protein mixtures containing recombinant polypeptides (yDr1 + Bur6, lanes 1–3; hDr1 + Bur6, lanes 4–6; hDr1 + DRAP1, lanes 7–9) were incubated with different antibodies as indicated at the top to coimmunoprecipitate yeast Dr1 (lanes 1–3), Bur6 (lanes 4–6), and DRAP1 (lanes 7–9). Antibodies used to detect the coimmunoprecipitated polypeptides in Western blot analysis are indicated at the bottom. Numbers on the left denote molecular weight markers, the immunoglobulin light chain (Ig), and the polypeptide analyzed in the coimmunoprecipitation.