FIG. 7.

Mapping the oligomerization domain on AMF1. (A) Interaction of VP16-AMF1 with LexA-AMF1 fusion proteins. Galactose-inducible expression vectors containing the LexA-AMF1 deletion fusions were transformed along with VP16-AMF1, AMF1, or the VP16 AD into DBY1 containing a lexA operator-lacZ reporter. Colonies were transferred to galactose-X-Gal plates and color formation was monitored. All constructs except LexA-AMF1(14–76) activate transcription of pSH18-34. A plus sign indicates earlier and more intense color formation in the presence of VP16-AMF1 than in the presence of the VP16 AD or AMF1. (B) In vitro binding of deletion mutant AMF1 and wild-type AMF1. Three of the AMF1 deletion mutants, AMF1(14–212), AMF1(14–130), and AMF1(14–76), were subcloned into vector pGEX2T (Pharmacia), and expressed in E. coli BL21::DE3(pLysS). Purified GST and GST-AMF1 fusion proteins were incubated with ³⁵S-labeled in vitro-translated full-length AMF1. Bound AMF1 was resolved by SDS–15% PAGE and analyzed with a Bio-Rad GS-250 molecular imager. The right side lane shows 10% of input AMF1.