Fig. 3. Yeast two-hybrid analysis of interactions between BP180 and BP230 and BP180 and ß4. (Top) Schematic representation of BP180. DSR, direct sequence repeat; TM, transmembrane region. (Bottom) Cotransformation of yeast host strain PJ69-4A with BP230^1-555 or ß4^1115-1666 cDNA constructs fused to the Gal4 (AD) domain (in pACT2) and cDNA constructs encoding various fragments of BP180 fused to Gal4 (BD) domain (in pAS2.1) as indicated. Transformation mixtures were spread on SC-LT and SC-LTHA plates and grown at 30°C. Plating efficiency on selective SC-LTHA plates is expressed as a percentage of the plating efficiency on non-selective SC-LT plates from the same transformation. Plates were scored after 6 and 10 days. All efficiencies listed represent an average of multiple independent transformations. ++, plating efficiency on SC-LTHA is 80% of the plating on SC-LT, colonies are fully developed on day 5; +, 40-80% of the plating on SC-LT, small and large colonies on day 5; ±, 50% of the plating on SC-LT at 10 days of growth; -, no colonies on selective plates after 10 days of growth. Note that the interaction between BP180 and BP230^1-555 requires a fragment of BP180 containing amino acids 145-230, whereas for the interaction of BP180 with ß4^1115-1666 other sequences are required. Identical results were obtained when, instead of BP230^1-555, BP230^1-1156 was used for assessment of the interactions with the different BP180 mutants.

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