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Fig. 4. Analysis of the effects of increased expression of domains of byr4. (A) Expression of the byr4(1-595) fragment from the nmt1 promoter was induced by growth in the absence of thiamine for 20 hours at 25°C in EMM2 medium. Cells were fixed and stained with DAPI and Calcofluor. (B) The byr4(1-595) fragment was expressed from the nmt1 promoter as in A. Cells also expressed the indicated tagged GFP-SIN fusion protein. The GFP signals were photographed in living cells. (C) Expression of the byr4(595-665) fragment from the nmt1 promoter was induced by growth in the absence of thiamine for 20 hours at 25°C in EMM2 medium. Cells were fixed and stained with DAPI and Calcofluor. (D) The byr4(595-665) fragment was expressed from the nmt1 promoter as in C, in cells expressing the indicated tagged GFP-SIN fusion protein. The GFP signals were photographed in living cells. (E) Protein extracts were prepared from either wild-type cells grown at 25°C (left panel) or mts2-1 cells incubated at 36°C for 4 hours (right panel), in which expression of the indicated fragment of byr4 from the nmt1 promoter had been induced, as described above. One lane, showing the effects of a truncation not discussed in this paper, has been removed from the gel. Bar, 10 µm (panels A-D).
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