Fig. 6. Phenotypes of Rgf3p overexpression. (A) Micrographs of Calcofluor White (Cfw) stained wild-type cells transformed with pREP3X (empty plasmid) or pREP3X-rgf3⁺ (rgf3⁺ overexpression) grown without thiamine for 20 hours. (B) In vitro glucan synthase (GS) activity assayed with the membrane fraction of wild-type cells (MS38) transformed with pREP3X, pREP4X-rgf3 (rgf3⁺ overexpression), pREP3X-rho1 (rho1⁺ overexpression) or both pREP4X-rgf3 and pREP3X-rho1 (rgf3⁺ and rho1⁺ overexpression). Extracts were prepared from cells grown in MM without thiamine at 32°C for 18 hours. Specific activity is expressed as milliunits per mg protein. Values are the means of at least three independent experiments with duplicated samples, and error bars represent standard deviations (s.d.). (C) Cell-wall composition in cells that overexpress rgf3⁺. The relative levels of [¹⁴C]-glucose radioactivity incorporated into each cell-wall polysaccharide are shown for the same strains as above: wild-type (MS38) transformed with pREP3X, pREP4X-rgf3 (rgf3⁺ overexpression), pREP3X-rho1 (rho1⁺ overexpression) or both at the same time. Cells were grown in the absence of thiamine for 18 hours and then [¹⁴C]-glucose was added 6 hours before harvesting the cells. Values are the means of three independent experiments with duplicate samples. Standard deviations for total carbohydrate values are shown.