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Fig. 1. Reconstitution of the core nematode PCD pathway in S. cerevisiae. (A) A semi-quantitative assay compares the effect of transgenes on yeast viability and growth. Yeast were transformed with the indicated plasmids, directing expression of either wild-type or mutant components of the core apoptosis pathway under the control of either a galactose-inducible promoter (GALS) or constitutive promoter (ADH). Suspensions of each transformant were prepared at standardised concentrations. Serial dilutions were made and spotted onto solid inducing minimal media (galactose) vertically down the plate. Colony size indicates growth rate and colony number reflects cell viability. Each dilution was also spotted onto a repressing plate (glucose) to verify that equivalent numbers of each transformant were spotted; only dilutions four and five are shown. (B) Fusion of a GFP tag to CED-4, a FLAG tag to CED-9, and a myc tag to EGL-1 did not affect function. The specified plasmids were transformed into yeast and assayed as described above. Immunoblotting was performed to visualise expression of epitope-tagged proteins. An asterisk indicates a non-specific yeast protein recognised by the anti-GFP antibody. Coomassie staining allowed visualisation of protein loading.
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