Fig. 4. Osmotic shock activation of Dd-STATc and of a Y to F mutant of Dd-STATc. GFP-STATc contains the entire Dd-STATc protein with GFP fused at its N-terminus, whereas in GFPSTATc(Y922F) the site of tyrosine phosphorylation at residue 922 is replaced with a phenylalanine residue (Fukuzawa et al., 2001). The two constructs were cloned into a vector containing a blasticidin cassette and transformed into Dd-STATc null cells generated using a hygromycin resistance cassette. The Dd-STATc gene displays a very high frequency of homologous recombination. Therefore, to prevent gene conversion/repair of the remnants of the endogenous Dd-STATc gene, the transformation was performed using REMI (restriction enzyme mediated integration); a technique that disfavours homologous recombination (Kuspa and Loomis, 1992). Western transfer using 7H3, the N-terminal-specific monoclonal antibody, showed that the GFP:STATc and GFP:STATc-YF cells express their respective fusion proteins at similar levels (data not shown). The GFP:STATc construct causes reversion of the Dd-STATc null phenotype; the transformant shows normal plaque morphology when plated on a bacterial lawn and loses its 'slugger' phenotype. By contrast, GFP:STATc-YF transformants remain as defective as their Dd-STATc null parent (data not shown). GFP:STATc and GFP:STATc-YF were subjected to shaken development as in Fig. 2 and then induced with 100 mM sorbitol for the indicated time.

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