Fig. 2. Identification of NESs within Dd-STATc. (A) Alignment of potential NES sequences. Dd-STATc (middle) contains, near its centre, a leucine/isoleucine rich region (red letters) (the EXP region). A part of Dd-STATa (top) that contains the 50 a.a. region homologous to EXP has been shown to function as an LMB-sensitive NES (see text) and is displayed here for comparison. Identities between Dd-STATa and Dd-STATc are shown in orange. The NES of rad24 is shown (bottom) for comparison, aligned below the leucine rich region near the C terminus of EXP (marked as B). Another candidate NES, i.e. a leucine-rich region, nearer the N terminus is also marked as A. (B) Nuclear accumulation of a Dd-STATc mutant protein lacking EXP. The GFP:STATc construct comprises the entire Dd-STATc protein, with GFP fused at its N terminus. It is a blasticidin-based, `single copy' vector (Fukuzawa et al., 2001). GFP:STATcEXP is an equivalent construct with an internal deletion (residues 505 to 554) that removes just the EXP region. Both these constructs were introduced into cells and stable transformant clones were selected. In order to rule out homologous gene conversion, a frequent event with Dd-STATc, the structure of the two GFP fusion proteins was checked by western blotting and were as expected. Cells transformed with these constructs were developed in shaken suspension for 4 hours and then analysed for nuclear GFP after a further 5 minutes of incubation in either the presence or absence of 100 nM DIF.

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