Regulation of the osmotic response of the rtoA gene
Masatsune Tsujioka and Jeffrey G. Williams+
School of Life Sciences, University of Dundee, Dow St., Dundee DD1 5EH
+ tel 44 1382 385220, fax 44-1382 34421
The rtoA gene is activated by hyper-osmotic stress. We identify a region upstream of the newly assigned ATG that is essential for stress-induced expression and suggest that it contains one or more basal promoter elements.
RtoA was identified, genetically, as being necessary for normal developmental patterning and for the linkage that exists between cell cycle position and choice of cell fate (Wood et al., 1996). Subsequent work showed that it has vesicle fusing properties, that it mediates cell cycle-dependent cytosolic pH regulation and that it affects calcium signaling (Azhar et al., 2001; Brazill et al., 2000). There is, therefore, a considerable body of knowledge concerning rtoA but there is no published study of its mode of transcriptional regulation.
Materials and Methods
Creation of mutant constructs
The rtoA gene and its promoter (defined as the region downstream of nt -604, numbered relative to the newly assigned ATG) were used to generate 3’ to 5’ deletion constructs by creating PCR products that were amplified using sequence specific primers (Fig 1). These were fused to lacZ, by subcloning them as XbaI/BglII fragments into the corresponding sites of the vector pDdgal17(Harwood and Drury, 1990). Dictyostelium cells, strain Ax-2, were grown axenically and transformed with these construct DNA as described previously (Araki et al., 2003).
Analysis of stress response
The assay for stress responsiveness was performed on cells developed in suspension for four hours and then exposed to 100mM sorbitol for 40 or 45 minutes (Araki et al., 2003). Total cellular RNA was extracted and expression of the lacZ reporter gene monitored at the RNA level, by Northern transfer. This was performed as described previously (Araki et al., 2003) and each gel lane contained 15µg of total RNA. The filter was hybridised with a BstXI/BstXI fragment from within the lacZ gene that was labelled with α-32PdATP using a Random Primed DNA Labeling Kit (Roche). The filter was hybridised in ‘ExpressHyb’ hybridization solution and washed in 1x SSC, 0.1% SDS as recommended by the manufacturer (Clontech).
An analysis of 3´to5´deletion lacZ fusion constructs of the rtoA promoter was performed by Northern transfer (Fig 1). Constructs A and B, and C remain stress inducible. However, construct D is inactive. The fusion of the rtoA promoter elements was directly to lacZ; no basal promoter elements were provided. Therefore we assume that the loss of expression in deleting from C to D reflects the loss of such elements and there is a perfect TATAAA box in the essential region. Clarification of this issue will require definition of the cap site.
Fig 1 Schematic representations of rtoA and analysis of the expression of 3´ to 5´ rtoA deletion constructs. Expression of rtoA:lacZ fusion genes, with the indicated end-points in rtoA, before and after 45 min of osmotic shock and analysed by Northern transfer.
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