e., they displayed cyclosporine A/paraquat-sensitivity comparable to the wild type strain (data not shown). Figure 6D shows GFP::AnRcnA germlings that were grown for 24 hs in MM+2% glycerol at 30°C and either incubated or not in the presence of calcium chloride 50 mM or EGTA 25 mM for 5 to 15 minutes. In all conditions, AnRcnA was mostly detected along the germling and did not accumulate Bafilomycin A1 cell line in the nuclei (Figure 6D and data not shown). The same results were observed when glucose was used as a single carbon source (data

not shown). These results show that AnRcnA cellular localization is not affected by the cellular response to calcium chloride. We overexpressed AnrcnA aiming to investigate genes that could be potentially regulated by the calcipressin-calcineurin pathway. Accordingly, we constructed an A. nidulans overexpression AnrcnA

strain by using the alcA promoter. We used real-time RT-PCR to test the mRNA levels of AnrcnA when the wild type and alcA::AnrcnA strains were grown in the presence of either glucose or glycerol+ethanol as carbon sources (Figure 7A). The AnrcnA gene showed about the same mRNA accumulation when the wild type strain was grown either in the presence of glucose or glycerol+ethanol (Figures 7A). However, when the alcA::AnrcnA strain was grown in the presence of glycerol+ethanol, the AnrcnA gene had a mRNA accumulation of about 16.0 CDK inhibitor times when compared its growth in the presence of glucose (Figures 7A). Surprisingly, AnRcnA overexpression in liquid medium (for 16 hours at 37°C) did not cause any growth inhibition in Axenfeld syndrome the presence of either calcium or cyclosporine (data not shown). AnRcnA overexpression (for 16 hours at 37°C) also had no effect on the ΔAncnaA phenotype in liquid medium (data not shown). Next, we observed the effects of overexpressing AnRcnA on calcineurin activity. Fosbretabulin Interestingly, calcineurin activity is dramatically increased when the wild type strain was grown in the presence of ethanol (Figure 7B). In the alcA::AnrcnA strain, calcineurin activity was reduced about 50% (Figure 7B), what it is again consistent with a role for Aspergilli RcnAs in the

inhibition of calcineurin activity. Both strains display the same calcineurin activity when grown in the presence of glucose (Figure 7B). Assuming an inhibitory role for AnRcnA on the calcineurin activity, it should be expected an increase in the calcineurin activity for the alcA::AnrcnA grown in the presence of glucose. However, the lack of reduction in the calcineurin activity is possibly due to the fact that the rcnA mRNA accumulation was not completely abolished in the alcA::AnrcnA grown in the presence of glucose (Figure 7A). Overexpression of the yeast protein Rcn1p or the human homologues also inhibited the activation of the transcription factor Crz1p and the inhibition of the H+/Ca2+ exchanger Vcx1p [33]. Figure 7 Overexpression of the A. nidulans AnrcnA gene.