We have previously suggested that both transcriptional and post-transcriptional mechanisms would contribute to these
differences [16]. Presently, we used Pb339, Pb3 and Pb18 in a controlled comparison of transcript accumulation in yeast cells cultivated to logarithmic phase in defined F12/glc medium. At similar cell concentrations for each culture, transcript accumulation was by far higher in Pb339, followed by Pb3 and Pb18 (Table 3). We have observed that differences were not apparent upon modulation LY2603618 cell line with primary nitrogen sources, i.e., PbGP43 transcript from Pb3, Pb18 and Pb339 were negatively modulated with ammonium sulfate at similar rates [22]. We presently tested two other types
of stimuli in cultures growing in F12 medium, specifically, fetal calf serum (FCS) and glucose. As observed in Figure 5, supplementation with 2% FCS was not able to modulate PbGP43 transcript accumulation in 30 min. On the other hand, an increase in selleck chemicals llc glucose concentration from 0.18% (present in F12 Apoptosis Compound Library mouse medium) to 1.5% for 30 min evoked a decrease in the relative amount of transcripts of about 70% (2,6-fold for Pb3, 4-fold for Pb18 and 3,5-fold for Pb339). This rate of modulation was similar in Pb339, Pb3 and Pb18, although the initial amount of transcripts varied considerably among them. This kind of negative expression modulation with glucose would be expected for glucanase genes [26]. Table 3 Real time RT-PCR showing PbGP43 transcript accumulation from three independent experiments, in which Pb339, Pb3 and Pb18 isolates were cultivated in F12/glc. Isolate Samples TA N° of cells/mL N° of days Pb339 Exp1 3860 ± 51,5 9,2 × 106 4 Exp2 4443 ± 25,6 1,1 × 107 4 Exp3 10106 ± 108 1,6 × 107 4 Pb3 Exp1 41,6 ± 3,9 8,9 Sucrase × 106 4 Exp2 55,5 ± 4,3 1 × 107 4 Exp3 51,66 ± 4,8 1,1 × 107 4 Pb18 Exp1 7,4 ± 0,8 1,4 × 107 6 Exp2 4,1 ± 0,5 1 × 107 6 Exp3 6,95 ± 0,5 1,2 × 107 6 TA, relative number of transcript copies when compared with α-tubulin. Culture densities and ages are indicated.
Figure 5 Accumulation of Pb GP43 transcript after 30 min of stimulus of P. brasiliensis yeast cells with glucose or fetal calf serum (FCS). Real time RT-PCR experiments showing the relative variation of PbGP43 transcript accumulation in Pb339, Pb18 and Pb3 cells stimulated with A, 2% FCS or B, 1,5% glucose. Control experiments were attributed value 1.0. The α-tubulin gene was used as standard. Discussion By using EMSA and a series of probes covering five regions within the upstream 326 bp of the PbGP43 ORF we managed to identify protein binding sequences between nt -134 to -103 and nt -255 to -215. Together, these regions abrogate three substitution sites characteristic of P. brasiliensis PS2 isolates: that might not be incidental, since one mutation at -230 seemed to alter binding affinity.