A — Nuclease S1 protection assays were performed using a 5′ end-labeled probe (the same used in Figure 3) and 50 μg of total RNA isolated from cells incubated at the following temperatures for 30 min: 27°C and 38°C (lane 1); 27°C and 42°C (lane 2); 27°C, 38°C, 27°C and 42°C (lane 3); 27°C, 38°C,

27°C, 42°C and 27°C (lane 4). B — Cells incubated at 27°C for 30 min and then with 250 μM CdCl2 for 60 min (lane 1); cells incubated at 27°C for 30 min, at 38°C for 30 min, at 27°C for 30 min, and then with 250 μM CdCl2 for 60 min (lane 2); cells incubated at 27°C for 30 min, with 250 μM CdCl2 for 60 min and then at 27°C for 60 min (lane 3); cells incubated at 27°C for 30 min, at 38°C for 30 min, at 27°C for 30 min, with 250 μM CdCl2 for 60 min and then at 27°C for 60 min (lane 4). selleck inhibitor Processing of gpx3 intron is inhibited by cadmium treatment To further verify the splicing inhibition by cadmium and its dose-dependent effect, SBI-0206965 purchase we selected another gene to evaluate

intron processing. The gpx3 gene encodes a Glutathione peroxidase and was chosen because its intron is 334-nt length, so unspliced mRNA could be easily selleck chemical differentiated from spliced mRNA in the Northern blot assays. The experiment was carried out using total RNA from B. emersonii cells submitted to heat shock (38°C), and cadmium (50 and 100 μM CdCl2). The unspliced form of gpx3 mRNA was detected only when cells were treated with cadmium, indicating a partial block in mRNA http://www.selleck.co.jp/products/AG-014699.html splicing (Figure 5). Inhibition of splicing was confirmed to be dose-dependent as a more pronounced inhibition was observed when B. emersonii cells were treated with the highest concentration of cadmium (100 μM). The unspliced form of gpx3 mRNA was not detected when cells were submitted to heat shock at 38°C, indicating that heat stress at this temperature produces no visible effect

in gpx3 mRNA splicing. Interestingly, we observed that the gpx3 gene is induced both in response to cadmium and heat shock, an indication that this gene probably plays an important role in the response of B. emersonii to these two environmental stresses. Figure 5 Analysis of gpx3 mRNA in cells exposed to heat shock and cadmium stress. A-Northern blot assay was performed using total RNA extracted from B. emersonii cells submitted to different cadmium concentrations or to heat shock. RNA extracted from cells 60 min after sporulation induction (lane 1). RNA extracted from cells submitted to heat shock (38°C) from 30 to 60 min (lane 2) after induction of sporulation. RNA extracted from cells 60 min after sporulation induction, incubated with 50 μM or 100 μM CdCl2 from 30 to 60 min (lanes 3 and 4, respectively) after sporulation induction. As a control of RNA levels, the 28S rRNA was shown. B — Relative transcript levels of gpx3 mRNA determined by densitometry scanning of the autoradiogram shown in A. The figure legend (1, 2, 3 and 4) is the same depicted above.