Figure 2.

Expression of ARlacZ1 and gene silencing effects. (A) Cells containing the ARlacZ1-expressing plasmid were treated with IPTG at increasing concentrations from 0 to 5 mM. β-galactosidase activities were measured after IPTG induction, and they are represented by bar graph. The indicated values are calculated from at least three independent experiments. The levels of ARlacZ1 analysed in Figure 2B are represented by line graph. (B) Total cellular RNA was prepared from IPTG-treated cells and subjected to northern blot analysis. ARlacZ1 afsRNA and 5S RNAs were probed with the oligonucleotides, ARlacZnp and 5S + 90R, respectively. RNA quantities are expressed relative to ARlacZ1 induced with 1 mM IPTG, using a semi-standard curve with serial dilutions of total cellular RNA from 1 mM IPTG-induced cells after normalization to 5S RNA. (C) The number of ARlacZ1 in a cell was estimated with serial dilutions of total cellular RNA and known amounts of in vitro transcribed ARlacZ1 as standards. Total cellular RNAs from the indicated numbers of ARlacZ1-expressing DJ480 ssrS::lacZ cells, treated with 1 mM IPTG, were subjected to northern blot analysis. Relative northern blot signals are indicated below each lane. About 0.6 pmole of ARlacZ1 was present in 1 × 10⁷ cells. (D). The lacZ mRNA was quantitated by qRT-PCR. Total cellular RNA from 3 × 10⁶ DJ480 ssrS::lacZ cells containing the vector, treated with 1 mM IPTG, was subjected to qRT-PCR (solid circle). For the standard curve, total cellular RNA prepared from the same number of DJ480 cells lacking lacZ mRNA was mixed with known amounts of LacZ370, an in vitro transcript consisting of 370 nucleotides from the 5′-end of ssrS-lacZ fusion mRNA, and also subjected to qRT-PCR (open circle). The abundance of lacZ mRNA was normalized to the amount of 5S RNA and depicted as relative levels. Values represent the average from three independent experiments. About 0.04 pmole of lacZ mRNA was present in 1 × 10⁷ cells.