Figure 5.

Production of extended hairpins and a crRNA in E. coli using the viroid-derived system. RNAs were extracted from the aliquots of E. coli cultures taken at 15.5 h post-inoculation, separated by denaturing PAGE, and gels were stained with ethidium bromide. (a) Hairpin RNAs were produced in the RNase III-deficient E. coli strain HT115(DE3) co-transformed with p15LtRnlSm and the different pLELVd-hairpin. Lane 1, RNA marker with the sizes of the standards indicated on the left in nt; lanes 2 to 6, RNAs from the E. coli transformed to express empty ELVd (lane 2) and the different ELVd forms that included hairpin RNAs of 40, 60, 80 and 100 nt, as indicated (lanes 3 to 6). The positions of the circular empty ELVd and the different chimeric ELVd-hairpin RNAs are indicated on the right. E. coli cultures were grown at 37 °C in TB medium. Each lane contains an aliquot of RNA that corresponds to 0.4 ml of culture. (b) crRNA was produced in E. coli BL21(DE3) co-transformed with p15LtRnlSm and pLELVd-crRNA. Lane 1, RNA marker with the sizes of the standards indicated on the left in nt; lanes 2 and 3, RNAs from the E. coli transformed to express empty ELVd and the chimeric ELVd-crRNA, respectively. The positions of the circular empty ELVd and the chimeric ELVd-crRNA are indicated on the right. E. coli cultures were grown at 37 °C in TB medium and cells harvested at 15.5 h post-inoculation. Each lane contains an aliquot of RNA that corresponds to 0.8 ml of culture.