Spliceozyme activity in E. coli cells.

(A) Schematic of the plasmid used for the expression of spliceozyme (Sz; black) and CAT pre-mRNA (CAT; red) containing an intron (blue) in E. coli cells. Restriction sites used in cloning of the plasmid are indicated. (B) Quantitation of E. coli cell growth on LB-agar plates containing 8 µg/mL chloramphenicol, by measuring the A600 of cell suspensions from washing the plates after incubation. The black column on the left shows the A600 from cells that express the wild type CAT gene without intron. Grey columns denote the A600 resulting from plasmids containing a CAT gene with an intron and wild-type spliceozymes. White columns denote the A600 from the same constructs as in the grey columns but with six mutations in the catalytic core of the spliceozymes. The black column on the right denotes the A600 resulting from cells containing the pUC19 plasmid without spliceozyme or CAT gene. Two different intron sequences with a length of 64 nucleotides were inserted into plasmid pUCSz2 for this assay (64-CiC2 and 64-SiC3; note that both of these introns were selected for their efficient removal by the spliceozyme; see materials and methods). Note the logarithmic scale for the A600. Error bars are standard deviations from three biological samples.