Effect of the internal intron sequence on spliceozyme activity in <i>E. coli</i> cells.

<p>(<b>A</b>) Secondary structure of the spliceozyme (black), with the substrate exons in red and the position of the randomized N₆₄ sequence indicated in the internal region of the intron (blue). (<b>B</b>) Quantitation of <i>E. coli</i> cell growth on LB-agar plates containing 10 µg/mL chloramphenicol. The resulting A₆₀₀ is given for 20 clones, ten of which were chosen before selection on LB-chloramphenicol plates (white columns), and ten of which were chosen after this selection step (grey columns). The name of each clone is given below, with ‘L’ indicating clones from the unselected library and ‘S’ indicating selected clones, 100 denoting the length of the intron, the letter i indicating that the internal region of the intron was randomized, and the number after ‘C’ denoting the clone number. Error bars are standard deviations from three experiments. (<b>C</b>) Sequences of 20 cloned intron sequences, sorted according to their activity in cells. The left column lists the clone names, using the same nomenclature as in (B). The middle column shows the internal sequence of that clone that resulted from the N₆₄ library, which was inserted between the constant regions of the intron (see (A)). The right column shows the growth activity, measured as A₆₀₀ of cell suspensions that resulted from washing LB-growth plates containing chloramphenicol, relative to growth on medium without chloramphenicol.