FIG. 10.
Effects of mutagenizing the downstream inverted repeat (nucleotides 824 to 841) on β-Gal activity and lacZ.st mRNA levels. A site-directed mutation to eliminate the inverted repeat was constructed by amplification of the 566–888 fragment into two separate fragments. Fragment 566–835 was amplified by using the universal −40 primer (on pTRK391) and a primer complementary to nucleotides 817 to 835 (A→G mutation at nucleotide 825). Fragment 842–888 was generated by using a primer consisting of nucleotides 842 to 858 and the lacZ primer (on pTRK391). Nucleotides 836 to 841 were replaced with a SalI site, which allowed fusion of the two fragments (566–835SalI and SalI842–888) to yield fragment 566–888S. Mutations made to the inverted repeat are indicated above the graph. β-Gal levels are the average of at least three separate assays. For each assay, time point determinations were performed in duplicate.