Tn1 Insertions in the 3' Untranslated Region of the ant Operon of Bacteriophage P22 Affect ant Gene Expression and Alter ant mRNA Stability: a Thesis

Abstract

Insertion of transposable elements within an operon has been known not only to abolish expression of the gene interrupted by the insertion, but also to exert a strong polar effect on the expression of downstream genes in the same operon. In this dissertation, I have shown that insertions of the transposable ampicillin-resistance element Tn1, either in the polar or nonpolar orientation, in the 3' untranslated region of the bacteriophage P22 antirepressor (ant) operon reduce the rate of upstream ant gene expression; insertions of Tn1 in the nonpolar orientation reduce the rate of ant gene expression more significantly than those in the polar orientation. This effect appears to be due to reduced stability of ant mRNA. Tn1 deletion mutants of one of the nonpolar Tn1 insertion mutations have been isolated. Two classes of Tn1 deletions are obtained. Class I retains a 68 bp Tn1 sequence that shows a potential 14 bp stem and 37 bp loop conformation, while class II retains 147 bp Tn1 sequence that shows a potential 69 bp stem and 6 bp loop conformation. These two classes of Tn1 deletions do not delete any P22 sequences. Class I but not class II Tn1 deletion mutants restore the rate of ant gene expression and ant mRNA stability. Six different Ant+ revertants of the class II Tn1 deletion mutant simultaneously restore the rate of ant gene expression and ant mRNA stability. They all have deletions that remove all or part of the class II Tn1 sequence. In one case, the Tn1 sequence retained shows a potential 15 bp stem and 8 bp loop conformation, in the other cases, no secondary structure is predicted to form. The results of the Tn1 deletion mutants suggest that the stem-and-loop structures and the length of stems potentially formed by the Tn1 sequences in mRNA may affect its stability.