PHYSICAL AND TRANSCRIPTIONAL CHARACTERISTICS OF ESCHERICHIA COLI PLASMIDS WITH DNA INSERTS OF POTENTIALLY ALTERABLE SECONDARY STRUCTURES (SUPERCOILED, CIRCULAR DICHROISM, SPECIFIC LINKING DIFFERENCE)
Abstract
The research covered in this thesis concerns the influence of alternating base sequence inserts on plasmid transcription in vitro and expression of resistance gene markers in vivo. Stretches of alternating base sequences (d(GC)(,n) and d(AG)(,n)) were inserted between the anti-tet promoter and the chloramphenicol resistance gene in pBR329, and between the lac Z' promoter (from pUC13) and the tetracycline resistance gene in derivatives of pBR329. The question was asked whether these inserts could influence transcription of the structural genes when under topological stress. The inserts were shown to alter the copy numbers of plasmids in recA- E. coli cells, as well as expression of the resistance genes in vivo (probably due to copy number changes). Two-dimensional gel electrophoresis was used to demonstrate a B-Z transition within a d(GC) stretch in supercoiled plasmids. Circular dichroism spectra of supercoiled plasmids were taken to show that such spectroscopy is useful in observing Z-DNA in plasmids. It was observed that total plasmid transcription increases with increasing negative superhelical density, but when transcription is initiated from the anti-tet promoter transcription decreases during a B-Z transition in the d(GC) insert. This effect was not observed for transcription initiated from the lac Z' promoter, a superhelical density dependent promoter.
Degree
Ph.D.
Subject Area
Molecular biology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.