Genes involved in alanine biosynthesis in {\it Escherichia coli} and the use of transposons in their analysis

Date of Completion

January 1989


Biology, Molecular|Biology, Genetics




Three genes involving alanine biosynthesis, avtA, alaA, and alaB were studied in this thesis. avtA, alaA and alaB were localized to 1.25 kb, 0.9 kb and 1.0 kb fragments, respectively, by $\gamma\delta$ (Tn1000) mutagenesis. The transcription direction of alaA and alaB were determined by alaA::lacZ protein fusion which was constructed using m $\cdot$ Tn3 insertion and alaB::cam gene fusion which was constructed in vitro. Using detailed Escherichia coli chromosome restriction map, the map positions of avtA, ilvE, tyrB, aspC and alaA were mapped or corrected.^ A new approach to determining the sequence of cloned DNA is described. Unique regions near each end of $\gamma\delta$ provide a pair of "portable" primer-specific sites for bidirectional sequencing by the dideoxy chain termination method. The avtA gene of E. coli K-12 was sequenced by this approach. Surprisingly, sequences downstream of the coding region which were interrupted by $\gamma\delta$ insertions or deletion were found to significantly reduce avtA expression. I suggest that these nondisruptive insertions or deletion probably change the DNA topology and thereby alter gene expression.^ The pBR322-related plasmids that are 2.3 kb to 5.1 kb were found predominantly as monomers, while plasmids that are 7.7 kb to 15.2 kb were found predominantly as dimers in rec$\sp+$ cells of E. coli K-12. Monomer size is not the sole determinant of the level of oligomerization, the level of oligomerization is physiologically adjusted. For cultures in which the cells contain both monomers and dimers, the equilibrium of plasmid types is found to be intracellular.^ The E. coli F factor mediates conjugal transfer of a plasmid such as pBR322 by replicative transposition of $\gamma\delta$ from F to the plasmid, to form a cointegrate. Although resolution yields a plasmid containing a single $\gamma\delta$ insertion, the occasional recovery of $\gamma\delta$-free plasmids after conjugal transfer has led to alternative hypotheses for F mobilization that do not involve $\gamma\delta$ transposition. I show here that $\gamma\delta$-free plasmids are found after F-mediated conjugal transfer only when the starting plasmid is a dimer and the recipient is Rec$\sp+$. These plasmids arise by homologous recombination in a heterodimer containing only one $\gamma\delta$ insert. ^