Date of Award


Degree Type


Degree Name

Doctor of Philosophy


This work involves the molecular cloning the {dollar}{lcub}\rm A\sp\ast{rcub}{dollar} gene of {dollar}\Phi{lcub}\rm X174{rcub}{dollar} and the rep gene of E. coli into plasmid vehicles that allow their expression to be regulated. These special plasmids contain controlling elements of phage {dollar}\lambda{dollar} that enable expression of cloned genes to be switched "on" by altering the temperature at which they are propagated.;The presence of the {dollar}{lcub}\rm A\sp\ast{rcub}{dollar} gene product is extremely toxic to E. coli. Therefore, to isolate and stably maintain a recombinant plasmid carrying this gene, its expression must be repressed. The use of an inducible expression vector allowed gene {dollar}{lcub}\rm A\sp\ast{rcub}{dollar} to be cloned and its expression to be induced to determine the effect on E. coli metabolism. The stably cloned {dollar}{lcub}\rm A\sp\ast{rcub}{dollar} gene also facilitated in vitro site-directed mutagenesis of this gene. Although expression of {dollar}{lcub}\rm A\sp\ast{rcub}{dollar} in E. coli inhibited DNA replication and cell division, elimination of {dollar}{lcub}\rm A\sp\ast{rcub}{dollar} expression during {dollar}\Phi{lcub}\rm X174{rcub}{dollar} infection had no major effects on the growth of phage that did not produce it.;Enhanced expression of Rep protein, although not deleterious to the cell, was desirable to increase the level of this protein, which is normally present at very low levels in E. coli. By cloning the rep gene into an inducible expression vector with a strong promoter is was possible to synthesize large quantities of Rep protein. In addition, the cloned rep gene was useful in constructing an E. coli strain that completely lacked rep function. The effects of increased levels of Rep protein, or its complete absence, on DNA replication were investigated.;The studies described in this thesis involve the use of expression vectors and other molecular and genetic techniques to manipulate the {dollar}{lcub}\rm A\sp\ast{rcub}{dollar} gene and rep gene to better understand their roles in phage and bacterial replication, respectively.



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