|Abstract or Summary
- The ability to faithfully replicate DNA is dependent upon the maintenance
and regulation of its precursors, the deoxyribonucleoside triphosphates.
Enzymes encoded by the bacteriophage T4 have been widely used as models
of biochemical processes. A body of evidence supports the concept that the
bacteriophage T4 enzymes involved in deoxyribonucleotide biosynthesis are
associated as a complex within the infected Escherichia coli. This dissertation
describes the continued examination of the protein-protein interactions
involved in deoxynucleotide biosynthesis of bacteriophage T4.
My studies on the protein-protein interactions involved in
deoxyribonucleotide biosynthesis focused on two unique phage proteins, the
dCMP hydroxymethylase enzyme and the translational regulator RegA. An
initial study was undertaken to determine if the generation of anti-idiotypic
antibodies would prove useful in the identification of direct interactions
between dCMP hydroxymethylase and other proteins of the
deoxyribonucleotide synthetase complex.
For the initial generation of anti-idiotypic antibodies, polyclonal rabbit
antibodies were generated to affinity purified anti-dCMP hydroxymethylase
polyclonal rabbit IgG. The anti-anti-dCMP hydroxymethylase antibody was
found to be specific in binding to the bacteriophage T4 dTMP synthase.
A second method to generate anti-idiotypic antibodies was attempted with
the translational regulator RegA. The generation of anti-idiotypic antibodies to
the RegA protein involved the purification of anti-RegA rabbit Fab fragments
and the generation of anti-anti-RegA polyclonal antibodies within mice. This
alternative method was determined to be inferior to the initial method for
generating anti-idiotypic antibodies. Additional studies involved the
examination of RegA protein-protein interactions using affinity chromatography.
A number of bacteriophage T4 early proteins were determined to associate
with an immobilized RegA column.
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