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Inhibition phenotype specific for oriλ replication-dependent phage growth, and a reappraisal of the Influence of λ P expression on escherichia coli cell metabolism : p-interference phenotype

dc.contributor.advisorHayes, Sidneyen_US
dc.contributor.committeeMemberXiao, Weien_US
dc.contributor.committeeMemberLoh, Lamberten_US
dc.contributor.committeeMemberHoward, S. Peteren_US
dc.contributor.committeeMemberGoldie, Hughesen_US
dc.contributor.committeeMemberCupples, Claireen_US
dc.contributor.committeeMemberBull, Harolden_US
dc.creatorHorbay, Monique Adelleen_US
dc.date.accessioned2005-12-22T14:52:57Zen_US
dc.date.accessioned2013-01-04T05:12:41Z
dc.date.available2006-12-22T08:00:00Zen_US
dc.date.available2013-01-04T05:12:41Z
dc.date.created2005-12en_US
dc.date.issued2005-12-16en_US
dc.date.submittedDecember 2005en_US
dc.description.abstractBacteriophage λ has been used as a model replicon system for forty years. While the basic λ replication initiation scheme has been elucidated for several decades, many aspects of the mechanisms are unclear. I wished to study two unanswered issues in λ replication initiation. Replication initiation of E. coli and λ each depend upon a protein generally called a licensing factor, which brings the DnaB helicase protein to the origin site to begin DNA synthesis. The licensing factors are the products of host gene dnaC and λ gene P. The synthesis of P from λ DNA in an E. coli cell can competitively interfere with DnaC activity needed for E. coli replication initiation. I wished to learn more about what happens to a host cell when exposed to extended P expression. Previous studies in this laboratory suggested that i) the continuous expression of P was tolerated by a subset of exposed cells and that ii) host defects mapping to dnaB could suppress the effect of extended P expression (P-lethality). I used DNA sequencing to determine if these suppressor mutations were within dnaB. I screened known host mutations for their influence on P-lethality. In summary: E. coli strains with GrpD55 and GrpA80 defects were found to each have two point mutations within their dnaB genes. I was unable to isolate mutations within P that suppressed P-lethality and instead obtained regulatory mutations preventing wild type P expression. Two of these sequenced mutations showed that a cI[Ts] lambda repressor was reverted to cI wild type, blocking P expression at all assay temperatures. P-lethality was reversible in cells exposed to P for up to five hours, causing me to suggest that P-Interference be used in place of the term P-lethality. A non-inducible allele of lexA prevented P-mediated cellular filamentation and enhanced P-Interference. This suggests that induction of the SOS response helps cells to tolerate extended P expression. A host strain containing a defective ClpXP protease significantly enhanced cellular sensitivity to P-Interference. This suggests an important role for the ClpXP chaperone-protease complex in degradation of P and cellular resistance to P expression. I present models to explain the P-Interference Phenotype. Recent reports have re-opened the possibility that the tO-oop-pO element influences λ DNA replication initiation. I have also been investigating this possibility. I found that a plasmid with tO-oop-pO (the terminator, nucleotide sequence and promoter for OOP RNA) and oriλ DNA sequence was inhibitory to the development of repλ phages, and designated this the Inhibition Phenotype (IP). In pursuing the mechanism for this inhibition, I mutated the tO-oop-pO and oriλ elements. I found that the expression of the 77nt OOP RNA transcript and the presence of four 18 base pair repeats (iterons) within oriλ were required for the IP. I isolated spontaneous phage mutants, resistant to the IP. I determined that singly infected cells were sensitive to the IP but that multiply infected cells escaped the IP. I propose that the IP to repλ phage development is directed to the initial or theta mode of λ replication initiation. I found that the theta-mode of λ replication initiation can be bypassed, likely via recombination between multiple phage genomes within a singe cell. I propose models to explain the IP and also suggest a role for OOP RNA in the regulation of λ DNA replication.en_US
dc.identifier.urihttp://hdl.handle.net/10388/etd-12222005-145257en_US
dc.language.isoen_USen_US
dc.subjectOOP RNAen_US
dc.subjectDNA replication initiationen_US
dc.subjectλ P proteinen_US
dc.subjectbacteriophage λen_US
dc.titleInhibition phenotype specific for oriλ replication-dependent phage growth, and a reappraisal of the Influence of λ P expression on escherichia coli cell metabolism : p-interference phenotypeen_US
dc.type.genreThesisen_US
dc.type.materialtexten_US
thesis.degree.departmentMicrobiology and Immunologyen_US
thesis.degree.disciplineMicrobiology and Immunologyen_US
thesis.degree.grantorUniversity of Saskatchewanen_US
thesis.degree.levelDoctoralen_US
thesis.degree.nameDoctor of Philosophy (Ph.D.)en_US

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