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      Influence of Bacteriophage Lambda Gene P Expression On Host Escherichia coli Cells

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      BANERJEE-THESIS.pdf (1.162Mb)
      Date
      2011-11-18
      Author
      Banerjee, Anirban
      Type
      Thesis
      Degree Level
      Masters
      Metadata
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      Abstract
      Bacterial viruses have been an important tool for molecular biological discoveries. Bacteriophage is a bacterial virus that has been intriguing researchers for over five decades. But still, we are yet to answer many questions about bacteriophage . genes, O and P play an important role in replication initiation. P outcompetes host DnaC and recruits and directs DnaB helicase to the origin of replication, ori . My study shows that P expression is lethal to the host cell. The P-survivor cells were examined and were found to have chromosomal mutations. This led to the possibility that P protein may be involved in elevating the level of random mutations of the host chromosome. These studies explore this possibility. The influence of P expression on the appearance of chromosomal mutations was assessed by using rifampicin resistance, and auxotrophy as chromosomal targets. Cellular expression of P from a cryptic prophage or from a ColE1-type plasmid was employed in this study. Insertional inactivation of P by recombineering knocked out P-lethality and the potential mutator phenotype among the 42°C survivors. The apparent mutator phenotype was also lost in 42°C survivors upon induction of a cryptic prophage with wild-type O and an insertion in P. When wild-type gene P on a ColE1 plasmid was replaced by a deleted P or a mutated P (P ) gene, the rate of rifampicin resistance among the 37°C cfu was significantly lowered. These observations suggest that there might be a relation between P expression and the appearance of mutations among the P-survivors. My data also support and extend laboratory findings that two missense mutations in host dnaB knock out P-lethality and suppress the observed potential mutator phenotype. ColE1 plasmid loss occurred among the P-survivor cells when P was expressed from a ColE1 plasmid in a wild-type cell. My data suggested that the plasmid-less or cured rifampicin resistant mutants that arose upon thermal induction of P and the 594 rifampicin resistant mutants that arose in the absence of P expression were resistant to P-toxicity. However, my data showed that a 9 base pair deletion in the rpoB gene (mutation in rpoB confers rifampicin resistance iii phenotype to the cells) that affected 4 amino acids was sensitive to P-toxicity which proved that the deletion itself did not make the cells resistant to P-toxicity.
      Degree
      Master of Science (M.Sc.)
      Department
      Microbiology and Immunology
      Program
      Microbiology and Immunology
      Supervisor
      Hayes, Sidney
      Committee
      Bull, Harold; Wilson, Joyce; Howard, Peter
      Copyright Date
      October 2011
      URI
      http://hdl.handle.net/10388/ETD-2011-10-195
      Subject
      Bacteriophage Lambda, Escherichia coli, ColE1 plasmid, potential mutator phenotype, rifampicin resistance, auxotrophy, P-lethality.
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