Bacteriophage D3112 is a Mu-like transposable bacteriophage of Pseudomonas aeruginosa. D3112 has a double-stranded DNA genome of approximately 38kbp with a genetic organization similar to that of coliphages Mu and D108. Genetic mapping and DNA sequence analysis has identified the left-end of the phage genome as encoding the transposase enzyme ( A) and the lysogenic (c) repressor. As genetic control of the lytic-lysogenic switch in D3112 has not been elucidated, characterization of the expression and activity in this region was undertaken. The c open reading frame (ORF), located at the left-most end of the phage genome, has four possible GTG initiation codons. Site-directed mutagenesis, along with an in vivo immunity assay, were used to determine that the second GTG is used as the translation-initiation codon. Northern blotting analysis, transcriptional lacZ fusions and primer extension analyses were used to define the promoter, Pc, to bp972-940 from the left-end of the phage genome. Overexpression, purification and activity analysis of the D3112 c repressor demonstrated that it binds to a 261bp Pvull fragment localized directly upstream of the c repressor ORF. We have located a previously unidentified Ner homologue in D3112. Using cloned fragments containing the D3112 promoter Pc fused to a lacZ reporter gene, beta-galactosidase expression was blocked when the fragment was lengthened from bp1099 to bp1439. A previously unidentified ORF was located from bp1033-1384 that demonstrated significant homology to the Ner proteins of coliphages Mu and D108. The ner promoter, Pner, has been located to bp957-992 using beta-galactosidase assays and primer extension analyses, and Northern blotting demonstrated that D3112 ner is part of a 2.2kb mRNA transcript. As the ner gene begins at bp1033 and the transposase gene begins at bp2539, this transcript is not large enough to encode for the A or B genes, as is seen in the early transcripts of coliphages Mu and D108. The ner prom
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.35549 |
| Date | January 1999 |
| Creators | Salmon, Kirsty Anne. |
| Contributors | DuBow, Michael S. (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Microbiology and Immunology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001681028, proquestno: NQ55377, Theses scanned by UMI/ProQuest. |
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