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Title: Restriction alleviation and modification enhancement by the ral gene of bacteriophage λ
Author: King, Gareth
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1995
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Unmodified λral+ phages are restricted by bacteria expressing type 1A R-M systems, presumably restriction occurs before the infecting phage has time to express the ral gene. In order to examine Ral activity in vivo, the degree of modification of progeny phages was measured after a single round of infection on a restriction-proficient strain, and the prediction that the progeny of ral+ phages are more likely to become modified than ral phages was confirmed. This modification would be advantageous to those phages that subsequently infect bacteria with an R-M system of the same specificity. Interestingly, in some hosts progeny phages were similarly modified irrespective of their ral genotype. Some strains of E. coli contain a defective prophage, termed Rac, that is known to encode a Ral-like antirestriction function, Lar. The inability to detect Ral activity was found to correlate with the presence of the Rac prophage; presumably lar is expressed in a sub-population of Rac+ hosts. The gene encoding lar was isolated by cloning DNA fragments from the Rac prophage in a plasmid vector and screening for anti-restriction activity. In this way, lar was localised to a 500 bp DNA fragment, and was further defined by site-directed mutagenesis in conjunction with an analysis of the phenotypes of the mutants, and the polypeptides produced. The predicted amino acid sequences of Ral and Lar are remarkably dissimilar, although a number of residues do align which may indicate important features for structure and/or function. The ral gene has been overexpressed and its product purified. Methylation assays, with purified Ral and the type IA EcoKI MTase failed to show any change in methylation activity in the presence of Ral.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available