Isolation and Characterization of Deletion Mutants of Bacteriophage Lambda

Citation

Huskey, Robert John (1968) Isolation and Characterization of Deletion Mutants of Bacteriophage Lambda. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/436X-JK20. https://resolver.caltech.edu/CaltechETD:etd-08102006-130739

Abstract

A method for the isolation of deletion mutants of lambda is reported utilizing thermal inactivation as a selective enrichment technique. Thermal inactivation of lambda is shown to depend upon the amount of DNA in the phage, the temperature of inactivation and the ionic make-up of the medium. A phenotypically heat resistant, less dense class of phage is reported which, upon growth, returns to the density and heat characteristics of wild type phage. The several hundred deletion mutants isolated have many properties in common with the lambda b2 mutant. All mutants are of lambda immunity and host restriction type. In these mutants, the amount of DNA deleted, calculated from their density, varies from 10 to 30%. All of the mutants like b2 form abortive lysogens. Preliminary experiments indicate that the mutants retain the recombination function of lambda. Some of the deletion mutants show reduced recombination for the region deleted, the middle segment of the genome. However, a number of the deletion mutants exhibit abnormally high recombination for the middle segment of the genome and in one of these mutants, lambda b130, a physical discontinuity in the DNA of the phage occurs at or near the locus which manifests high recombination. This discontinuity resembles in structure the cohesive ends of lambda DNA. In an attempt to explain the properties and bizarre structure of the chromosome of b130, a model for lambda integration and excision is proposed. As a result of this model, the high recombination of certain lambda deletion mutants is thought to be due to the inclusion of a small region of host DNA into the middle segment of the DNA of these mutants. It is proposed that this included region resembles closely the cohesive ends of lambda leading to the production of a new set of cohesive ends and localized high recombination. The model introduces several new notions concerning the integration of lambda into the chromosome which are experimentally testable.

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