Isolation and Characterization of Temperature-sensitive Protein Synthesis Mutants of Escherichia Coli by Directed Mutagenesis of the Defective Bacteriophage Lambda Fus2
PhD (Doctor of Philosophy)
Date of Award
Mutagenesis of the defective transducing bacteriophage lambda fus2 was used to isolate a collection of temperature-sensitive mutants of E. coli in the major ribosomal protein gene cluster. Four mutants were examined in detail. Two of the mutants were resistant to the ribosomal antibiotics neamine and spectinomycin. Another mutant was defective in 50S ribosomal subunit assembly at 42(DEGREES)C. The 30S subunit proteins S17 and S19 were changed in two different mutants. Each protein migrated as a more basic species in two-dimensional gels of ribosomal proteins. Ribosomes from each of the four mutants examined showed a temperature-dependent reduction in translational activity in cell-free assays. The kinetic assays showed declines in both the rate and extent of translation at three temperatures. Ribosomes from three of the four mutants were also found to have an increased rate of heat inactivation at 45(DEGREES)C compared to control particles. Mixed subunit assays idendtified a t.s. subunit in each mutant. A defect in reassociation at high temperature was found for the subunits from one mutant. Another mutant showed significantly high levels of misreading at 32(DEGREES)C and 42(DEGREES)C. Two mutants showed a decreased ability to bind 14C-phenylalanine tRNA at the two temperatures tested. The increased efficiency and utility of this mutagenesis method for the isolation of protein synthesis mutants is discussed.
Dissertation - unrestricted
Lohman, Kenton L., "Isolation and Characterization of Temperature-sensitive Protein Synthesis Mutants of Escherichia Coli by Directed Mutagenesis of the Defective Bacteriophage Lambda Fus2" (1985). Electronic Theses and Dissertations. Paper 2722. https://dc.etsu.edu/etd/2722