Development of an in vivo Assay for Mistranslation: Inducing Activity of Pollutants and Characterization of Amino Acid Substitutions.
Annual scientific rept. 1 Aug 82-31 Jul 83,
OHIO STATE UNIV COLUMBUS DEPT OF MICROBIOLOGY
Pagination or Media Count:
In experiments directed toward development of a simple, quantitative in vivo assay for mistranslation-inducing activity of pollutants, we have established the natural level of cysteine misincorporation into the bacteriophage T7 encoded 0.3 protein. We have also shown that this level can be increased by altering the environment of the translation machinery. This can be accomplished either by growing cells in the presence of mistranslation-inducing antibiotics or by inducing mutations which cause defective ribosomal proteins into the cells being studied. The above results were obtained using purified 0.3 protein. Additional experiments directed toward the first objective have led to a second procedure for quantitating cysteine misincorporation into 0.3 protein. A radioimmune precipitation RIP assay was developed which used polyclonal antibodies to 0.3 protein, SDS-polyacrylamide gel electrophoresis SDS-PAGE, and scanning densitometry. We are currently preparing monoclonal antibody to 0.3 protein to obviate the need for SDS-PAGE and scanning densitometry. Experiments directed toward the second overall objective have provided interesting preliminary results. Trypsinization of cysteine-labeled 0.3 protein analysis of fragments by SDS-PAGE have shown that new peptide fragments are produced.