Accession Number : ADA137069

Title :   Development of an in vivo Assay for Mistranslation: Inducing Activity of Pollutants and Characterization of Amino Acid Substitutions.

Descriptive Note : Annual scientific rept. 1 Aug 82-31 Jul 83,


Personal Author(s) : Reeve,J N ; Rice,J B

PDF Url : ADA137069

Report Date : 23 Nov 1983

Pagination or Media Count : 68

Abstract : 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.

Descriptors :   *Proteins, *Cysteine, In vivo analysis, Molecules, Antibodies, Ribosomes, Radioimmunoassay, Precipitation, Mutations

Subject Categories : Biochemistry

Distribution Statement : APPROVED FOR PUBLIC RELEASE