Development and Use of Anucleated Bacterial Cells to Assay the in vivo Activity of Pollutants
Final rept. Apr 1981-Jul 1985
OHIO STATE UNIV COLUMBUS DEPT OF MICROBIOLOGY
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There were 2 objectives in this research project development of an in vivo assay for mistranslation-inducing activity of pollutants and characterization of amino acid substitutions. The first objective proved to be the more difficult. The T7 0.3 gene product 0.3 protein was purified by a modification of the published procedure, and used to raise rabbit antibody to this protein. A radioimmune precipitation assay was developed which could be used to estimate increased misincorporation of cysteine into 0.3 protein. A sample assay involving only counting of the radioimmune precipitate was not achieved because we were not able to obtain the 0.3 protein free of contaminating proteins in the RIP. The 2nd objective proved more fruitful. We have been able to successfully identify the cysteine substitution sites in the N-terminal 42 positions of 0.3 protein. Cleavage of 0.3 protein with trypsin to identify cysteine for arginine substitutions showed that the major sites of cysteine misincorporation were at residues other than arginine. Sequencing of 35S cysteine-labeled 0.3 protein showed that the most frequent substitution was at residue 15 tyrosine and other substitutions were at a positions 9 asparagine, 12 aspartate, 41 alanine and 42 aspartate. Mistakes at these positions were unexpected and show that context greatly affects misincorporation and that misreading of 2 bases in the triplet at a time occurs relatively frequently.