Development and Use of Anucleated Bacterial Cells to Assay the in vivo Activity of Pollutants

摘要

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 trypsion 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 (aspartqate). 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.