Degree Type

Dissertation

Date of Award

1985

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

Abstract

Mitochondrial (mt) ribosomes and ribosomal subunits were prepared from HeLa S(,3) (chloramphenicol (CAP) sensitive) and 296-1 (CAP resistant) cell lines and used in in vitro protein synthesis activity studies. Mt ribosomes isolated in low salt (100 mM KCl, 30 mM MgCl(,2)) were active in poly(U)-directed protein synthesis when supplemented with E. coli tRNA and E. coli supernatant fraction (S-100). Washing these ribosomes with high salt-containing buffers (500 mM KCl, 5 mM MgCl(,2)) results in the loss of protein synthesis activity. Activity by high salt washed mt ribosomes was restored by the addition of a mt supernatant fraction (mt S-100);Mt S-100 was shown to aminoacylate both E. coli and mt tRNA with phenylalanine. The efficiency of this aminoacylation was not sufficient to support protein synthesis; E. coli S-100 was required in the protein synthesis system to provide sufficient aminoacyl-tRNA synthetase activity;Mt S-100 was found to inhibit protein synthesis by E. coli ribosomes. This inhibition was not due to contamination by lysosomes in mt preparations, nor was it affected by RNasin, a ribonuclease inhibitor. The cause of this inhibition remains unknown;Mt ribosomal subunits were prepared by dissociation in high salt and separation on sucrose gradients. Ribosomes, reconstituted from subunits, were active in polynucleotide-directed protein synthesis. Using hybrid ribosomes reconstituted from S(,3) and 296-1 subunits in poly(U,C)-directed protein synthesis, it was found that the cytoplasmicly inherited mutant gene conferring CAP resistance to the 296-1 cell line, alters a component of the mt ribosomal large subunit.

DOI

https://doi.org/10.31274/rtd-180813-6123

Publisher

Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/

Copyright Owner

Thomas James Girard

Language

en

Proquest ID

AAI8514402

File Format

application/pdf

File Size

133 pages

Included in

Biochemistry Commons

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