Hampel, Arnold E.
M.S. (Master of Science)
Department of Biological Sciences
A temperature sensitive mutant of the Chinese Hamster Ovary cell line has been biochemically and morphologically characterized. Initial studies of the mutant implicated the leucyl-tRNA synthetase as the affected gene product (Thompson et al., 1973). The activity of the enzyme was examined by the rate of esterification of labelled leucine to tRNA in vitro. The assay conditions were optimized as follow: 0.01 M Tris-HCl (pH 8.6), 5 mM Na-EDTA, 8 mM MgCl2 , 5 mM ATP, 0.35 mM CTP, and appropriate amounts of radioactive leucine, non-radioactive amino acids minus leucine, tRNA, and enzyme. The mutant leucyl-tRNA synthetase was not temperature sensitive, but its activity was reduced from that of the wild type. The values for the leucine Km of mutant and wild type enzymes were compared. The of the mutant enzyme was 2 to 4 times higher and thus suggested a reduced affinity for leucine. The species of tRNALeu, tRNAIle, and tRNAVal aminoacylated by mutant and wild type enzymes were identical. Several preliminary enzyme purification methods have been investigated. The surface morphology of the mutant and wild type cells has been examined by scanning electron microscopy. Synchronized cells were obtained by a modification of the mitotic selection procedure of Tobey et al (1967). At low cell density, the cells remained rounded throughout the cell cycle and showed blebs and microvilli. A distinctive feature of cells in sparse culture is the appearance of large cytoplasmic extensions (macropodia). At high cell density, both cell types show cell cycle dependent characteristics similar to those described by Porter et al (1973a). Macropodia are not routinely found in dense cultures. The mutant cells appear to differ from wild type in four ways: i) macropodia are more extended and contorted, ii) microvilli are seen arising from blebs, iii) ruffles on the main cell mass are suppressed, iv) blebs and microvilli appear to alternate their appearance during the cell cycle.
Haars, Laura J., "Biochemical and morphological characterization of a mammalian cell line mutant" (1975). Graduate Research Theses & Dissertations. 1644.
viii, 122 pages
Northern Illinois University
Rights Statement 2
NIU theses are protected by copyright. They may be viewed from Huskie Commons for any purpose, but reproduction or distribution in any format is prohibited without the written permission of the authors.