Studies on Protein Synthesis after Heat Shock in Drosophila melanogaster

Citation

Chomyn, Anne (1979) Studies on Protein Synthesis after Heat Shock in Drosophila melanogaster. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/ree4-cz17. https://resolver.caltech.edu/CaltechETD:etd-05222003-103429

Abstract

The synthesis of the heat shock proteins on cytoplasmic rather than mitochondrial ribosomes has been shown. The time course of the synthesis of heat shock proteins in prepupal and pupal stages of Drosophila melanogaster has been analyzed. Prepupae were heat shocked at 37.5[degrees]C for 20 minutes and pupae, either at 37.5[degrees]C for 20 minutes or at 40.2[degrees]C for 40 minutes. In prepupae, all of the heat shock proteins are synthesized immediately after the shock and continue to be synthesized for two to three hours. By three hours after the shock, the synthesis of normal proteins has resumed. In pupae, the time course is similar after the milder shock. However, the more severe shock causes a drastic reduction in total protein synthesis for at least one hour after the shock. It has been shown previously that this shock causes the production in pupae of stage specific phenocopies at high penetrance, an effect which is not observed after a shock at 37.5[degrees]C for 20 minutes. The drastic decrease in total protein synthesis and the subsequent occurrence of anomalies in the resumed program of gene expression and their possible relation to phenocopy production are discussed. The increase in activity of phenol oxidase after a shock of 40[degrees]C for 40 minutes has been investigated to determine whether a component of the enzyme is a heat shock protein. This hypothesis was tested by measuring the extent of co-banding of [superscript 35]S-methionine labeled proteins from heat shocked and non-heat shocked cells with partially purified phenol oxidase in a sucrose gradient. The results do not support such a role for any heat shock protein; a mechanism whereby phenol oxidase activity could increase after heat shock is discussed. The 84,000 dalton heat shock protein has been purified by ammonium sulfate fractionation, chromatography on hydroxylapatite, and one- or two-dimensional gel electrophoresis. This purified protein has been used to produce antibodies in rabbits. The antibodies have been used to show, by indirect immunoprecipitation experiments, that this heat shock protein is normally synthesized in at least three D. melanogaster tissues. Indirect immunofluorescence experiments using these antibodies indicate that the 84,000 dalton protein is also present on the chromosomes of normal and heat shocked salivary glands at the interband regions. Evidence is presented to show that this binding pattern may simply reflect the high concentration of this protein in the cell. In a separate investigation, the effects of heat shock on the protein synthesis pattern in HeLa cells have also been analyzed.

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