The regulation of protein synthesis in animal cells

Gilmour, R. Stewart (1968) The regulation of protein synthesis in animal cells. PhD thesis, University of Glasgow.

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The technique of DNA-RNA molecular hybridization was adopted for investigating the regulation of protein synthesis at the genome level in differentiated animal cells. In preliminary experiments, attempts were made to assay mammalian DNA-RNA complexes by means of methylated albumin-kieselguhr chromatography, Sephadex G-200 chromatography and by selective adsorption on nitrocellulose filters. The latter technique proved to be the most convenient for the present studies, especially when the DNA was immobilised to the nitrocellulose filter prior to hybridisation with RNA. Using this technique for the hybridization of in vivo labelled 14C- rat kidney RNA to homologous DNA it was found that under conditions of maximum hybridization only 7% of the DNA formed hybrids with the RNA. This result was taken as evidence for the restriction of DNA template activity in animal cells. In an attempt to detect tissue specific RNAs in various rat organs, comparisons were made of the rapidly 32P-labelled RNA factions of rat liver, kidney and pancreas by competitive hybridisation against unlabelled rat kidney RNA. Differneces were observed in the hybridization kinetics of the RNAs from each organ. As it was not possible to determine the real specific activity of rapidly labelled RNA from in vivo labelling experiments, it was concluded that the observed differences in competitive hybridization kinetics of these RNAs could be due either to absolute differences in RNA types or due to differences in the specific activities of the RNAs isolated from each organ. The difficulties associated with the use of in vivo labelled RNAs could be avoided by synthesising the RNAs in vitro from chromatin primers with the RNA polymerase of Micrococcus lysodeikticus. Experiments in which the RNA synthesised in vitro from calf thymus and rabbit thymus and bone marrow chromatins was hybridized to homologous DNA, showed that the template activity of the isolated chromatin was restricted to 5-10% of the total DNA. Competitive hybridization experiments showed that the RNA synthesised in vivo from calf thymus chromatin was qualitatively identical to that synthesised in the in vitro system. This was also confirmed with the RNAs synthesised in vivo and in vitro from rabbit bone marrow chromatin. It was concluded that the restriction of DNA template activity found in vivo in animal cells is also a property of the isolated chromatin. In further competitive hybridization experiments, the RNAs synthesised in vivo from rabbit thymus and bone marrow were compared separately with the RNAs synthesised in vitro from rabbit thymus and bone marrow chromatins. Evidence was provided for the existence of qualitative differences in the RNA populations of the two tissues. This suggests that the restriction of DNA template activity in differentiated cells is organ specific. Investigations into the nature of the specific restriction of template activity in calf thymus chromatin suggested that protein ionically bound to DNA is responsible for the restriction. Under specified conditions it was found possible to reconstitute chromatin from crude DNA, histone and non-histone fractions. The uncleohistone obtained by reconstituting DNA with the histone fraction alone, could not direct the in vitro synthesis of any RNA capable of hybridising to calf thymus DNA. On the other hand, nucleohistone reconstituted from DNA in the presence of both histone and non-histone fractions resulted in a specific part of the DNA being available for transcription. The magnitude of this unrestricted portion was found to be the same as that of whole chromatin. These results suggest that there exists in calf thymus chromatin a non-histone fraction responsible for conferring the specific restriction of DNA template activity.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Biochemistry
Date of Award: 1968
Depositing User: Enlighten Team
Unique ID: glathesis:1968-72674
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 11 Jun 2019 11:06
Last Modified: 11 Jun 2019 11:06

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