The isolation and characterisation of specific RNA molecules and RNA-DNA complexes in animal cells

Morrison, Marcelle R (1971) The isolation and characterisation of specific RNA molecules and RNA-DNA complexes in animal cells. PhD thesis, University of Glasgow.

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Abstract

1. The identification, isolation and characterisation of a mammalian mRNA required choice of a cell type which synthesises only one or a few proteins. The mammalian reticulocyte synthesises globin almost exclusively. This cell type can be isolated in large quantities and has low endogenous ribonuclease activity, thus making it a suitable system for the isolation of large amounts of undegraded RNA. Preliminary experiments indicated that the reticulocyte RNA component sedimenting at 9s had many of the characteristics expected of the globin mRNAs. 2. The characterisation of an mRNA which can only be labelled to a small extent in vivo and which is only about l of the total RNA requires the development of large-scale isolation procedures. Preliminary experiments designed to circumvent the difficulty of labelling reticulocyte 9s RNA in vivo involved the isolation of uridine-9s RNA from cultures of 14 day mouse embryo liver cells. The technique of preparative polyacrylamide gel electrophoresis was adapted for the isolation of pure 9n RNA from total 14 day mouse embryo liver nucleic acids. The 14 day mouse embryo liver may contain cell types synthesising 90 mPNAs for other proteins. Contamination of any globin 9s DNA with breakdown products of other RNA species was also likely since the livers contain some endogenous ribonuclease. 30 9s RNA was therefore isolated from mouse reticulocytes. The development of techniques for separation of large amounts of DNA on 173. the zonal ultracentrifuge combined with the use of diethyl pyrocarbonate as an RNase inactivator permitted the isolation of large amounts of 9s RNA. Preliminary experiments used the M.S.E. B-XV zonal rotor for the isolation of a fraction enriched in the 148 mIINP particle dissociated from polysomes by EBT.A. The RNA isolated from the 14s mRNP particle was further purified by preparative gel electrophoresis. A 9s RNA fraction was also isolated from total reticulocyte RNA. Since isolation of undegraded RNA was more routine using this method, conditions were developed for the isolation of a 9s RNA. fraction, uncontaminated with RNAs of other size classes, after a single fractionation of the total RNA in the M.S.E. B-XIV zonal rotor. 4. The RNA was analysed for purity on 2.6% polyacrylamide gels. The microheterogeneity of the RNA in the 9s RNA region was analysed on 6% polyacrylamide gels. There were two major and several minor bands in the 9s region. There was some variation in the relative amounts of the minor bands ii. the 9s RNAs isolated from different batches of reticulocytes. 5. The molecular weight of the 9s RNA was determined by analytical ultracentrifugation. The value obtained was 170,000, equivalant to about seventy nucleotides greater than the theoretical estimate for BNAs coding for the a or p globins. Determination of the molecular weight by relative electrophoretic mobilities on analytical polyacrylamide gels gave a higher value, 225,000, thus demonstrating that the 9s RNA had different migration properties compared to the rRNA 174 marker species. 6. Isolation of large amounts of pure 9s RNA made possible the study of the hybridisation of this messenger - enriched RNA fraction to DNA. Since 9s DNA is only labelled to a small extent in vivo, it was chemically methylated with tritiated dimethyl sulphate. In this way, specific activities of up to 5,000 cpm/mug were obtained. Techniques were developed for the hybridisation of dimethyl sulphate-labelled RNA to DNA. Such chemically-labelled RNA gave a higher background sticking to blank filters. This was reduced to less than 0.01% of the input RNA by hybridising at low temperatures in the presence of formamide. Background levels were further reduced by pre-incubating all filters in Denhardt's medium and by extensive purification of the RNA. 7.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: John Paul
Keywords: Biochemistry
Date of Award: 1971
Depositing User: Enlighten Team
Unique ID: glathesis:1971-73874
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Jun 2019 08:56
Last Modified: 14 Jun 2019 08:56
URI: http://theses.gla.ac.uk/id/eprint/73874

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