Possible RNA processing enzymes in HeLa cell nuclei

Strachan, T (1979) Possible RNA processing enzymes in HeLa cell nuclei. PhD thesis, University of Glasgow.

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Abstract

1. HnRNP particles were isolated from monolayer cultures of HeLa cells and characterized with respect to their sedimentation in sucrose density gradients (s values from 0 to>250s)5 their buoyant density in CsC1 density gradients following aldehyde fixation of the particles (1.390 g.cm-3), their heterogeneous complement of polypeptides as analysed on SDS-polyacrylamide gels (species from 38,000 to >150,000 daltons) and the heterogeneous sedimentation of their rapidly labelled RNA component (s values from 0 to >40s). 2. Various potential RNA processing enzyme activities were investigated in relation to their possible association with HeLa hnRNP particles or related subnuclear fractions of chromatin and nucleosol. (a) Exoribonuclease activity which was dependent on Mg2+ ions was found to be largely confined to a nucleosol frciction and, to a lesser extent, to the chromatin fraction. No exoribonuclease activity could be detected in association with HeLa hnRNP par ticles. (b) Endoribonuclease activity of poor substrate specificity was detected in association with HeLa hnRNP particles, chromatin and nucleosol fractions. The hnRNP particle-associated activity was stimulated by Mg2+ but did not appear to be strictly dependent on the presence of Mg2+. The same activity permitted degradation of HeLa hnRNA to molecular species of lower average sedimentation coefficient than present in mRNA but without any accompanying production of acid-soluble components. (c) No double-strand RNA-specific RNase activity could be detected in association with HeLa hnRNP particles. Instead such activity appeared to be largely confined to the nucleosol fraction. (d) RNA guanylyItransferase activity could not be detected in association with HeLa hnRNP particles. Again such activity appeared to be confined to the nucleosol fraction. (e) RNA ligase activity similar to the bacteriophage T4 activity could not be detected in any of the HeLa subnuclear fractions tested, (f) No poly A synthetase activity could be detected in association with HeLa hnRNP particles. Rather, such activity was prominent in a HeLa nucleosol fraction and, to a lesser extent, in the corresponding chromatin fraction. The nucleosol-located activity was optimally stimulated in the presence of a poly A primer and appeared to be more dependent on the presence of exogenously provided RNA primer than the corresponding chromatin-associated activity.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Roy H Burdon
Keywords: Biochemistry
Date of Award: 1979
Depositing User: Enlighten Team
Unique ID: glathesis:1979-73363
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/73363

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