Crampton, Julian M. (1978) The control of RNA synthesis in vitro. PhD thesis, University of Warwick.

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Abstract

Conditions for the isolation and incubation of Xenopus cultured cell nuclei were optimised for maximal synthesis of RNA. The Xenopus nuclei showed all three RNA polymerase activities and appeared to process rRNA to 18S and 28S species, and possibly to initiate new RNA chains.

The rate of RNA synthesis in isolated XTC-2 nuclei was changed when they were incubated with Xenopus oocyte and egg cell extracts in an optimised assay system (which included a preincubation step), the oocyte being stimulatory and the egg inhibitory. Cell extracts from other developmental stages assayed in the same way indicated that extracts of early cleavage stages were also inhibitory, whilst stages 7 ½ to 13 became increasingly stimulatory. Some possible trivial causes of these effects were eliminated.

Four main stimulatory factors were isolated from mature oocytes and were purified to varying degrees by gel filtration and ion exchange chromatography. Three were found to be relatively non-specific in their action; the fourth specifically stimulated RNA polymerase I activity and the synthesis of rRNA. The activity of this factor was found to vary during oogenesis and early development (though possibly because of poor recovery), correlating reasonably with the known rates of rRNA synthesis in vivo. The factors were not spec les specific.

A fifth minor stimulatory factor was found mainly to affect RNA polymerase II activity. The factor’s activity changed little between extracts from different developmental stages, however, this was the only purified factor which also stimulated RNA synthesis in isolated Xenopus blood nuclei.

The factors stimulated elongation and possibly initiation also. All four triphosphates were required for factor stimulation to occur. Activity of the rRNA-specific factor was dependent on continued polymerase II activity.

The factors stimulated RNA synthesis when assayed with Xenopus chromatin and added homologous polymerases. They were ineffective, however, when assayed with homologous purified DNA and RNA polymerases.

Experiments involving the factors in vivo were inconclusive; their relevance and that of the nuclear assay system is discussed.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QL Zoology Q Science > QP Physiology
Library of Congress Subject Headings (LCSH):	Xenopus, RNA -- Synthesis
Official Date:	November 1978
Dates:	Date Event November 1978 Submitted
Institution:	University of Warwick
Theses Department:	Department of Biological Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Woodland, Hugh R.
Extent:	xiii, 104 leaves
Language:	eng

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