Control of DNA synthesis in L cells

Lindsay, J (1970) Control of DNA synthesis in L cells. PhD thesis, University of Glasgow.

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Regenerating liver, rabbit kidney cortex cells cultured in vitro and PHA-stimulated lymphocytes have been widely employed to study the sequence of metabolic events which is required for the onset of DNA synthesis and cell division when resting cells are stimulated to renewed proliferation. A parallel resting cell system has been established with a permanent cell line by maintaining cultures of L 929 cells at high population density. Such a system is particularly suitable for study because of the ease of culture manipulations and the relatively high degree of synchrony obtained. Released-stationary cells begin to synthesise DNA after a lag period of 14 hr. and by 20 hr. 700 of the cells are in S phase. Increases in cell number are observed by 25 hr. In contrast to primary resting systems no change in the rate of RNA synthesis is detected after release from stationary phase. RNA synthesis during the lag phase is required for subsequent DNA synthesis. Changes in the activity of DNA polymerase in nuclear and supernatant fractions of L cells have been investigated following release from stationary phase and particularly during S phase. The results of previous investigators have been confirmed and extended. Nuclear preparations of L 929 cells show a 2.5-fold preference for native DNA primer while the supernatant DNA polymerase activity is correspondingly more active with denatured DNA. The general characteristics of the DNA polymerase(s) in these fractions have been investigated and methods for releasing the enzyme from isolated nuclei studied. Preliminary purification of the enzyme was undertaken although difficulties were encountered because of the small amounts of material available from tissue culture cells. A 7-8-fold purification was achieved by pH 5 precipitation and Sephadex G200 chromatography and 950 of the DNase activity removed as judged by the relative capacity of the fractions to hydrolyse native or heat-denatured-DNA to acid-soluble fragments. After purification DNA polymerase activity, primed by denatured DNA, was rapidly lost. on freeze/thawing of.the solution. DNA polymerase activity in extracts of L 929 cells was found to be heterogeneous by fractionation on Sephadex G200, DEAF-cellulose and polyacrylamide gels. As similar results have recently been reported for the rat liver enzyme the possible significance of this result in relation to in vivo replication is considered. The nuclear location of a fraction of the DNA polymerase activity and its preference for native DNA primer makes it a possible candidate in this respect. Sephadex-purified nuclear and supernatant fractions have been used to synthesise Nd DNA on their preferred primers and the characteristics of the DNA products examined. Both products were found to be resistant to degradation by exonuclease x suggesting that the newly-synthesised DNA was not present in a single-stranded form. Analysis of the DNA products on neutral and alkaline morose gradients reveals that the pq DNA is not covalently attached to the DNA primer. Our findings on the characteristics of the DNA products are compared to previous data on the DNA products formed by the calf thymus and Escheriohia coli DNA polymerase.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: C J Adams
Keywords: Biochemistry
Date of Award: 1970
Depositing User: Enlighten Team
Unique ID: glathesis:1970-73200
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Jun 2019 08:56
Last Modified: 14 Jun 2019 08:56

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