Studies on the methylation of eukaryotic DNA

Browne, Michael J (1976) Studies on the methylation of eukaryotic DNA. PhD thesis, University of Glasgow.

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Abstract

The DNA of eukaryotes has been shown to contain the 'minor' base 5-methyl cytosine. This arises by enzymic modification of cytosine already incorporated into DNA. The balance of evidence suggests that this base is involved in the control of expression of information encoded in the genome. The work presented here is an attempt to elucidate the relationship of DNA methylation to cellular differentiation using a variety of approaches. Preliminary experiments using a model differentiating system (the Friends Erythroleukaemic cell line) showed the techniques used to measure gross changes in the DNA content of 5 MeC to be inadequate; alternative approaches were sought. Pairs of cell lines derived from the same species, e.g. the human HeLa and Chang Liver cell lines, were used as models for intertissue comparisons and Hamster, Mouse, Human, Xenopus and Soya Bean Cotyledon cell lines were used in interspecies comparisons. The characteristics of DNA methylation in these cells were determined by; (i) DNA fingerprinting. This approach showed the distribution of 5-methyl cytosine in pyrimidine oligonucleotides to be identical in cell lines derived from the same species and to exhibit only slight variations between vertebrate species, virus transformation of cells did not alter the pattern of raethylation (the plant cell line was markedly different). (ii) Fingerprinting of DNA methylated vitro by a purified DNA methylase showed that the enzyme is able to methylate DNA in a manner remarkably similar to that found vivo. This result, taken with the observed lack of variation in distribution of 5-methyl cytosine in the vertebrate cell lines, suggests that all vertebrate cells contain only one DNA raethylase and that the characteristics of the DNA methylase and nucleotide sequence methylated have been evolutionarily conserved. (iii) Large variations in the 5 MeC content of some of the cell lines used suggest that there are 'control' elements acting upon the ba,sic DNA methylase system mentioned in (ii). (iv) Isopycnic centrifugation and thermal elution of DNA from hydroxylapatite showed the distribution of 5-methyl cytosine, with respect to the G+C and A+T rich sections of the genome, to be species specific., This may reflect the methylation of satellite DNAs. (v) Rapidly reannealing DNA of HeLa cells was shown to be enriched for 5 MeC content, as has been shown for rodent cells - evolutionary conservation is implicated. (vi) Digestion of DNA using the Hpa II restriction endonuclease, which will not cleave the sequence S' C-C-G-G 3' when methylated, provided an alternative method for measuring the level of 5-methyl cytosine in eukaryotic DNA and furnished evidence for the clustering of unmethylated Hpa II restriction endonuclease cleavage sites. The features of the interaction between the DNA methylase and DNA, and the control of this interaction in vertebrate cells are discussed.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: R H Burdon
Keywords: Biochemistry
Date of Award: 1976
Depositing User: Enlighten Team
Unique ID: glathesis:1976-72347
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 24 May 2019 15:12
Last Modified: 24 May 2019 15:12
URI: http://theses.gla.ac.uk/id/eprint/72347

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