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Date of Award

10-1993

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry

Supervisor

Evert Nieboer

Abstract

The experimental approach adopted had three components. The first involved an examination of the effects of the Ni(II) ion on simple DNA and synthetic polydeoxynucleotides of defined sequence and base composition. In the second part, the interaction of the Ni(II) ion with chromosomes in intact nuclei was investigated. A final component assessed the genotoxic effects of a variety of nickel compounds in a mammalian cell line (AS52) by the characterization of damage at a specific gene locus. A comparison of the effects of Ni(II) to those of other metal ions suggested that the interaction of the Ni(II) ion with DNA or polydeoxynucleotides is to some extent base (or basepair) and sequence-context specific. A clear affinity for G/C bases was evident, which was also reflected in the magnitude of the binding constants. All DNAs and polydeoxynucleotides examined featured two binding sites, differing in affinity by approximately two orders of magnitude. The interaction of the Ni(II) ion with chromosomes in intact nuclei was assessed utilizing UV/visible spectroscopy, resistance to nuclease digestion and electron microscopy. As monitored by spectroscopic techniques, the Ni(II) ion induced a chromosomal condensation which was nickel specific and observable even when Mg(II) ions were present in the buffer. Susceptibility to nuclease digestion decreased in a concentration-dependent manner following an incubation for 96 h with Ni(II). By contrast, no decrease was observed following incubations of 2 or 24 h duration. Ultrastructural changes in chromosome structure were also observed in electron micrographs for the longer exposures. Molecular analysis of the AS52 cell line identified and characterized two independent sites of integration of a bacterial gpt gene insert. Sequence analysis revealed that the locus designated as the pseudogene (gpt') shared homology with the 5' and 3' ends of the gpt gene but also involved a unique insert, part of which derived from the β-lactamase gene of the parent transformation plasmid (pSV₂gpt). The pseudogene was not transcribed as deduced from reverse transcriptase (RT) PCR and Northern hybridization analysis. Analysis of nickel-generated mutants yielded three empirically distinct PCR profiles: (1) amplification of both the gpt and gpt' loci; (2) absence of the gpt band (no amplification); and (3) absence of both the gpt and gpt' bands. The illustrated ability of the Ni(II) ion to induce conformational changes in DNA and chromosomal condensations in nuclei, and its recognized capability of generating oxygen-derived chemicals clearly point to its potential involvement in epigenetic pathways of carcinogenesis. The confirmation by PCR analysis that nickel compounds are mutagenic and can induce point mutations and apparent loci deletions indicates that genotoxic mechanisms are also plausible. (Abstract shortened by UMI.)

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