Groves, Richard P.; (1992) DNA amplification in mammalian cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

DNA amplification has been observed in mammalian cells derived from tumours and also in tissue culture cells. The initial amplification event is thought to be random and In some cases the amplified DNA contains a gene whose consequent over-expression can confer a growth advantage or reduced sensitivity to a cytotoxic compound. In tissue culture systems, specific drugs can be used to isolate cells which have previously amplified a specific gene. The mechanisms responsible for these DNA abnormalities have been difficult to study as the initial events are rare and the amplification products could previously only be visualised many cell generations after their formation. During this time secondary mechanisms may alter the structure and appearance of the amplified arrays making an evolutionary interpretation of the amplification difficult. Until recently, only indirect methods were available to study the structure of amplified arrays but advances in fluorescent in situ hybridisation of metaphase chromosomes have allowed direct visualisation of very early gene amplification events. Part of this thesis describes an attempt to construct a model cell line in which a single predetermined locus could be amplified simultaneously throughout a cell population in a controlled manner. Cosmids covering the monkey CAD locus were available and one was used to construct a homologous replacement fragment containing an SV40 replication origin and a dominant selectable marker. The fragment was transfected into ts-COS cells and if a cell line could have been isolated in which the fragment had integrated by homologous recombination, it may have been possible to over-replicate the locus by a shift to the permissive temperature to mimic the 'onionskin' model of gene amplification. Other chapters of this thesis are concerned with the evolution and stability of amplified DNA and also the analysis of amplified DNA by in situ hybridisation on human metaphase chromosomes. Amplified DNA is lost very rapidly from PALA- resistant mutants selected in a single selection step but in mutants selected in several steps of increasing drug concentration the amplified genes are lost more slowly, if at all. PALA resistance in human cells has not been extensively investigated and, following recent advances in the understanding of early events in gene amplification provided by in situ hybridisation analysis of PALA-resistant BHK ceils, a human system would provide a more accessible genome for further experiments.

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