The temperature-dependence of cell cycle parameters and chromosonal DNA replication in tissue cultures of xenopus laevis
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Chapter I. Pulse/chase labelling and DNA fibre autoradiography have been used to study the durations of the stages of the cell cycle, and the manner of chromosomal DNA replication, in Xenopus cells in tissue culture at 18°C, 23°C and 28°C. Cultures were grown in modified Eagle's basal medium, containing salts at concentrations appropriate to Amphibia, plus glutamine and foetal calf serum. The subculturing procedures were carried out every fortnight, with a medium change every week. For studying the durations of the stages of the cell cycle, cells were labelled with low specific activity tritiated thymidine (3H-TdR) for 30 min or 1 hour, then left to continue growth in non-radioactive medium, and fixed at regular intervals thereafter. Whole-cell autoradiographs, stained in Giemsa, were prepared from these fixations. For studying DNA replication, tissue cultures were treated with flurodeoxyuridine (FUdR) to arrest cells at the beginning of the S-phase, then labelled with high specific activity 3H-TdR for various times. In the case of pulse/stepdown labelling, the first period of labelling was followed by a further period in the presence of 3H-TdR at one quarter of the original specific activity. DNA fibre autoradiographs were prepared from such labelled tissue cultures. Chapter II. An analysis of the durations of the cell cycle stages obtained from pulse/chase labelling experiments gave the following results: (1) at 18°C G1 lasts for 31 hrs, S for 29.5 hrs, G2 for 8.5 hrs, M for 3 hrs and the total generation time is 72 hrs; (2) at 23°C G1 lasts for 14-3 hrs, S for 15-5 hrs, G2 for 5.7 hrs, M for 0.5 hrs and the total generation time is 36 hrs, and (3) at 28°C G1 lasts for 11.3 hrs, S for 13.5 hrs, G2 for 4.8 hrs, M for 0.4 hrs and the total generation time is 30 hrs. Pulse labelling followed immediately by fixation, and subsequent Giemsa staining, enables a quick and convenient assessment to be made of the relative durations of the cell cycle stages. In such preparations nuclei in S-phase are labelled, nuclei in G1 are small and unlabelled and nuclei in G2 are large and unlabelled. Chapter III. Pulse/stepdown labelling shows that DSTA replicates bidirectionally in the Xenopus cells. Origin to origin distances (initiation intervals) vary, but the range of and the mean initiation intervals at all three temperatures are much the same. The mean interval between initiation points is of the order of 60 to 66 mum. Staggering of initiations is evident at all three temperatures, and may be disproportionately greater at 28°C than at 23°C and 18°C. Evidence against the existence of replication termini is provided. Chapter IV. The rates of progress of DITA replication forks cannot be determined from pulse/stepdown preparations, so these rates had to be estimated from pulse labelled cultures. They are 5.5 mum/hr at 18°C, 10 mum/hr at 23°C and 16 mum/hr at 28°C
Thesis, PhD Doctor of Philosophy
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