Title page for ETD etd-08242005-123051

Type of Document Master's Thesis
Author Auckland, Ian
Author's Email Address iaucklan@vt.edu
URN etd-08242005-123051
Title Quantitative Analysis of a Cell Cycle Checkpoint in Xenopus laevis Cell-Free Egg Extracts
Degree Master of Science
Department Biology
Advisory Committee
Advisor Name Title
Sible, Jill C. Committee Chair
Tyson, John J. Committee Member
Walker, Richard A. Committee Member
  • cell cycle
  • Xenopus laevis
  • nucleocytoplasmic ratio
  • cyclin-dependent kinases (Cdk)
Date of Defense 2005-08-10
Availability unrestricted
In somatic cells, checkpoint pathways trigger cell cycle arrest in response to

unreplicated or damaged DNA by inhibiting the activity of cyclin-dependent kinases

(Cdks). In the Xenopus laevis embryo, checkpoints are not operational until the

midblastula transition (MBT). Studies in cell-free egg extracts indicate that a threshold

concentration of nuclei, which approximates the MBT concentration, is required to elicit

a checkpoint. The checkpoint response to unreplicated DNA in the extract prevents

transition into mitosis by inhibiting Cdk1/cyclin B, causing an increase in the minimum

amount of cyclin B necessary to enter mitosis, termed the cyclin threshold. Once the

threshold of cyclin is maintained or exceeded, the system will proceed into mitosis after a

lag time. We have investigated the relationship between nuclear concentration and cell

cycle regulation in the extract. By precisely regulating the concentration of cyclin B and

nuclear content in extract samples, we have found 1) the concentration of nuclei affects

cyclin B thresholds and lag time of entry into mitosis, 2) elevated cyclin thresholds

caused by DNA replication blocks are further increased by increasing the concentration

of nuclei, and 3) double-stranded DNA breaks in the extract system do not affect cyclin

thresholds or lag time of entry into mitosis within the range of nuclear concentrations that

can be efficiently replicated. This data provides evidence of the importance of the

nucleocytoplasmic ratio in normal cell cycle progression and its importance for

checkpoint acquisition during early Xenopus laevis development.

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