20+-+Cell+Cycle+II - BME 418 Quantitative Cell Biology...

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BME 418, Quantitative Cell Biology Alan J. Hunt Lecture #19: Cell Cycle II Early evidence of regulatory molecules came from cell fusion experiments: - Two cells can be fused experimentally by disrupting their adjacent membranes with electricity (electroporation), viruses or polyethylene glycol. - Produce a heterokaryon. - If one cell is in S, and one is in G1, the G1 nucleus quickly initiates DNA synthesis. - If S and G2 are fused DNA synthesis is suppressed. This suggests that the S phase cell contains a substance(s) in its cytosol or membrane that induces DNA synthesis, and the G2 cells contain a substance that suppresses DNA synthesis.
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BME 418, Quantitative Cell Biology Alan J. Hunt More evidence from frog oocytes - If cytoplasm from a mature (fertilization competent) frog egg is injected into an immature (pre-meiosis) oocyte, meiosis is triggered. Using this response as an assay for biochemical purification led to the isolation of maturation- promoting factor (MPF). - MPF has been found in a broad range of eukaryotes, and appears to play a critical role in promoting the onset of mitosis (thus MPF now stands for "mitosis-promoting factor"). - Interestingly, yeast MPF can substitute for human despite 2-3 billion years of evolutionary divergence. Cell cycle progression is regulated at "checkpoints".
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BME 418, Quantitative Cell Biology Alan J. Hunt - Progression through a checkpoint is defined by structural and biochemical changes. For example, upon entry into S phase DNA replication begins, and the synthesis of histones that stabilize the structure of the newly synthesized DNA is up regulated. - Most checkpoints were initially identified by molecular genetics in yeast (cerevisiae and pombe, respectively pioneered by Hartwell and Nurse) - In yeast a critical checkpoint is located at the G1-S transition is called "start". The standard molecular genetic approach: 1) Design screen 2) Select interesting phenotypes 3) Use molecular genetics to map genetic locus. 4) Use gene library to clone by “rescue” of wild-type. For example, look for mutants involved in chromosome segregation, possibly motor proteins, by identifying strains that exhibit increased frequency of loss of an artificial chromosome during cell division. In yeast an artificial mini-chromosome can be constructed from centromeric DNA sequences and some sort of readily identifiable marker (such as Beta-Galactosidase).
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