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Title: Studies on mammalian cyclin A
Author: Adamczewski, Jorg Peter
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 1994
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This thesis describes studies on cyclin A, a member of a family of proteins centrally involved in the regulation of the eukaryotic cell cycle. Cyclins are activating subunits of the cdc2 family of protein kinases. I constructed a chimera consisting of staphylococcal protein A fused to bovine cyclin A and expressed and purified the resulting protein from bacteria. The cyclin A fusion protein could bind to and activate both p33cdk2 and p34cdc2 when added to a variety of cell free extracts. I purified both types of protein kinase holoenzymes and compared their activities towards various substrates. It was also possible to produce an active cyclin A:GST-cdk2 complex from components expressed in bacteria, but a protein kinase that phosphorylates p33cdk2 is required to activate the complex. I constructed several mutant forms of cyclin A that were unable to bind or activate p33cdc2 or p34cdc2 and served as negative controls. In contrast to analogous cyclin B constructs the binding of pA-cyclin A to p34cdc2 does not seem to promote the inhibitory phosphorylation of tyrosine 15 in p34cdc2. Cyclin A can also facilitate the activation of cyclin B dependent kinase in both egg and oocyte extracts. I shared the cyclin A protein and antibodies with a number of other groups in order to study properties of cyclin A dependent kinases. Cyclin A could promote nuclear envelope breakdown and chromosome condensation in Xenopus interphase extracts; it inhibited vesicle fusion in HeLa cell free extracts; and it could stimulate DNA synthesis in both Xenopus and HeLa extracts. I found that the protein kinase activity associated with SV40 large T antigen is partly due to an association with cyclin A-p33cdk2.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available