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Title: Production and characterisation of specific antibodies to regions of the C-terminal domain of cytoplasmic dynein heavy chain
Author: Fan, J.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2000
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Cytoplasmic dynein (CD) is a multisubunit microtubule (MT) motor protein that transports vesicles and other particles along cellular microtubules (MTs). The aim of the work in this thesis is to better understand the relationship between dynein's molecular structure and function, especially the mechanics of CD-MT interactions. Two approaches have been used. (1) Using recombinant techniques, a number of overlapping domains (each around 30 KDa) of DHC, downstream of the coiled coil stalk, were expressed in bacteria and their in vitro MT-binding properties were characterised by cosedimentation with MTs. (2) Using immuno-techniques, a number of specific antibodies to the expressed proteins or to synthetic peptides have been produced and characterised. Two good anti-peptide antibodies were further used to assay their effects on native dynein-driven MT motility in vitro. The results showed that one expressed protein, termed CD-H2, bound to MT's in vitro and that an antibody against 16 residues outside this region did not. The conclusion is that the region of the sequence involved in forming the surface that interacts with MTs probably extends at least 500 residues downstream of the known MT-binding sites on the coiled-coil stalk and plays an essential role in MT-DHC interaction during motility. Some preliminary studies by immuno-electron microscopy indicate that it will be possible to locate this and other functional regions on the observed structure of native CD molecules.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available