CPF1 (<strong>c</strong>entromere <strong>p</strong>romoter <strong>f</strong>actor 1) of Saccharomyces cerevisiae is a multifunctional protein. This multifunctionality may be associated with the different forms of the protein. To date, no clear function has been attributed to this protein. The general roles currently attributed to CPF1 are: maintenance of proper chromosome segregation or centromere function; methionine prototrophy and chromatin modulation. A series of recent papers have suggested the possibility of CPF1 being involved in protein-protein interactions. This thesis shows that CPF1 has an additional protein-protein interaction domain which is a coiled-coil. A truncation of CPF1 coding for only the C-terminal dimerization motif was cloned into a 17 expression vector and the protein, Dd, was produced in and purified from E. coli. Characterisation of the conformation of the C-terminal dimerization domain of CPF1 show the presence of a leucine zipper in Dd and that Dd forms a dimer under physiological conditions. Sequence alignment analysis of CPF1 and other bHLHZIP proteins identified a conserved serine residue in the basic domain of CPF1 and a conserved asparagine residue in the leucine zipper. Point mutations were introduced separately mutating these residues into either alanine or glutamate. The mutant alleles were cloned into yeast expression vectors. The cloned alleles were used to transform YAG93, a cpf1 deletion strain. Characterisation of the transformants obtained indicate the possibility of CPF1 being involved in the spindle assembly checkpoint control, possibly as a tethering protein. Finally, attempts were made to identify interacting partners of CPF1 and a purification scheme was designed to purify a putative CPF1 complex. Initially, a GST-CPF1 chimera was used to screen radioactively labelled crude yeast extracts for interacting partners of CPF1. This strategy identified five or six potential proteins which might form a multiprotein complex with CPFl. Attempts were then made to purify this putative complex from S. cerevisiae. The partially purified extracts were tested for CDEI activity, indicative of the presence of CPF1 in the samples and. tjhen analysed for protein content. The samples were also tested in a Holiday junction binding assay and in a histone acetyltransferase activity assay. In conclusion, CPF1 is a bHLHZIP protein that has a role in nutrient signalling and checkpoint control, possibly by acting as an intermediary protein that recruits essential factors to gene promoters or the centromere by protein-protein interactions.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:390529 |
| Date | January 1998 |
| Creators | Nissom, Peter Morin |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:93828daa-84e6-419b-ac80-1b86108a414f |
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