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The use of the yeast two-hybrid system as a means of identifying protein interactors of the human protein, BMI-1

Holgate, Robert George Edward; (1999) The use of the yeast two-hybrid system as a means of identifying protein interactors of the human protein, BMI-1. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

In Drosophila melanogaster, the establishment of the expression patterns of the Homeotic complex (HOM-C) genes, involved in the anteroposterior organisation of the developing embryo, are initiated by maternal-effect genes and segmentation genes. These expression patterns defining differentially determined states need to be maintained over many cell generations after initiation and, to ensure this, two classes of genes have evolved, the Trithorax-Group and the Polycomb-Group (Pc-G), which act to maintain the spatially restricted expression patterns in a positive and negative manner respectively. Similar mechanisms to those found in Drosophila are thought to exist in vertebrates to regulate expression of the vertebrate Hox genes. BMI-1 is a 45KDa nuclear protein with several features in common with other nuclear proteins which suggest a role in transcriptional regulation. Furthermore, BMI-1-specific motifs are conserved in a related gene in Drosophila, posterior sex combs (Psc), a member of the Polycomb-Group of proteins. The mode of action of the Polycomb-Group proteins is thought to be through the formation of higher-order chromatin structures, and so it was postulated that BMI-1 would interact with several other proteins, with other Pc-G proteins seeming suitable candidates. In order to identify such interactors, a truncated BMI-1 fragment was used as bait to perform several library screens in a GAL4-based yeast two-hybrid system. From these screens, several clones were identified as potential interactors. In particular, two non-Polycomb-Group clones were each identified from two different species, and were deemed suitable candidates for further analysis. One encodes a putative novel protein of unknown function, while the other encodes a protein, MCM6, involved in the control, or 'licensing' of DNA replication. Further work has been aimed at characterising these interactions further, and corroborating the interactions using two different techniques: GST-affinity capture and co-immunoprecipitation. In addition, further analysis of the novel protein has also been performed. At present, the balance of evidence suggests that the novel protein is a false positive, and thus not a real interactor of BMI-1. Further work is required to investigate the MCM6-BMI-1 interaction and, if proved, could provide valuable insights into Polycomb-Group-mediated inheritance of repressive chromatin states. In addition, the interaction may also help in the understanding of how replication of DNA is able to proceed, despite the presence of higher order chromatin structures.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: The use of the yeast two-hybrid system as a means of identifying protein interactors of the human protein, BMI-1
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences
URI: https://discovery.ucl.ac.uk/id/eprint/10120371
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