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Title: The role of Mre11-Rad50-Nbs1 CTBP-Interacting Protein (MRN-CtIP) in providing resistance against camptothecin
Author: Ezeh, Ikechukwu
ISNI:       0000 0004 5350 1984
Awarding Body: Bangor University
Current Institution: Bangor University
Date of Award: 2014
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MRN -CtIP protein complexes in humans have been implicated in the resistance of cancer cells to camptothecin (CPT). The experiment investigated randomly mutated ctp1 gene, in S. pombe, which is a homolog of CtIP, to discover mutations on ctp1 DNA sequence that lead to sensitivity to CPT but not to other genotoxins. Cre -lox RMCE was performed in vitro to recombine cassettes carrying mctp1 into cre expression plasmid pAW8 -ccdb, and transformed into E. coli to generate a plasmid library. Plasmid library were transformed into S. pombe and cre expression on plasmid switched on to catalyze in situ RMCE, which recombined mctp1 into endogenous gene location in S. pombe base strain, in place of ura4+ marker. In order to create library of mutants for screening colonies that showed sensitivity to drug agents, ura4-cells possessing mctp1 cells were selected for by the action of 5FOA and propagated to screen CPT (4μM&8μM), MMS (0.004% & 0.008%) and phleomycin (2.5μg/ml&3.5μg/ml). A total of eight plate-isolates sensitive to one or more genotoxins were discovered, of which one isolate showed sensitivity to CPT alone. This reiterated that defects in ctp1 results in sensitivity to genotoxins, due to inability of defective mctp1 to activate Mre11 complex and bring about DSB resection and repair. However, screening for isolates is a statistical effort that requires continuous building up of libraries to generate more data that could provide more outcomes. Thereby underlying molecular effects of unique isolate that showed phenotype to CPT could not be analyzed to draw more conclusions.
Supervisor: Hartsuiker, Edgar Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available