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Title: DNA replication initiation in the protozoan parasite Giardia lamblia
Author: Grignard, Lynn
ISNI:       0000 0004 2724 366X
Awarding Body: St George's, University of London
Current Institution: St George's, University of London
Date of Award: 2012
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In this thesis the Giardia lamblia origin recognition proteins, GiOrc1/cdc6 and GiOrc4 (GiORC) are under investigation. We set out to do a basic molecular characterisation of the GiORC proteins as well as determining origins of DNA replication in the protozoan parasite. The sequences for both GiORC were successfully identified from genome databases and analysed using bioinformatics tools. Furthermore, the GiORC proteins were successfully over-expressed in a bacterial system. In a first strategy, pure protein with or without an affinity tag was used for antibody production, for the use in western blotting, immuno precipitation (interacting partners), chromatin immuno precipitation (identification of origins of DNA replication) and microscopy (protein localisation). Three antibodies against the GiORC were generated. Unfortunately, none of the antibodies recognised the native GiORC. GST-tagged GiOrc4 for GST pull down experiments was used to identify interacting partners, without success. In a second strategy, the GiORC were cloned into Giardia over-expression vectors. Two novel vectors were constructed, pV5.pac and ProtORCtet. Both vectors had not been used previously in the parasite. In addition GiORC sequences were cloned into pGFP.pac, a GFP fusion vector. Six different constructs, three for each GiORC sequence, were generated. In four of the constructs, GiORC/pGFP.pac and GiORC/pV5.pac, expression could not be detected in transgenic cell lines. In ProtOrc4tet, expression of V51HA tagged GiOrc4 was detected by western blot and immuno fluorescence microscopy. In a third strategy, the interaction of GiOrcl/cdc6 and GiOrc4 was shown by yeast two hybrid. We were also interested in the effects of GiORC gene knockdown by RNA interference. The results from the DNA based RNAi vector, however, were inconclusive. In summary, we managed to compile information on the DNA replication proteins m Giardia lamblia and to set up additional tools to further study DNA replication initiation in the parasite.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available