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Title: Nucleosome-independent permanently condensed chromosome : investigation of novel nuclear organisation in the dinoflagellates
Author: Hu, I. Ian
ISNI:       0000 0004 8501 083X
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
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Dinoflagellates hold vast diversities and are major contributors to overall marine primary production. Close relatives to the apicomplexan parasites and ciliates, the dinoflagellates, however, have a shockingly different nuclear biology. The dinoflagellates have massively inflated genomes (2-245 Gbps, up to 80X that of humans), permanently condensed liquid crystalline chromosomes throughout the life cycle, minimal histone proteins expression, and loss of nucleosome-mediated chromosome organisation. These changes co-occur with adoption of a novel nuclear protein termed DVNP (Dinoflagellate/Viral NucleoProtein). DVNP is highly positively charged, nucleus-located, and is found in only dinoflagellates and numerous marine large DNA viruses. I will address two issues in this thesis, 1) what are the properties of the protein DVNP, and can we infer if and how DVNP manages the permanently condensed chromosome state; and 2) how do other proteins accommodate this drastically different organisation of genetic material, more specifically how does transcription work in a cell with permanently condensed chromosomes? To elucidate the location of transcription, specific probe were designed and synthesised to locate RNA polymerase and newly synthesised nascent RNA in relation to the compacted chromosomes, and result showed that both are distributed at the periphery but not the inside of the chromosomes. On the other end, DVNPs are expressed and purified to perform biophysical and biochemical characterisation. In addition, using single-molecule optical tweezers microscopy, DVNP was observed to compact and change the mechanical properties of DNA. Most interestingly, the DVNP/DNA aggregates showed a propensity to travel along the DNA strand en masse. I then endeavoured to solve the NMR structure of DVNP. The results suggest that DVNP plays a central role in chromatin packaging in dinoflagellates. Finally, to marry the results obtained from both experiments, at the end of the thesis I propose a model of a novel nucleosome-independent chromatin management in dinoflagellates and how its transcription could proceed.
Supervisor: Waller, Ross Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Dinoflagellate ; permanently ; condensed ; chromosome ; DVNP ; optical tweezers ; NMR ; microscopy ; biophysics ; biochemistry ; single-molecule spectroscopy ; evolutionary biology ; gremlin ; transcription ; protein structure