Owen, Helen Jane;
(2004)
Characterisation of a phospholipase C-delta from sea urchin eggs and embryos.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
At fertilisation the sea urchin egg is activated by an increase in calcium (Ca2+), largely generated by an increase in inositol (1,4,5) trisphosphate (IP3). Increases in intracellular IP3 and Ca are also observed during the first cell cycle. The precise role of phospholipase Cs (PLCs) during these events is unknown. To date, PLC-γ is the only PLC isoform identified in sea urchin egg cytosol, yet evidence suggests the presence of additional PLC activity. Here, immunoblot analysis suggests that a PLC-δ isoform is also present in sea urchin egg cytosol and remains present in fractions with PLC activity after heparin column and gel filtration fi-actionation. This thesis investigates this previously unidentified PLC-δ activity. Strongylocentrotus purpuratus egg, embryo and testes cDNA databases and macro-arrays were used to identify and clone a PLC-δ, named PLC-δsu, from sea urchin embryos. PLC-δsu consists of 2.44 kb, has a molecular weight of 84.δ kDa and is most similar to zebrafish PLC-δ. Production of a recombinant PLC-δsu fusion protein proved PLC-δsu to be a catalytically-active PLC. In addition, RNA transcripts of full-length and truncated PLC-δsu were used to study the function of PLC-δsu in mouse and sea urchin eggs. Data suggests that PLC-δsu may play a role in producing apoptosis in the developing sea urchin. This thesis also shows that the PH domain of PLC-δsu preferentially binds 3-phosphorylated phosphoinositides on membranes spotted with phospholipids. The localisation of the PLC-δsu PH domain in mouse and sea urchin eggs during fertilisation has been investigated by the construction and use of GFP-tagged PH domain fusion proteins and confocal microscopy. Different localisation patterns are observed in mouse and sea urchin eggs.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Characterisation of a phospholipase C-delta from sea urchin eggs and embryos |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Thesis digitised by ProQuest. |
| Keywords: | Pure sciences; Phospholipase |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10097648 |
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