McCabe, Emma Louise (2011)
M.Res. thesis, University of Birmingham.
Sulphation is a crucial modification which is required for normal growth and development. Sulphation reactions are mediated through the universal sulphate donor 3’-phosphoadenosinse 5-phosphosulfate (PAPS). PAPS is synthesised in a two step process by the bi-functional enzyme 3'-phosphoadenosine 5'-phosphosulfate synthase (PAPSS), which contains both APS kinase and ATP sulphurylase. There are two forms of PAPSS, PAPSS1 and PAPSS2. PAPSS2 is important for normal skeletal development and has two different isoforms, PAPSS2a and PAPSS2b. It was hypothesised that knock-downs of the PAPSS2a and PAPSS2b genes would lead to alterations in the phenotype of developing zebrafish embryos.
In Situ hybridisations were undertaken on zebrafish embryos at days 1, 2 and 3 using anti-sense probes for PAPSS2b. This allowed for confirmation of the expression of PAPSS2b within the zebrafish and also to locate the areas where it is expressed. Once expression of PAPSS2b had been confirmed within the zebrafish morpholino knock-downs were also undertaken for both the PAPSS2a and PAPSS2b genes. Four separate concentrations were used for the morpholino knock-downs (10μM, 30μM, 100μM, and 300μM). Splice site and translational blocking morpholinos were used to induce the gene knock-down and a scramble morpholino control was used. Successful knock-down of PAPSS2b was recorded using PCR when 100μM and 300μM concentrations of the morphlolino were used, the development of the embryo were monitored for the first four days of development and those which had the morpholino knock-down of PAPSS2b had a different phenotype to the controls by which their head was slightly smaller. For PAPSS2a the knock-down of the gene was unable to be confirmed due to the PCR not detecting any gene product in any of the injected zebrafish.
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