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Title: The first genome-wide screen for animal peroxins using RNAi in Drosophila melanogaster
Author: Drake, Peter
ISNI:       0000 0004 2736 2845
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2013
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Peroxisomes are spherical, membrane-bound organelles found ubiquitously in Eukaryotes. They have very important metabolic functions, the loss of which has severely morbid consequences for human patients. Genes involved in peroxisome biosynthesis are evolutionarily conserved. There are now more than thirty known proteins (peroxins) involved in peroxisome biosynthesis (including peroxisomal matrix protein import, peroxisome multiplication and inheritance). Higher organisms are expected to have many more peroxins than yeasts and plants that have well established peroxisome families. Yet fewer peroxins are known of and consequently their interactions are poorly understood. Recent technological advances in RNA interference (RNAi) and high-throughput screening have meant entire organisms can now be screened in negative gene function assays. The inauguration in 2010 of the Sheffield RNAi Screening Facility (SRSF) has meant that an on-site facility at Sheffield University can facilitate genome-wide screening of Drosophila melanogaster, an organism for which technologies into genetic manipulation are well established. The use of cell-culture and high-content, automated epifluorescence microscopy means that D. melanogaster cells can be screened for peroxins using well-established fluorescence assays. Two genome-wide screens were performed on S2R+ cells in an attempt to uncover novel peroxins in fruitflies which may elucidate further peroxisomes in mammals. Both screens used fluorescent peroxisomal matrix markers. The first was transfected transiently and the second stably, improving the expression profile in the cellular population. A small number of aberrant phenotypes were quickly identified and many knockdown phenotypes had a milder presentation. The most statistically significant aberrant candidates were chosen to be compiled into a custom designed microtitre plate. We made many copies of this and we repeated the assay in triplicate. We were able to identify 3 newly aberrant phenotypes of knockdown of which 2 appear to effect peroxisomes directly. One of these appears to behave in a PEX16 like manner and we decided to investigate this further.
Supervisor: Hettema, Ewald Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available