RNAi-mediated knockdown of chromatin modifier proteins and their effect on long-term memory in Drosophila : a thesis presented to Massey University in partial fulfillment of the requirements for the degree of Master of Science in Genetics
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Date
2008
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Massey University
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Abstract
Memory formation in Drosophila melanogaster is composed of two pathways that are
genetically distinct, and functionally independent of each other. These are short-term
and long-term memory. Short-term memory is a transient phenomenon, located in the
cytoplasm of the neuronal cells, which requires no alteration of gene expression. The
formation of long-term memory requires a change in gene expression, therefore
chromatin-modifying complexes may play an integral part. The mushroom-bodies of
Drosophila are a distinct bilateral brain structure and are essential for the formation and
recollection of long-term memory. Therefore, an alteration in gene expression within
the mushroom bodies is essential to the formation of long-term memory. Disruption of
a gene within the mushroom-bodies that resulted in an alteration in the formation of
long-term memory would indicate that the gene is involved in long-term memory.
In order to investigate the role of the two chromatin-modifying proteins, HDACX and
pr-Set7, whose role in memory function is unknown, RNA interference was used to
knockdown expression of their respective mRNA. Published GAL4 lines were used to
drive down expression in the mushroom bodies. The efficacy of the knockdown on
levels of mRNA was measured by quantitative RT-PCR. The effect of these
knockdowns on the formation of long-term memory was assayed using conditioned
courtship. Additionally, the actual spatial and temporal expression of the GAL4 drivers
was investigated using fluorescent proteins, and analysed using fluorescent microscopy.
Both pr-set7 and HDACX appear to play a role in long-term memory function. The
RNAi-induced knockdown of the individual mRNAs caused impairment in long-term
memory formation, although the exact mode of action is still to be elucidated. The
levels of mRNA from these knockdowns were reduced within the head, although not to
the extent expected. The fluorescent microscopy analysis indicated that the expression
of mushroom-body specific GAL4 drivers was more widespread than previously
reported.
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Keywords
Genetics, Gene expression, Drosophila melanogaster, Common fruit fly, Long-term memory formation, Chromatin-modifying complexes, Memory