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The brain of living drosophila is a powerful model system to study neural circuits and memory formation. The large number of genetic tools available allow for modifications of biochemical properties. The small size makes imaging less daunting.
On the other hand, the small size makes handling and sample preparation of the brain more challenging. The fly brain is infamous for it's strong auto-fluorescence. The cuticle makes imaging without opening the head nearly impossible.
We are tackling these challenges using two photon microscopy (NLO) and linear unmixing.
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Stowers Links:
Stowers Institute: Si Lab
Adv. Instr. & Physics: Technology
Web Links:
Flybrain: Online Atlas of the Drosophila Nervous System
FlyBase: Database of the Drosophila Genome
FlyMove: Embryonic Development of Drosophila
J*Fly: How to dissect Drosophila Brains
Download movie: Dissecting adult brain
Download movie: Dissecting brain w/ body
Download movie: Dissecting whole CNS
Download movie: Dissecting larval CNS
Download movie: Dissecting pupal antenna
J*Fly: Images of the Drosophila Brains
Literature:
Si, K., S. Lindquist, et al. (2003). "A Neuronal Isoform of the Aplysia CPEB Has Prion-Like Properties." Cell 115(7): 879-891.
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Gene transcription by RNA polymerase II, responsible for the production of cellular mRNA, can be a critical target for regulating which complement of the cellular genome is expressed. An understanding of the mechanisms underlying the various stages of transcription will be crucial to being able to find solutions to the problems caused by misexpression of genes, problems that may give rise to a host of human diseases.
Regulation of RNA polymerase II activity can occur both during the initiation and elongation stages of transcription. A protein which affects transcription elongation of RNA polymerase II, eleven-nineteen lysine-rich in leukemia (ELL), was initially identified as a gene fusion partner of the mixed lineage leukemia (MLL) gene in some patients with acute myeloid leukemia. In vitro studies have shown that the ELL protein is capable of interacting with RNA polymerase II and increasing its catalytic rate of transcription elongation.
ELL has been found associated with several proteins. Simone et al. identified EAF1 (ELL-associated factor 1) via a yeast two-hybrid screen with ELL. As with the MLL-ELL gene-fusion, an MLL-EAF1 gene fusion is able both to transform primary cells, and to induce a leukemia state in mice when expressed in injected bone marrow cells.
Although ELL and RNA Polymerase II have been shown to coimmunoprecipitate, it is not clear whether ELL might be recruited to RNA Polymerase II during initiation or elongation and whether the proteins remain bound during elongation. The role of EAF1 is also unclear, and although an ELL/EAF1 heterodimer affects RNA polymerase II activity, it is not clear whether ELL and EAF1 interactions are concurrent with those of ELL and polymerase. I will use fluorescence correlation spectroscopy to determine when fluorescent labeled ELL and EAF1 are recruited to the transcription complex during an in vitro transcription assay.
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Stowers Links:
Stowers Institute: Conaway Lab
Adv. Instr. & Physics: FCS
Literature:
Kong, S. E., C. A. S. Banks, et al. (2005). "ELL-associated factors 1 and 2 are positive regulators of RNA polymerase II elongation factor ELL." PNAS 102(29): 10094-10098.Download PDF
Conaway, J. W., A. Shilatifard, et al. (2000). "Control of elongation by RNA polymerase II." Trends Biochem Sci 25: 375-80.Download PDF
Shilatifard, A., R. C. Conaway, et al. (2003). "The RNA polymerase II elongation complex." Annul Rev Biochem 72: 693-715.Download PDF
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