Transcriptional regulation of the Drosophilafushi tarazu gene by its proximal enhancer.
Item
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Title
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Transcriptional regulation of the Drosophilafushi tarazu gene by its proximal enhancer.
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Identifier
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AAI9510670
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identifier
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9510670
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Creator
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Han, Wei.
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Contributor
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Adviser: Leslie Pick
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Date
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1994
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Molecular
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Abstract
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The expression of the Drosophila segmentation gene fushi tarazu (ftz) is controlled at the level of transcription. The ftz proximal enhancer, a 794-bp fragment located at {dollar}\sim{dollar}3.4 kb upstream of the transcription start site, directs reporter gene expression in seven ftz-like stripes in transgenic embryos. We have taken a biochemical approach to unravel the molecular basis for the enhancer function. Nine footprint sites were identified using DNaseI footprinting and methylation interference experiments with staged Drosophila embryo nuclear extracts (NE). A 323-bp fragment containing all nine sites directed lacZ fusion gene expression in seven ftz-like stripes. Site-specific substitution mutations of sites 6, 8 and 9 abolished reporter gene expression. Twelve different sequence-specific DNA-binding complexes (fEBC1-12) interacted with eight of the nine sites. Twelve proteins were purified from NE with site 4 and 6 affinity chromatography. Reliable peptide sequences were obtained for P1 to P8. The cDNAs encoding P1 were isolated from an embryo cDNA library. P2 is a known transcription factor, Adf1. Both Escherichia coli expressed P1 and Adf1 bound to multiple footprint sites specifically. Adf1 and FTZ-F1 are involved in the formation fEBC8 with footprint sites 6, 8, and 9, as suggested in gel retardation assays with antibodies. Both P1 and Adf1 have potential transcription activation function when tested in a yeast system. P5 is another known transcription factor, TTK. It binds to five of the nine footprint sites. We propose that ftz expression is initiated and maintained by transcriptional activators identified in these studies, including FTZ-F1, P1, and Adf1, along with FTZ protein. These proteins interact with multiple sites in the ftz proximal enhancer. Potential repressors, including TTK, compete with the activators for binding to the same DNA sequences to actively repress ftz autoregulation and turn off ftz expression.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
