Abstract
We combine immunofluorescence and single-molecule fluorescence in situ hybridization (smFISH), followed by automated image analysis, to quantify the concentration of nuclear transcription factors, number of transcription factors bound, and number of nascent mRNAs synthesized at individual gene loci. A theoretical model is used to decipher how transcription factor binding modulates the stochastic kinetics of mRNA production. We demonstrate this approach by examining the regulation of hunchback in the early Drosophila embryo.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Alpha-Amanitin / chemistry
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Animals
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Cell Nucleus / metabolism
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Crosses, Genetic
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DNA / chemistry
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Drosophila Proteins / metabolism
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Drosophila melanogaster / embryology
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Drosophila melanogaster / genetics
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Gene Expression Regulation, Developmental*
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Image Processing, Computer-Assisted
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In Situ Hybridization, Fluorescence
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Microscopy, Confocal
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Microscopy, Fluorescence
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Models, Theoretical
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Normal Distribution
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Pattern Recognition, Automated
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Proteins / chemistry*
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RNA, Messenger / chemistry*
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RNA, Messenger / metabolism
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Species Specificity
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Stochastic Processes
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Transcription Factors / metabolism
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Transcription, Genetic*
Substances
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Alpha-Amanitin
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Drosophila Proteins
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Proteins
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RNA, Messenger
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Transcription Factors
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DNA