||Mammalian tissue culture cell
||Darzacq X, Shav-Tal Y, de Turris V, Brody Y, Shenoy SM, Phair RD, Singer RH. In vivo dynamics of RNA polymerase II transcription. Nat Struct Mol Biol. 2007 Sep14(9):796-806 p.800 left column bottom paragraphPubMed ID17676063
||Parameter optimization of FRAP data in U2OS 200 copy gene array. "A method for the in vivo labeling of mRNA transcripts containing a series of repeated stem-loops (from phage MS2), which are specifically bound by an MS2 coat protein fused to green fluorescent protein (GFP) [ref 16]. The assay consists of a human cell line harboring a gene array into which these stem-loops have been integrated [ref 17]." FRAP- Fluorescence Recovery After Photobleaching, takes advantage of the light emitting property of dyes. A dye will emit light of a certain wavelength after being exposed to light of another wavelength. However, having being illuminated beyond a certain intensity it will lose its ability to emit light, a phenomenon known as photobleaching. In FRAP a limited area (often of a cell membrane) of known fluorescence is photobleached. Fluorescent molecules outside of this area will diffuse into it, resulting in fluorescence recovery. By constant tracking of fluorescence level, fluorescence recovery is measurable by two parameters: 1)The time for recovery to have occurred (the shorter the time, the faster the diffusion of neighboring molecules). 2)The percent of original fluorescence before (photobleaching) restored after recovery has reached saturation. Photoactivation is the absorption of energy from a photon in raising a molecule (or chromophore) from the ground state.
||"The average polymerase velocity over this time (517 s) for the 3.3-kb
gene would be approximately 378 bases min^–1, much slower than has been reported
[ref 37]." See also fast component: maximal polymerase velocity, 71.6 nts/sec BNID 100662. YFP-Pol II is amanitin resistant allele.