RRC ID 50308
Author Ando R, Hama H, Yamamoto-Hino M, Mizuno H, Miyawaki A.
Title An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein.
Journal Proc. Natl. Acad. Sci. U.S.A.
Abstract We have cloned a gene encoding a fluorescent protein from a stony coral, Trachyphyllia geoffroyi, which emits green, yellow, and red light. The protein, named Kaede, includes a tripeptide, His-Tyr-Gly, that acts as a green chromophore that can be converted to red. The red fluorescence is comparable in intensity to the green and is stable under usual aerobic conditions. We found that the green-red conversion is highly sensitive to irradiation with UV or violet light (350-400 nm), which excites the protonated form of the chromophore. The excitation lights used to elicit red and green fluorescence do not induce photoconversion. Under a conventional epifluorescence microscope, Kaede protein expressed in HeLa cells turned red in a graded fashion in response to UV illumination; maximal illumination resulted in a 2,000-fold increase in the ratio of red-to-green signal. These color-changing properties provide a simple and powerful technique for regional optical marking. A focused UV pulse creates an instantaneous plane source of red Kaede within the cytosol. The red spot spreads rapidly throughout the cytosol, indicating its free diffusibility in the compartment. The extensive diffusion allows us to delineate a single neuron in a dense culture, where processes originating from many different somata are present. Illumination of a focused UV pulse onto the soma of a Kaede-expressing neuron resulted in filling of all processes with red fluorescence, allowing visualization of contact sites between the red and green neurons of interest.
Volume 99(20)
Pages 12651-6
Published 2002-10-1
DOI 10.1073/pnas.202320599
PII 202320599
PMID 12271129
PMC PMC130515
MeSH Amino Acid Sequence Animals Anthozoa Cloning, Molecular Cytosol / metabolism DNA, Complementary / metabolism Diffusion Electrophoresis, Polyacrylamide Gel Green Fluorescent Proteins HeLa Cells Hippocampus / cytology Hippocampus / embryology Humans Hydrogen-Ion Concentration Light Luminescent Proteins / chemistry* Luminescent Proteins / genetics Luminescent Proteins / metabolism Molecular Sequence Data Neurons / metabolism Normal Distribution Rats Rats, Wistar Scattering, Radiation Sequence Homology, Amino Acid Spectrum Analysis Time Factors Ultraviolet Rays
IF 9.504
Resource
DNA material Kaede/pRSETB (RDB15231)