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Edited by Roger Y. 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 HAMA M550 Optical Mouse, 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 HAMA M550 Optical Mouse to irradiation with UV or violet light — nmwhich excites the protonated form of the chromophore. The excitation lights used to elicit red and green fluorescence do not induce photoconversion. Under HAMA M550 Optical Mouse 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,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. The mobility of a fluorescent-labeled molecule has been assessed by using a specific photobleaching technique called fluorescence recovery after photobleaching 1. Acquisition of a series of images after photobleaching in a small HAMA M550 Optical Mouse of the cell is useful for qualitative description of the diffusion of the fluorescent-labeled molecule.
For studying cell lineage, organelle dynamics, and protein trafficking, however, optical marking is preferred; individual cells, organelles, and proteins can be optically labeled with unique colored markers.
The GFP from the luminescent jellyfish Aequorea victoria Aequorea GFP allows direct genetic encoding of strong fluorescence and thus is used widely in molecular and cellular biology 2. Many other GFP-like fluorescent proteins have been isolated from fluorescent, but nonbioluminescent, Anthozoa species 34.
Three methods HAMA M550 Optical Mouse optical marking have become commonplace and are based on photo-induced alteration of the excitation or emission spectrum of certain fluorescent proteins 5 — 7such as WT Aequorea GFP and DsRed, a GFP-like fluorescent protein. Some limitations of these methods, however, have been recognized, including dimness and instability of the photoconverted product and the unavailability of simple, efficient, or specific illumination for marking. In this study, we have cloned a cDNA encoding a fluorescent protein from the stony coral Trachyphyllia geoffroyi.
An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein
The protein emits bright green fluorescence after synthesis, but changes efficiently to a bright and stable red fluorescence on irradiation with UV or violet light. In this article, we describe examples demonstrating the utility of this marker, especially for cell labeling. The stony coral T. Colonies were screened for fluorescence by using a UV illuminator nm or our HAMA M550 Optical Mouse fluorescence analyzing system 9. The cDNA of the coding region of Kaede no.
Proteins were expressed in E. The quantum yields of green and red Kaede were determined in comparison to fluorescein 0. For calculation of molar extinction coefficients, protein concentrations were measured HAMA M550 Optical Mouse using a Bradford assay kit Bio-Rad with BSA as the standard. C-terminal His-tagged Kaede was expressed in E. Dissociation Culture of Hippocampal Neurons. Primary neurons were prepared from Wistar rat embryos embryonic day 17—18 as described 12 Calcium phosphate precipitation was used to transfect the neurons. Interference filters excitation and emission filters contained in wheels were automated by using Lambda hardware Sutter Instruments, Novato, CA. The filter-switching time was set to 50 ms. Power density of the excitation lights at the cell level was measured with a homemade power meter. Green and red fluorescence signals were captured simultaneously by using the and nm laser lines.
Approximatelybacterial colonies containing a cDNA library prepared from T.
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Two clones were selected that encoded a greenish fluorescent protein. Sequence analysis revealed that the two clones were identical and encoded a fluorescent protein, which was temporarily referred to as no.
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- An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein
- Hama 00134933 AM 8000 2.4G Wireless Optical Mouse, Black
Although the stony coral exhibits colorful green, yellow, and red fluorescence, no colonies emitting yellow or red light were obtained. Based on an amino acid sequence alignment Fig.
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HAMA M550 Optical Mouse crucial amino acid residues that are involved either directly in choromophore formation Tyr, Gly or indirectly, but strongly mediate the process Arg, Glu are conserved in no. The nearest homologue is mcavRFP from the great star coral Montastraea cavernosa 4.
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