GFP background
Aequorea victoria are brightly luminescent jellyfish, with glowing points around the margin of the umbrella. Light arises from yellow tissue masses that each consist of about 6000-7000 photogenic cells. The cytoplasm of these cells is densely packed with fine granules that contain the components necessary for bioluminescence [1:2] . In other bioluminescent coelenterates these have been characterised as 0.2 micron diameter particles enclosed by a unit membrane, and have been termed lumisomes [4] . The components required for bioluminescence include a Ca++ activated photoprotein, aequorin, that emits blue-green light, and an accessory green fluorescent protein (GFP), which accepts energy from aequorin and re-emits it as green light [5] . GFP is an extremely stable protein of 238 amino acids [6] .
The fluorescent properties of the protein are unaffected by prolonged treatment with 6M guanidine HCl, 8M urea or 1% SDS, and two day treatment with various proteases such as trypsin, chymotrypsin, papain, subtilisin, thermolysin and pancreatin at concentrations up to 1 mg/ml fail to alter the intensity of GFP fluorescence [7] . GFP is stable in neutral buffers up to 65oC, and displays a broad range of pH stability from 5.5 to 12.
Stacks Image 814
The protein is intensely fluorescent, with a quantum efficiency of approximately 80% and molar extinction coefficient of 2.2 x10(4) cm-1 M-1 [5] (after correction for the known molecular weight). GFP fluoresces maximally when excited at 400 nm with a lesser peak at 475 nm, and fluorescence emission peaks at 509nm .

The intrinsic fluorescence of the protein is due to a unique covalently-attached chromophore which is formed post-translationally within the protein upon cyclisation and oxidation of residues 65-67, Ser-Tyr-Gly [6:8,9] . Several genomic and cDNA clones of gfp have been obtained from a population of A. victoria [6] . The gfp gene contains at least three introns, and the coding sequence derived from one of the cDNA clones, pGFP10.1 has been used for protein expression, first in Escherichia coli, Caenorhabditis elegans [10:9] and Drosophila melanogaster [11] . Fluorescent protein has now been produced in a number of heterologous cell types and there appears to be little requirement for specific additional factors for post-translational modification of the protein, which may be autocatalytic or require ubiquitous factors.

GFP has been successfully expressed at high levels in tobacco plants using the cytoplasmic RNA viruses potato virus X [12] and tobacco mosaic virus [13] . In these experiments, the gene was directly expressed as a viral mRNA in infected cells, and very high levels of GFP fluorescence were seen. However, poor or no fluorescence was seen when the gfp cDNA was transformed into isolated cells or transformed plants of Arabidopsis [14-16] . We have shown that expression of the gfp cDNA is curtailed by aberrant mRNA splicing in Arabidopsis. We have engineered mutant forms of gfp, to restore and improve expression of the fluorescent protein. The modified gene now contains (i) altered codon usage to remove a cryptic plant intron, (ii) added peptide sequences to allow targeting of the protein to the lumen of the endoplasmic reticulum, and mutations which (iii) improve folding of the apoprotein during post-translational maturation (V163A, S175G), and (iv) provide equalised UV and blue light excitation (I167T) [17] . This highly modified variant (mgfp5-ER) is proving useful as a safe and bright marker in transgenic plants [18] . We expect that the mgfp5-ER gene and its derivatives will also be useful in work with transgenic fungi and animals, where at least some similar problems may be encountered. Image from cover of Nature Biotechnology, F. Yang, L.G. Moss & G.N. Phillips 14:1246-1251, 1996.

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