A complementary DNA for the Aequorea victoria green fluorescent protein (GFP) produces a fluorescent product when expressed in prokaryotic (Escherichia coli) or eukaryotic (Caenorhabditis elegans) cells. Because exogenous substrates and cofactors are not required for this fluorescence, GFP expression can be used to monitor gene expression and protein localization in living organisms.

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria is an unusual protein with strong visible absorbance and fluorescence from a p-hydroxybenzylidene-imidazolidinone chromophore, which is generated by cyclization and oxidation of the protein's own Ser-Tyr-Gly sequence at positions 65-67. Cloning of the cDNA and heterologous expression of fluorescent protein in a wide variety of organisms indicate that this unique posttranslational modification must be either spontaneous or dependent only on ubiquitous enzymes and reactants. We report that formation of the final fluorophore requires molecular oxygen and proceeds with a time constant (-4 hr at 22°C and atmospheric PO2) independent of dilution, implying that the oxidation does not require enzymes or cofactors. GFP was mutagenized and screened for variants with altered spectra. The most striking mutant fluoresced blue and contained histidine in place of Tyr-66. The availability of two visibly distinct colors should significantly extend the usefulness of GFP in molecular and cell biology by enabling in vivo visualization of differential gene expression and protein localization and measurement of protein association by fluorescence resonance energy transfer.Proteins are often labeled with fluorescent tags to detect their localization and sometimes their conformational changes both in vitro and in intact cells. Such labeling is essential both for immunofluorescence and for fluorescence analog cytochemistry, in which the biochemistry and trafficking of proteins are monitored after microinjection into living cells (1). Traditionally, fluorescence labeling is done by purifying proteins and then covalently conjugating them to reactive derivatives of organic fluorophores. The stoichiometry and locations of dye attachment are often difficult to control, and careful repurification of the proteins is usually necessary. If the proteins are to be used inside living cells, a final challenging step is to get them across the plasma membrane via micropipet techniques or various methods of reversible permeabilization.An alternative would be to devise molecular biological means to generate fluorescent proteins. The natural UV fluorescence of tryptophan residues is of little use except in the simplest in vitro samples, because the wavelengths are too short and tryptophan is too ubiquitous for any one protein to stand out in a complex biological mixture. A more promising strategy would be to concatenate the gene for the nonfluorescent protein of interest with the gene for a naturally fluorescent protein and express the fusion product. The most highly fluorescent proteins known are the phycobiliproteins (2), but their fluorescence depends entirely on correct enzymatic insertion of a difficult-to-obtain tetrabilin chromophore into a large apoprotein (3). The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a much smaller molecule of 238 amino acids, whose natural function seems to be to convert the blue chemiluminescence of the Ca2+-sensit...

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The green f luorescent protein (GFP) from the jellyfish Aequorea victoria is finding wide use as a genetic marker that can be directly visualized in the living cells of many heterologous organisms. We have sought to express GFP in the model plant Arabidopsis thaliana, but have found that proper expression of GFP is curtailed due to aberrant mRNA processing. An 84-nt cryptic intron is efficiently recognized and excised from transcripts of the GFP coding sequence. The cryptic intron contains sequences similar to those required for recognition of normal plant introns. We have modified the codon usage of the gfp gene to mutate the intron and to restore proper expression in Arabidopsis. GFP is mainly localized within the nucleoplasm and cytoplasm of transformed Arabidopsis cells and can give rise to high levels of f luorescence, but it proved difficult to efficiently regenerate transgenic plants from such highly f luorescent cells. However, when GFP is targeted to the endoplasmic reticulum, transformed cells regenerate routinely to give highly f luorescent plants. These modified forms of the gfp gene are useful for directly monitoring gene expression and protein localization and dynamics at high resolution, and as a simply scored genetic marker in living plants.