The isolation and expression of the cDNA for the green fluorescent protein (GFP) from the bioluminescent jellwsh Aequorea victoria has highlighted its potential use as a marker for gene expression in a variety of cell types (Chalfie et al.: Science 263: 802-805, 1994). The longer wavelength peak (470 nm) of GFP's bimodal absorption spectrum better matches standard fluorescein filter sets; however, it has a considerably lower amplitude than the major absorption peak at 395. In an effort to increase the sensitivity of GFP with routinely available instrumentation, Heim et al. (Nature 373:663-664, 1995) have generated a GFP mutant (serine-65 to threonine; S65T-GFP) which possesses a single absorption peak centered at 490 nm. We have constructed this mutant in order to determine whether it or wild-type GFP (wt-GFP) afforded greater sensitivity when excited near their respective absorption maxima. Using the conventionally available 488 nm and ultraviolet (W) laser lines from the argon ion laser as well as the 407 nm line from a krypton ion laser with enhanced violet emission, we were able to closely match the absorption maxima of both the S65T and wild-type forms of Aequorea GFP and analyze differences in fluorescence intensity of transiently transfected 293 cells with flow cytometry. The highest fluorescence signal was observed with 488 nm excitation of S65T-GFP relative to all other laser line/GFP pairs. The wt-GFP fluorescence intensity, in contrast, was significantly higher at 407 nm relative to either 488 nm or W. These results were consistent with parallel spectrofluorometric analysis of the emission spectrum for wt-GFP and S65T-GFP. The relative contribution of cellular autofluorescence at each wavelength was also investigated and shown to be significantly reduced at 407 nm relative to either W or 488 nm. In vivo, GFP is excited by energy transfer from the Ca2+ -dependent blue light emission of the photoprotein aequorin ( 1 1, 20). A similar mechanism involving energy transfer from a photoprotein to a GFP has been demonstrated in a number of other bioluminescent coelenterates (5,(17)(18)(19). The covalently-boundp-hydroxybenzylidene-imidazolidinone chromophore responsible for the fluorescence of Aequorea GFP is derived from the cyclization of the amino acid residues Ser-dehydroTyr-Gly (4). This 0,-dependent cyclization reaction proceeds via a poorly understood mechanism which appears to be either autocatalytic, or at least catalyzed by ubiquitous enzymes, as hnctional GFP can be expressed in both prokaryotic and eukaryotic cells (3, 10). The GFP spectrum displays a major absorption peak at 395 nm and a minor peak at 470 nm. The fluorescence emission spectrum exhibits a major peak at 509 nm with a shoulder at 540 nm (18, 20,29).Although Aequoreu GFP has been identified and studied extensively since the late 1960s, recent isolation of the cDNA (22) and its subsequent expression in heterologous cell types (3, 10) has opened a new chapter for
Aequorea green fluorescent protein (GFP) has been expressed in a variety of cell lines and host organisms. A recent report (Heim et al.: Proc Natl Acad Sci USA 91:12501–12504, 1994) has documented that a GFP mutant with a single amino acid substitution (tyrosine 66 to histidinc; Y66H‐GFP) elicits altered spectral properties. Whereas wild‐type GFP emits with a maximum at approximately 509 nm (green fluorescence), Y66H‐GFP fluoresces with a maximum at approximately 448 nm (blue fluorescence). In this study we employed available argon and krypton ion laser lines to investigate the impact of laser excitation wavelength on the detection of Y66H‐GFP by flow cytometry. Using transiently transfected 293 cells, a cellular subpopulation with elevated blue fluorescence was detectable with excitation at 407 nm, but not with ultraviolet (UV), 458 nm, or 488 nm excitation. The blue‐fluorescing cells were further documented to express Y66H‐GFP by immunoblot analysis of sorted cells. Finally, we demonstrated the simultaneous analysis of both wild‐type and Y66H‐GFP in cotransfected 293 cells using 407 nm excitation while collecting blue fluorescence at 460 ± 20 nm (Y66H‐GFP) and green fluorescence at 525 ± 25 nm (wild‐type GFP). These studies illustrate the potential for assessing differential gene expression by simultaneously analyzing multiple GFP species with multiparameter flow cytometry. © 1996 Wiley‐Liss, Inc.
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