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Cytosolic expression of Green Fluorescent Protein (GFP) and its derivatives ,,, in the yeast Saccharomyces cerevisiae: Detection in vivo using ,,, the Varian Cary Eclipse

l fluorescence imaging procedures, which often damage or kill cells, and are not generally feasible for in vivo studies. GFP has allowed the real time study of cellular events in live, intact cells and organisms, allowing a better understanding of biological mechanisms in physiological models

Fused to a protein of interest, one of the first applications of GFP was as a reporter of gene expression and protein localisation. It was used to illuminate cellular organelles, the cytoskeleton, and the secretory pathway (reviewed by Gerdes and Kaether2). Since then, a rapidly increasing variety of applications for GFP and its derivatives have been developed. Mutational studies of the chromophore and surrounding amino acids have produced new GFPs with altered excitation and emission spectra. These include blue (BFP), cyan (CFP), and yellow (YFP) fluorescent proteins, all with enhanced maturation rates and fluorescent intensities3. The color spectrum was recently extended by Matz and co-workers4, who isolated a red fluorescent protein from the Discosoma coral (DsRed) with 30% sequence homology to Aequoria GFP. This wide variety of spectrally distinct fluorescent proteins provides distinguishable markers for the simultaneous study of multiple cellular events, as well as allowing more advanced applications such as the study of protein-protein interactions using the phenomena of fluorescence resonance energy transfer (FRET).

The fluorescent properties of the GFP family vary in terms of spectral profiles, intensity and susceptibility to photobleaching. Therefore it is important to optimise detection and measurement protocols for GFP and its derivatives prior to experimental analysis in each experimental
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