- Email: [email protected]
Techniques update
TECHNIQUES Contributed by Stephen Cronin and Randolph Hampton, Dept of Biology, UCSD, 9500 Gilman Drive, La Jolla, CA 0347, USA. E-mail: rhampton@ biomail.ucsd.edu
Techniques update A genetics-friendly GFP assay Green fluorescent protein (GFP) has become a popular reporter protein for research in cell biology. When used in yeast, GFP expression is usually monitored by fluorescence microscopy or flow cytometry1. However, the lack of a colony-level assay for GFP expression has somewhat limited the genetic applications of this powerful tool. We have developed a straightforward technique for direct examination of GFP fluorescence in living yeast colonies on solid medium. To examine colony fluorescence, agar plates must be illuminated with intense light of the appropriate wavelength, and the emitted fluorescence must be detected separately from the very intense background of reflected excitation light. For the S65T version of GFP, the excitation wavelength is 488 nm. A standard slide projector (Kodak CarouselTM 4400) provides luxuriant illumination when fitted with a 488 nm, narrow-bandpass filter. Such filters can be custom-made by Omega Optical (www.omegafilters.com). The 488 nm field produced by the filtered projector allows simultaneous examination of multiple agar plates. The green fluorescence emitted from the colonies is visualized by viewing the illuminated plates through a long-bandpass cutoff filter that only blocks passage of the excitation light. For this, we use a Kodak Wratten No. 12 filter, available in any camera store, which can be taped to a clear face mask, goggles or glasses. We use the GFP reporter to study the regulated degradation of the essential enzyme HMG-CoA reductase (HMGR). This protein is an integral-membrane resident of the endoplasmic reticulum that undergoes degradation as part of cellular control of sterol synthesis. One of the ways in which we study the degradation of the yeast HMGR isozyme Hmg2p is to use a fusion protein of Hmg2p and GFP that undergoes physiologically regulated degradation and is detectable by a variety of optical techniques2. The degradation rate of the Hmg2p–GFP determines the in vivo steady-state levels of the protein, and hence,
the cellular fluorwt hrd1-1 (a) escence. We are using the GFP GFP colony assay to screen for mutant yeast (b) strains with alterCells ations in steadystate level. For example, in FIGURE 1 hrd1-1 mutants, (a) Green fluorescent protein (GFP) Hmg2p–GFP degradation is fluorescence in wild-type (wt) and hrd1-1 blocked, and the cells. (b) Same field as (a) but viewed by in vivo levels of direct illumination. (Reproduced, with the reporter are permission, from Ref. 2.) higher than in a strain with a wild-type (wt) HRD1 gene3. This difference in cellular fluorescence is readily apparent at the colony level when viewed with the projector illuminator. Fig. 1 shows patches of otherwise identical yeast strains expressing the Hmg2p–GFP, in which degradation is normal (wt) or deficient (hrd1-1). Although the colonies look identical by direct illumination (Fig. 1b), the difference in fluorescence is readily apparent when examined for fluorescence (Fig. 1a). We are using the colony-fluorescence assay in a variety of genetic approaches to identify colonies with greater or lesser levels of Hmg2p–GFP. It is important to emphasize that the approach clearly depends on the levels and distribution of the GFP reporter being studied. However, we believe that this simple technique will have many applications in yeast as well as in other organisms that are amenable to GFP studies.
References 1 Gardner, R. G. et al. (1998) Mol. Biol. Cell 9, 2611–2626 2 Cronin, S. and Hampton, R. Y. Methods Enzymol. (in press) 3 Hampton, R. Y., Gardner, R. G. and Rine, J. (1996) Mol. Biol. Cell 7, 2029–2044
http://www.elsevier.com/locate/tto u http://www.elsevier.nl/locate/tto
New Technical Tip articles published recently in Technical Tips Online include: j
Shimamoto, N. et al. (1998) Efficient solubilization of proteins overproduced as inclusion bodies by use of an extreme concentration of glycerol Technical Tips Online (http:www.elsevier.com/locate/tto) T01576
New products featured in Technical Tips Online: PanoramaTM E. coli Gene Arrays, from Genosys Biotechnologies Inc. A new tool for genome-wide analysis. These arrays provide a rapid means to determine global gene expression during cellular responses to developmental cues, external stimuli or a variety of stresses.
36
0962-8924/99/$ – see front matter © 1999 Elsevier Science. All rights reserved. PII: S0962-8924(98)01421-4
trends in CELL BIOLOGY (Vol. 9) January 1999
Techniques update A genetics-friendly GFP assay Green fluorescent protein (GFP) has become a popular reporter protein for research in cell biology. When used in yeast, GFP expression is usually monitored by fluorescence microscopy or flow cytometry1. However, the lack of a colony-level assay for GFP expression has somewhat limited the genetic applications of this powerful tool. We have developed a straightforward technique for direct examination of GFP fluorescence in living yeast colonies on solid medium. To examine colony fluorescence, agar plates must be illuminated with intense light of the appropriate wavelength, and the emitted fluorescence must be detected separately from the very intense background of reflected excitation light. For the S65T version of GFP, the excitation wavelength is 488 nm. A standard slide projector (Kodak CarouselTM 4400) provides luxuriant illumination when fitted with a 488 nm, narrow-bandpass filter. Such filters can be custom-made by Omega Optical (www.omegafilters.com). The 488 nm field produced by the filtered projector allows simultaneous examination of multiple agar plates. The green fluorescence emitted from the colonies is visualized by viewing the illuminated plates through a long-bandpass cutoff filter that only blocks passage of the excitation light. For this, we use a Kodak Wratten No. 12 filter, available in any camera store, which can be taped to a clear face mask, goggles or glasses. We use the GFP reporter to study the regulated degradation of the essential enzyme HMG-CoA reductase (HMGR). This protein is an integral-membrane resident of the endoplasmic reticulum that undergoes degradation as part of cellular control of sterol synthesis. One of the ways in which we study the degradation of the yeast HMGR isozyme Hmg2p is to use a fusion protein of Hmg2p and GFP that undergoes physiologically regulated degradation and is detectable by a variety of optical techniques2. The degradation rate of the Hmg2p–GFP determines the in vivo steady-state levels of the protein, and hence,
the cellular fluorwt hrd1-1 (a) escence. We are using the GFP GFP colony assay to screen for mutant yeast (b) strains with alterCells ations in steadystate level. For example, in FIGURE 1 hrd1-1 mutants, (a) Green fluorescent protein (GFP) Hmg2p–GFP degradation is fluorescence in wild-type (wt) and hrd1-1 blocked, and the cells. (b) Same field as (a) but viewed by in vivo levels of direct illumination. (Reproduced, with the reporter are permission, from Ref. 2.) higher than in a strain with a wild-type (wt) HRD1 gene3. This difference in cellular fluorescence is readily apparent at the colony level when viewed with the projector illuminator. Fig. 1 shows patches of otherwise identical yeast strains expressing the Hmg2p–GFP, in which degradation is normal (wt) or deficient (hrd1-1). Although the colonies look identical by direct illumination (Fig. 1b), the difference in fluorescence is readily apparent when examined for fluorescence (Fig. 1a). We are using the colony-fluorescence assay in a variety of genetic approaches to identify colonies with greater or lesser levels of Hmg2p–GFP. It is important to emphasize that the approach clearly depends on the levels and distribution of the GFP reporter being studied. However, we believe that this simple technique will have many applications in yeast as well as in other organisms that are amenable to GFP studies.
References 1 Gardner, R. G. et al. (1998) Mol. Biol. Cell 9, 2611–2626 2 Cronin, S. and Hampton, R. Y. Methods Enzymol. (in press) 3 Hampton, R. Y., Gardner, R. G. and Rine, J. (1996) Mol. Biol. Cell 7, 2029–2044
http://www.elsevier.com/locate/tto u http://www.elsevier.nl/locate/tto
New Technical Tip articles published recently in Technical Tips Online include: j
Shimamoto, N. et al. (1998) Efficient solubilization of proteins overproduced as inclusion bodies by use of an extreme concentration of glycerol Technical Tips Online (http:www.elsevier.com/locate/tto) T01576
New products featured in Technical Tips Online: PanoramaTM E. coli Gene Arrays, from Genosys Biotechnologies Inc. A new tool for genome-wide analysis. These arrays provide a rapid means to determine global gene expression during cellular responses to developmental cues, external stimuli or a variety of stresses.
36
0962-8924/99/$ – see front matter © 1999 Elsevier Science. All rights reserved. PII: S0962-8924(98)01421-4
trends in CELL BIOLOGY (Vol. 9) January 1999