Worm Breeder's Gazette 14(1): 21 (October 1, 1995)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
|1||HHMI, Dept. of Biology, MIT, Cambridge, MA 02139 (email@example.com)|
|2||* Address from January 1996: Department of Biology, Sinsheimer Laboratories, University of California, Santa Cruz, CA 95064|
The use of GFP has enabled the visualization of biological processes in living animals (Chalfie et al., 1994; see reviews in TIG 11 320-329). Recently, several mutations in GFP were found to alter the wavelength spectrum of excitation and emission of the protein and to increase the intensity of fluorescence (Heim et al. 1994; 1995). In particular, Ser65Cys or Ser65Thr, and Ile167Thr mutations resulted in an increased intensity of fluorescence and in red-shifting of the excitation and emission wavelengths. We have made two GFP variants containing mutations at both amino acid 65 and 167: GFP-TT with Ser65Thr and Ile167Thr, and GFP-CT with Ser65Cys and Ile167Thr. These GFP variants are still fluorescent. The intensity of fluorescence of these new GFP variants appeared stronger than that of either the original wild-type GFP or the GFP containing single mutations such as Ser65Cys and Ser65Thr when the expression of each GFP was driven by the promoters of the unc-25 and vab-3 genes. These new GFP variants seemed to have a broader spectrum of excitation and emission than the wild-type GFP and the GFPs with single mutations, since we could detect fluorescence using the rhodoamine filter from these new GFP variants but not from the wild-type or other GFPs when driven by the unc-25 promoter; the maximal intensity was still obtained using the fluorescein filter. These double mutant GFPs also gave stronger signals than the wild-type GFP in premorphogenesis embryos (Andrew Chisholm, personal comunication). We have not performed any biochemical or biophysical analyses of these new GFP proteins. Although GFP-CT and GFP-TT result in similarly strong fluorescence, when fused to the VAMP protein for targeting to synaptic vesicles (Nonet, 1995), GFP-CT produced a much weaker signal than GFP-TT or the wild-type GFP, which suggests that local factors, such as pH, may not be optimal for fluorescence from a Ser65Cys mutant GFP in synaptic vesicles. These GFP variants are available. We would appreciate feedback from anyone who uses these constructs. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. and Prasher, D. C. (1994). Green fluorescent protein as a marker for gene expression. Science 263, 802-805. Heim, R., Cubitt, A. B. and Tsien, R. Y. (1995). Improved green fluorescence. Nature 373, 663-664. Heim, R., Prasher, D. C. and Tsien, R. Y. (1994). Wavelength mutations and posttranslational autoxidation of green fluorescent protein. PNAS 91, 12501-12504. Nonet, M. L. (1995). Visualization of presynaptic terminals using GFP. WBG 13 #5, 40.