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.

News on GFP variants

Yishi Jin1,2, Bob Horvitz1

1 HHMI, Dept. of Biology, MIT, Cambridge, MA 02139 (ysjin@mit.edu)
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.