Content area
Full Text
We generated a mutant of the red fluorescent protein drFP583. The mutant (E5) changes its fluorescence from green to red over time. The rate of color conversion is independent of protein concentration and therefore can be used to trace time-dependent expression. We used in vivo labeling with E5 to measure expression from the heat shock-dependent promoter in Caenorhabditis elegans and from the Otx-2 promoter in developing Xenopus embryos. Thus, E5 is a "fluorescent timer" that can be used to monitor both activation and down-- regulation of target promoters on the whole-organism scale.
Green fluorescent protein (GFP) from the luminescent jellyfish Aequorea victoria is an important tool in molecular and cellular biology as a transcriptional reporter, fusion tag, or biosensor (1). The recent discovery of GFP-like fluorescent proteins from nonbioluminescent Anthozoa species (2), in particular the red fluorescent protein drFP583, has opened new horizons for multicolor labeling and fluorescence resonance energy transfer applications.
An earlier report (2) suggested that the red fluorophore of drFP583 requires an additional autocatalytic modification of a GFP-like fluorophore. We thus generated mutants of drFP583 using error-prone polymerase chain reaction (PCR) (3) and screened for mutants exhibiting a green intermediate fluorescence (4).
Mutations resulted in proteins with varying fluorescent properties, such as faster maturation, double emission (green and red), or exclusive green fluorescence. Of particular interest was the E5 mutant, which changes its fluorescence over time. This mutant changed from initial bright green fluorescence to yellow, orange, and finally red over time (Fig. 1, A and B). Yellow and orange fluorescence indicate that the protein species with green and red fluorophores are both present (Fig. 1B, color insert). The existence of a green-- emitting intermediate suggests that E5 maturation involves the modification of a GFP-- like fluorophore to give the red fluorophore. Changing the temperature had the same effect on the rates of decay of green fluorescence and growth of red fluorescence, which suggests that these processes reflect the same chemical reaction (Fig. 1B). In addition, the overall reaction speed was independent of the initial concentration of E5 protein in the range from 10 (mu)g/ml to 1 mg/ml (as in Fig. 1B). It was also insensitive to variations in ionic strength in the range from 10 mM to 1 M NaCI,...