Worm Breeder's Gazette 16(3): 16 (June 1, 2000)
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.
Carnegie Institution of Washington, Department of Embryology, 115 West University Parkway, Baltimore, Md. 21210
Intrinsically fluorescent proteins have become a remarkably ubiquitous and effective tool to follow molecular entities in living cells. To date, most applications of this approach have used the coding region for the Aequora victoria green fluorescent protein (gfp ), and derivatives thereof. Although gfp variants with different spectra have been produced (e.g. [2,3,4]), these variants (so far) all have fluorescence properties that place them in the blue-cyan-green-yellow portion of the emission spectrum. It would be useful for many studies to have an additional reporter that would emit red light and thus be easily distinguished from all of the gfp isoforms. In October, 1999, Matz and colleagues at the Russian Academy of Sciences published a characterization of an intrinsically fluorescent protein from Discosoma sp . This protein, with definitive (although limited) homology to Aequora gfp, has a an enticing fluorescence spectrum, with absorption and emission properties that are similar to Rhodamine. We sought to test the effectiveness of the coding region for the Discosoma sp. red fluorescent protein ("dsRED") as a reporter for experiments in C. elegans. Fusions of several promoters to the dsRED coding sequence were produced and used to make transgenic animals by standard injection methods (see  for a description of standard protocols). We found that strong promoters such as myo-2 and mec-7 could drive a sufficient quantity of the dsRED gene product to produce a strong red signal, visible in both compound and dissecting fluorescent microscopes using standard rhodamine filters. Amongst viable progeny of injection, fluorescence produced using these promoters was most strongly visible in late larvae and adults. We don't know whether the lack of a strong signal in embryos and earlier larvae results from a simple requirement for quantity of fluorescent protein or (alternatively) whether the dsRED protein might require a long period of time to fold and assemble the red fluorochrome (as is the case for wild-type GFP... see below). High-expression constructs for dsRED (such as the myo-2 construct) appeared to produce considerable toxicity, so that numerous arrested embryos and larvae were produced (some of which became very red over time). As with other toxic DNAs, derivation of lines was only possible by diluting the DNA (keeping a constant level of the selectable marker DNA). Although we have not performed direct comparisons with GFP in these experiments, some indirect comparisons suggest that dsRED may pose substantially greater toxicity problems than GFP in C. elegans. We also tested a promoter (hlh-1) which was more modestly active in the adult. The resulting dsRED fusions were only marginally visible, and were seen in only a small number of cells in the adult. Mesodermal precursors in embryos (which show strong expression of the native hlh-1 gene and of hlh-1 fusions to lacZ ) showed no fluorescence in transgenic lines carrying hlh-1::dsRED fusions. The lack of fluorescence activity in embryos is reminiscent of observations that had been made early in the application of wild-type (unmodified) gfp to C. elegans . GFP was subsequently shown to require long times for folding and acquisition of fluorescence . Eventually, the "low-activity" problems with gfp were solved (as incorporated into the standard set of C. elegans gfp vectors ) using modified gfp versions that had been selected in bacteria by Heim et al  to have faster acquisition of fluorescence and greater photostability. We tested a comparable mutation in dsRED (Q64C) and found that it didn't stimulate fluorescence, but instead eliminated fluorescent character. We had also observed in earlier studies with other reporters (gfp, lacZ) that the expression signals could be improved substantially by adding synthetic introns to punctuate the coding region [9,10]. We have tested versions of the dsRED coding region with 1-3 synthetic introns and found no improvement in overall fluorescence activity. So far, we have no data to rule out the possibility (our working model at this point) that toxicity of high amounts of the dsRED protein sets up a selection for low expression in transgenic lines that are produced. Other explanations are still conceivable, including the possibility that some other aspect of the dsRED coding sequence is problematic in C. elegans, or the possibility that some feature of the constructs that we produced was incompatible with expression. Given the interest in fluorescent proteins in the academic and commercial community, it seems likely that the Discosoma coding region will be subject to the same level of intensive mutagenesis and structural analysis that has been so successfully carried out for gfp. Thus we are optimistic that more generally applicable versions of dsRED will become available. We thank J. Yanowitz and J. Fares for their suggestions, and acknowledge support from the NIH (R01-GM37706) and Carnegie Institution. References: 1. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W., and Prasher, D. (1994) Green Fluorescent protein as a marker for gene expression. Science 263:802-805. 2. Heim, R., Prasher, D. and Tsien, R. (1994) Wavelength mutations and posttranslational autooxidation of green fluorescent protein. Proc. Natl. Acad. Sci. 91, 12501-12504. 3. Heim, R., Cubitt, A., and Tsien, R. (1995) Improved Green Fluorescence. Nature 373, 663-664. 4. Heim, R., and Tsien, R.Y. (1996). Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Current Biology 6:178-182. 5. Matz, MV, Fradkov, AF, Labas, YA, Savitsky, AP, Zaraisky, AG, Markelov, ML, Lukyanov, SA (1999) Fluorescent proteins from nonbioluminescent Anthozoa species. Nat Biotechnol 17:969-973 6. Mello, C. and Fire, A. (1995) "DNA transformation". in Methods in Cell Biology 48: Caenorhabditis elegans, Modern Biological Analysis of an Organism, H.F. Epstein and D.C. Shakes eds. pp. 451-482 7. Krause, M., Fire, A., Harrison, S.W., Priess, J., and Weintraub, H. (1990). CeMyoD accumulation defines the body wall muscle cell fate during C. elegans embryogenesis. Cell 63, 907-919 8. Fire, A., J. Ahnn, G. Seydoux, S. Xu (1994) Vectorology II: Green Fluorescent Protein (or "It Isn't Easy Being Green"). Worm Breeder's Gazette 13(4): 30 (October 1, 1994) 9. Fire, A., Ahnn, J., Kelly, W., Harfe, B., Kostas, S., Hsieh, J., Hsu, M., and Xu, S. (1998) GFP applications in C. elegans. in GFP Strategies and Applications, M. Chalfie and S. Kain eds, John Wiley and Sons, NY. pages 153-168. 10. Fire, A. and S. Xu (1994) Vectorology I: New lacZ Vectors ("Building a Better Gene Trap"). Worm Breeder's Gazette 13(4): 29 (October 1, 1994)