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 [1]), 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 [5]. 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 [6] 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 [7]) 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 [9]. GFP was subsequently shown to require long times for
folding and acquisition of fluorescence [2]. Eventually, the
"low-activity" problems with gfp were solved (as incorporated into the
standard set of C. elegans gfp vectors [9]) using modified gfp versions
that had been selected in bacteria by Heim et al [3] 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.
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