* Institute of Bioorganic Chemistry RAS, Moscow, Russia
Institute of Biochemistry RAS, Moscow, Russia
Shirshov Institute of Oceanology RAS, Moscow, Russia
Whitney laboratory, University of Florida, St. Augustine
|| Laboratory of Bioinformatics and Gene Expression, INTAUniversity of Chile, Santiago, Chile
¶ Harbor Branch Oceanographic Institution, Fort Pierce, Florida
# Department of Molecular Genetics and Microbiology, University of Florida, Gainesville
Correspondence: E-mail address: matz{at}whitney.ufl.edu.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: DsRed chromophore coral color nidogen convergent evolution
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In 1999 GFP homologs were cloned from nonbioluminescent Anthozoa species (Matz et al. 1999). These proteins exhibited an unexpected color diversity that, in addition to green, also included yellow and red fluorescent and purple-blue nonfluorescent colors (Labas et al. 2002). Recent analysis of GFP-like proteins in the great star coral Montastraea cavernosa (Kelmanson and Matz 2003) concluded that proteins of a cyan subtype of the green class have a separate evolutionary history, suggesting that the cyan coloration may serve a specific function. As for the function of fluorescence, the hypothesis originally proposed by Kawaguti (1944), who suggested that fluorescent proteins may be photoprotective, has received the most experimental support thus far with respect to photoprotection of endosymbiotic algae (Salih et al. 2000). Still, the function remains controversial. In many cases the efficacy of intrinsic photoprotection mechanisms of the algae (Gorbunov et al. 2001) surpasses the expected effects offered by fluorescent proteins (Mazel et al. 2003). Moreover, under the photoprotection hypothesis, the reason behind the existing diversity of colors remains elusive. It is possible that photoprotection is one, but not the only, function of fluorescent proteins in Anthozoa. Among possible alternatives are photosynthesis aid (Salih et al. 2000) and photoreception functions, although no evidence for either has yet been found in related studies (Gorbunov and Falkowski 2002; Gilmore et al. 2003). Still another possibility is a straightforward pigment function: the generation of color effects aimed at the outside observer (Ward 2002). Fluorescent GFP-like proteins seem to be quite suitable for such a task (Mazel and Fuchs 2003); however, it is difficult to imagine where the selective advantage of appearing colorful might lie in the case of Cnidaria.
To date the number of GFP-related, Anthozoa-derived sequences in Genbank is about 100a striking contrast to just three sequences that were cloned from class Hydrozoa. We recently described the diversity and phylogenetic relationships among anthozoan GFP-like proteins (Labas et al. 2002), but it was impossible to relate this information to GFPs from other sources or even to root the Anthozoan tree, because of the lack of data from other systematic groups.
Another unexpected finding came in 2001 (Hopf et al. 2001), when it was shown that bilaterian animals also harbor an unmistakable GFP-like domain that comprises part of the so-called G2F fragment ("globular fragment two") of extracellular matrix proteins called nidogens (entactins) and fibulins. This GFP-like domain of the G2F fragment (which we later call G2FP domain) has a clearly homologous fold, but is less than 10% identical to GFPs by amino acid sequence. G2FP domains are found in all bilaterian genomes sequenced thus far and are neither colored nor fluorescent, but instead they serve as protein-binding modules that participate in control of the extracellular matrix formation during development (Willem et al. 2002; Tunggal et al. 2003). Although G2FP domains can be successfully aligned with fluorescent GFP-like proteins by structure tracing methods (see online Supplementary Material), it was previously impossible to determine the phylogenetic relationships between these two major groups with suitable confidence, because of the great sequence divergence between them and the lack of other comparably disparate groups in the dataset.
In the present work we describe four new sequences of GFP-like proteins from Hydrozoa, including the yellow fluorescent and the nonfluorescent purple ones. Six green-fluorescent GFP homologs from planktonic Copepoda of the Pontellidae family (phylum Arthropoda, class Crustacea) are also described. The expansion of the Hydrozoa clade and the addition of an entirely new clade of bilaterian fluorescent proteins into the data set provided an opportunity to resolve deep-level phylogenetic relationships within the superfamily for the first time.
The following are the GenBank accession numbers:
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
cDNA Cloning and Protein Analysis
The organisms were fixed in RNAlater solution (Ambion) and stored at 20°C for 12 months before processing. Total RNA was isolated from single copepod specimens using a NucleoSpin RNA II kit (Clontech). cDNA was synthesized and amplified with a SMART PCR cDNA Synthesis kit (Clontech) and cloned into PCR-Script vector (Stratagene). About 5 x 104 recombinant clones for each library were visually screened using a fluorescent stereomicroscope. See Supplementary Material online for details of protein purification and spectroscopic and biochemical analyses.
Phylogenetic Analysis
The alignment of cDNA coding regions was analyzed using the Bayesian maximum likelihood method implemented in MrBayes 3.0 (Huelsenbeck and Ronquist 2001) and also by nonparametric bootstrap that used maximum likelihood, minimum evolution, and maximum parsimony criteria in PAUP* version beta 4.10 (Swofford 2002). Protein alignment was analyzed under maximum likelihood criterion using TreePuzzle (Schmidt et al. 2002). See online Supplementary Material for the list of accession numbers of the aligned sequences, details of alignment construction, and phylogenetic methods.
![]() |
Results and Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Chromoprotein anm2CP
The anm2CP absorption spectrum was very similar to that of Anthozoa chromoproteins (Gurskaya et al. 2001a). It was suggested (Chudakov et al. 2003) and recently confirmed during analysis of the first crystal structure of the blue chromoprotein Rtms5 from the coral Montipora efflorescens (Prescott et al. 2003) that the chromophore in chromoproteins may be a DsRed-type in a nonplanar trans conformation. Another chromophore was reported in chromoprotein asulCP from the sea anemone Anemonia sulcata (Martynov et al. 2001). Although the exact structure still awaits confirmation by crystallography, distinction from the DsRed-type chromophore is evident when absorption spectra of denatured asulCP and DsRed are compared (Gross et al. 2000; Martynov et al. 2001) (see fig. 1HK). In contrast, spectral properties of anm2CP in a denatured state clearly indicated that it possesses a DsRed-type chromophore (fig. 1HK), which then must be isomerized similarly to Rtms5.
Earlier we showed that amino acids at positions 148, 165, and 203 are the key determinants of spectral differences between fluorescent proteins and chromoproteins (Lukyanov et al. 2000; Gurskaya et al. 2001a; Bulina et al. 2002; Chudakov et al. 2003). In anm2CP, positions 148, 165, and 203 are occupied by Glu, Cys, and Val, respectively. Although such a combination was never observed in GFP-like proteins, it is more similar to a chromoprotein-like than to a fluorescent protein-like arrangement. One can speculate that bulky Glu148 and Val203 should cause steric hindrance of the chromophore cis conformation while small Cys165 should favor its trans conformation.
Copepoda GFPs
We identified six closely related (more than 60% identity) GFP-like proteins in copepods: two from Pontellina plumata, one from Labidocera aestiva, two from cf. Pontella meadi, and one from an unidentified species. All proteins demonstrated green fluorescence and possessed similar, but not identical, spectra (fig. 1E). Copepoda GFPs represent the most remote clade of fluorescent GFP-like proteins (see below for more discussion on phylogeny); however, the chromophore-forming Tyr66 and Gly67, as well as Arg96 and Glu222 that are presumably involved in autocatalysis, are conserved. A characteristic feature of Copepoda GFP is the absence of tryptophane residues (only laesGFP contains Trp179), which explains their weak excitation at 280 nm.
Absorption spectra of the acid- and alkali-denatured ppluGFP2 exactly matched the denatured GFP spectra (not shown). pH titration of the ppluGFP2 solution in the acidic region (pH 5.0 and below) demonstrated gradual transformation of the 482 nm absorption peak into the peak at 376 nm with an isosbestic point at 415 nm, a behavior characteristic of GFP and many of its mutants. Therefore, ppluGFP2 most likely possesses an imidazolidinone chromophore identical to that of A. victoria GFP in the deprotonated state.
Possible Functions of Novel GFP-like Proteins
GFPs of anthomedusae may be linked to bioluminescence, similarly to GFP from Aequorea victoria. However, none of our subjects was obviously luminous and the localization of fluorescence in these medusaeextendable parts, such as tentacles and/or manubriumdid not resemble known luminosity patterns, which are confined to the bell (Herring 1978). It is possible that fluorescence in these medusae represents a daylight functional analog of bioluminescence (similar to "blanching" in comb jellies [Mackie 1995]). It may be speculated that upon mechanical stimulation medusa would retract its previously extended tentacles and/or manubrium, thereby rapidly concentrating the fluorescent substance in a small area, which might produce a flash-like visual effect. The function of the yellow fluorescent protein, phiYFP, from Phiallidium is more difficult to explain. Although the protein seems to colocalize with luminous regions, it is unlikely to be involved in bioluminescence, because the emission color of this medusa is green (Herring 1978), suggesting the involvement of GFP (that apparently escaped our cloning efforts) rather than YFP.
In the case of Copepoda it would also be tempting to suggest that their GFPs are involved in bioluminescence, because many copepods possess this capability, if not for the fact that no luminous species are known from the Pontellidae family (Herring 1988). Moreover, the fluorescent areas in our specimens are not associated with any visible glands and are certainly quite unlike the luminous gland regions in other copepods (Herring 1988). The Pontellidae family includes many species that are colored blue or purplish by carotenoproteins. Among our specimens, Labidocera aestiva exhibited this kind of blue coloration, which did not show any obvious spatial correlation to the fluorescent regions. It is possible that this blue as well as green fluorescent coloration may serve as counter-shading (blue for open ocean, green fluorescence for near shore waters) or as disruptive coloration directed at near-surface predators that use color vision. It can also be reasoned, given the notable dissimilarity of fluorescent patterns in analyzed species, that these patterns may be used by copepods themselves to recognize conspecific individuals.
The role of nonfluorescent proteins in general is the most mysterious. In Anthozoa they are usually confined to extremities of the organism, such as branch or tentacle tips, and are not correlated to any of the tested physiology parameters (Takabayashi and Hoegh-Guldberg 1995). Finding of such a protein in a hydromedusa only adds more intrigue to the story. Among other possibilities, the protein may serve a shadowing role for the jellyfish's photoreceptors, which would enable the animal to sense the direction of light. Although this function in most animals is served by melanins, there are variations, including a peculiar case of hemoglobin recruited for this role (Burr et al. 2000). The nonfluorescent protein from Hydrozoa represents a third case of independent evolution of a nonfluorescent color (see discussion below).
Evolution of the Superfamily
GFP Homologs Comprise a Superfamily
The group of structural homologs of GFP that all share the GFP-like "beta-can" fold should be regarded as a superfamily following the criteria proposed by the Protein Information Resource (http://pir.georgetown.edu/pirwww/otherinfo/sfdef.html). The main reason for such a classification is that this group unites at least two clearly definable protein families. The first one consists of G2FP domains, which are incapable of autocatalytic chromophore synthesis and are found within multi-domain proteins of the extracellular matrix. The second one includes fluorescent and/or colored proteins capable of synthesizing the chromophore autocatalytically and which are not found in a multi-domain context. We compiled an HMM profile that recognizes all of the currently known members of the superfamily (see online Supplementary Material) and has the potential to identify novel ones.
Deep-level Phylogeny
The evolutionary route of the GFP superfamily could be imagined as two separate lineages that diverged by descent, whereby one, in Cnidaria, gained fluorescent properties, while another, in Bilateria, specialized in protein binding and became part of extracellular matrix proteins. It is also possible, however, that these two lineages originated as a result of an ancient gene duplication preceding the separation of Cnidaria and Bilateria, so that both G2FPs and fluorescent GFPs could be found within a single genome, both in Cnidaria and Bilateria. The position of Copepoda fluorescent proteins within the tree helps to discriminate between these two possibilities. Under the first scenario ("G2FPs for Bilateria, GFPs for Cnidaria"), Copepoda proteins can either be G2FPs that evolved fluorescence independently of GFPs, or products of horizontal gene transfer from one of the Cnidarian lineages. Under the second scenario ("ancient duplication"), Copepoda should be seen as an outgroup with respect to Cnidaria, but still much more related to them than to bilaterian G2FPs. Results of phylogenetic analysis (fig. 2A) clearly confirm the latter variant. First, there is very strong statistical support for monophyly of the clade uniting deuterostome and protostome G2FPs (P < 0.0001 in AU, KH, and SH tests; Kishino and Hasegawa 1989; Shimodaira and Hasegawa 1999; Shimodaira 2002), which indicates that Copepoda GFPs were already separate at the time of the common ancestor of deuterostomes and protostomes. Second, Copepoda GFPs did not arise within any of the known Cnidaria lineages either, ruling out the possibility of horizontal gene transfer from there. Third, Copepoda sequences are significantly more similar to cnidarian GFPs than to G2FPs; the average maximum likelihood distance between Copepoda and Cnidaria cDNA sequences is 2.2 replacements/nucleotide, which is about four times less than the average distance between Copepoda GFPs and bilaterian G2FPs (8.7 replacements/nucleotide).
Accepting the "ancient duplication" scenario implies that the common ancestor of Cnidaria and Bilateria had both G2FP and GFP proteins. Moreover, by that time the GFP lineage may have already evolved fluorescence; Copepoda GFPs follow the same structural solution to fluorescence as Cnidaria proteins, which is most parsimoniously explained by the appearance of the solution only once before Cnidaria and Bilateria separated 700900 MYA (Benton and Ayala 2003). If the green fluorescence appeared so early, it must not have been related to bioluminescence or coloration with the purpose of producing visual effects, because the visual systems were (most probably) not yet developed to the necessary extent (Knoll and Carroll 1999; Erwin and Davidson 2002). Notably, this early origination of fluorescence implies that proteins that are members of the fluorescent GFP lineage are likely to be found in virtually any animal.
The only remaining alternative explanation of the presence of fluorescent GFPs in copepods is horizontal gene transfer from some yet unknown non-cnidarian source. Although current data do not warrant such an assumption, it is interesting to note that horizontal gene transfer may indeed have contributed to the distribution of GFP-like proteins, at least among Anthozoa; it seems to be the only possible way to explain the striking sequence similarity of some 60 recently described chromoproteins from Indo-Pacific corals and corallimorphs (Karan et al. 2002).
Color Evolution in Anthozoa
Several phylogeny reconstruction methodsincluding parsimony, neighbor-joining, and Bayesian maximum likelihood inferencesuggested a position of the root for Anthozoa proteins within the branch leading to the pennatulacean GFPs, although support for this topology is not very high, except in the neighbor-joining bootstrap (fig. 2B). We tend to accept this topology as the best guess, especially because it is in agreement with the phylogeny of Anthozoa obtained using some other methods (Won, Rho, and Song 2001). The four major gene lineages of Zoantharia proteins identified previously, clades A, B, C, and D (Labas et al. 2002), were also observed in the new analysis. However, the deep relationships between these lineages remain poorly resolved (fig. 2B).
Color diversity originated independently within different lineages. We inferred the color origination events from the phylogenetic tree topology following the maximum parsimony principle. Green was assumed to be the basal ("background") color because of two considerations: First, green is the only color known in the Copepoda outgrop. Second, according to the current state of knowledge, green proteins have the simplest chromophore structure and represent a kind of "default" state; proteins of all other colors (including the cyan subtype) tend to become green as a result of random mutagenesis (Baird, Zacharias, and Tsien 2000; Terskikh et al. 2000; Wiehler, von Hummel, and Steipe 2001; K. Lukyanov, unpublished data).
From the current data a total of 15 color diversification events can be inferred (fig. 3). Of these, there are six independent events leading to the appearance of red color, four of cyan color, three of nonfluorescent color, and two of yellow color. In most cases the parsimony principle dictates that a nongreen color evolved from green along a terminal branch of the tree. This fact may indicate the recent origin of color diversity. However, we believe that in this particular case such a pattern is mostly the result of biased sampling of taxa; to date, sampled species from each of the major clades were rarely related more closely than at the order level. Still, there are few cases when deep nongreen nodes can be inferred. Thus, within clade A, the node joining the group of chromoproteins and the red fluorescent protein equaRFP is likely to be either a chromoprotein or a red fluorescent protein (fig. 3) rather than green, because such a scenario would require only one unlikely conversion from a green to nongreen color (Labas et al. 2002) instead of two. Within clade D, as an alternative to the scenario depicted on figure 3, it is equally parsimonious to assume three ancestral nodes along the stem of the clade to be cyan instead of green, which would lead to the inference of some "backward" cyan to green color conversions in their descendants. Theoretically, an origination of green from nongreen is possible and can even be anticipated (Labas et al. 2002), because a nongreen protein may become green simply as a result of the accumulation of random mutations, requiring no color-related selection pressure to achieve the color change.
|
It would be very interesting to study the pathways of color diversity evolution by means of the reconstruction and recreation of ancestral proteins, which correspond to the nodes within the tree (Chang and Donoghue 2000). It is tempting to speculate that independent origination of color diversity of the reef Anthozoa was the result of some environmental and/or ecological factors that were experienced by all these organisms. Therefore, there is an exciting possibility that identifying such factors would provide a key to the biological significance of the colorfulness of coral reefs in general.
Nongreen Proteins: Convergent Molecular Evolution of Complexity
Red fluorescent proteins perform three consecutive autocatalytic reactions during chromophore synthesis, in contrast to just the first two required for cyans and greens, resulting in an extended chromophore structure (Gross et al. 2000; Wall, Socolich, and Ranganathan 2000; Yarbrough et al. 2001) and therefore representing a higher-complexity level of organization. The fact that red fluorescence evolved from green on several independent occasions must be considered an example of convergent evolution of complexity at the molecular level. Different structural solutions evolved in different clades, substantiating a case for true convergent evolution rather than parallelism. In clade B, the red-emitters DsRed and dis2RFP possess wide and skewed emission spectra and show barely detectable green fluorescence during maturation. Zoan2RFP from clade C and equaRFP from clade A show emission spectra similar to DsRed, but they proceed through a brightly fluorescent green stage during maturation ("fluorescent timer" phenotype). Finally, red-emitters from clade D (tgeoRFP, mcavRFP1, rfloRFP and dendRFP [the latter was originally described as green by Labas et al. 2002]) have a peculiar narrow emission spectrum, exhibit "timer" phenotype, and require long-wave UV-A light to complete maturation (Ando et al. 2002; M. Matz, unpublished data). It has recently been demonstrated that the chromophore of one of their representativestgeoRFP, or Kaedeis indeed structurally different from the one in DsRed (Mizuno et al. 2003). Thus far, there are only a few documented cases of convergent molecular evolution (Zakon 2002), and to our knowledge, none of these cases can be claimed as convergent evolution of complexity. We believe that red-emitting proteins can become an excellent model for studying basic principles of the evolution of complexity at the molecular level, especially since the complex functional feature in question (red color of fluorescence) is so easily tractable in mutagenesis studies.
Yellow fluorescent proteins, of which only two are known at present, clearly represent another case of convergent evolution, because they attained the yellow fluorescent feature by different structural means: zoanYFP apparently has unique chromophore structure, whereas phiYFP relies on modification of the molecular environment of a green-type chromophore. Chromoproteins, which have three independent origins, may represent another case of functional convergence, because different chromophore structures have been reported in different lineages (Martynov et al. 2001; Prescott et al. 2003). Another indication of convergent evolution of chromoproteins is the fact that the Hydrozoa protein anm2CP has a unique arrangement of the spectroscopically relevant residues. In wild-type cyan fluorescent proteins (in contrast to the genetically engineered cyan variant of GFP from Aequorea victoria), the chromophore is the same as in greens, so the phenotype is dependent on the chromophore's molecular environment (Gurskaya et al. 2001b). This fact makes it difficult at present to evaluate whether the structural features that are responsible for cyan color are the same or different in independent cyan lineages and, therefore, whether these lineages represent cases of parallel or convergent evolution of the cyan color.
Implications for Biotechnology
The fact that different organisms yield GFP-like proteins that may be of the same color, but based upon different structural principles, makes the study of natural GFP-like proteins of great value for biomedical imaging technology. First, there is always hope that a new natural protein would possess some very useful feature not seen before in other proteins of the same color, such as the natural timer phenotype (Labas et al. 2002) or photoactivation (Ando et al. 2002) in red fluorescent proteins originating from lineages other than the first-found DsRed (Matz et al. 1999). The second advantage lies in creating a basis for future protein engineering. Sophisticated in vivo imaging applications relying on GFP and its homologs often demand specific properties from a reporter protein of a certain color, which are not inherent to the original protein and need to be engineered, such as sensitivity to external factors, absence of oligomerization, or specific photochemical behavior. Proteins demonstrating different structural solutions to achieving the same color may become independent starting points in such engineering efforts, which would significantly raise the chances of success.
Conclusions
The gene tree of the GFP superfamily consists of two major lineages, one uniting all fluorescent proteins and another leading to colorless GFP-like protein-binding domains of the nidogens and related Bilaterian proteins of the extracellular matrix. Representatives of the fluorescent lineage were found not only in Cnidaria, but also in Bilateria (Arthropoda, Crustacea, Copepoda), suggesting that fluorescent GFP-like proteins evolved before separation of Bilateria and Cnidaria and therefore may be more widely utilized as pigments in the animal world than previously thought. In Cnidaria, descendants of the fluorescent lineage formed a multigene family of paralogs very early in evolution, preceding the separation of Anthozoa subclasses. These paralogous lineages underwent extensive duplications and losses throughout the evolution of Anthozoa, leading to intricate sorting of the gene lineages among extant taxonomic groups. Color diversity and the corresponding complexity of the protein organization evolved independently in several Anthozoa lineages, as well as in Hydrozoa. Apparently, the first color to separate from the ancestral green was non-fluorescent purple-blue, followed by cyans, reds and yellows. Apart from being a key to the origins of the color diversity of contemporary coral reefs, phylogeny of fluorescent GFP-like proteins may represent an excellent model for studying such theoretical issues of molecular evolution such as the origin of complex features, roles of natural selection in evolution of paralogous gene families and evolution of functions in gene families addressed by means of recreating ancestral genes.
![]() |
Supplementary Material |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ando, R., H. Hama, M. Yamamoto-Hino, H. Mizuno, and A. Miyawaki. 2002. An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proc. Natl. Acad. Sci. USA 99:12651-12656.
Baird, G. S., D. A. Zacharias, and R. Y. Tsien. 2000. Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc. Natl. Acad. Sci. USA 97:11984-11989.
Benton, M. J., and F. J. Ayala. 2003. Dating the tree of life. Science 300:1698-1700.
Bulina, M. E., D. M. Chudakov, N. N. Mudrik, and K. A. Lukyanov. 2002. Interconversion of Anthozoa GFP-like fluorescent and non-fluorescent proteins by mutagenesis. BMC Biochem 3:7.[CrossRef][Medline]
Burr, A. H. J., P. Hunt, D. R. Wagar, S. Dewilde, M. L. Blaxter, J. R. Vanfleteren, and L. Moens. 2000. A hemoglobin with an optical function. J. Biol. Chem. 275:4810-4815.
Chalfie, M. 1995. Green fluorescent protein. Photochem. Photobiol. 62:651-656.[ISI][Medline]
Chang, B. S. W., and M. J. Donoghue. 2000. Recreating ancestral proteins. Trends Ecol. Evol. 15:109-114.[CrossRef][ISI][Medline]
Chudakov, D. M., V. V. Belousov, A. G. Zaraisky, V. V. Novoselov, D. B. Staroverov, D. B. Zorov, S. Lukyanov, and K. A. Lukyanov. 2003. Kindling fluorescent proteins for precise in vivo photolabeling. Nat. Biotechnol. 21:452-452.
Cody, C. W., D. C. Prasher, W. M. Westler, F. G. Prendergast, and W. W. Ward. 1993. Chemical structure of the hexapeptide chromophore of the Aequorea green-fluorescent protein. Biochemistry 32:1212-1218.[ISI][Medline]
Cormack, B. P., R. H. Valdivia, and S. Falkow. 1996. FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173:33-38.[CrossRef][ISI][Medline]
Eddy, S. R. 1998. Profile hidden Markov models. Bioinformatics 14:755-763.[Abstract]
Erwin, D. H., and E. H. Davidson. 2002. The last common bilaterian ancestor. Development 129:3021-3032.
Gilmore, A. M., A. Larkum, A. Salih, S. Itoh, Y. Shibata, C. Bena, H. Yamasaki, M. Papina, and R. Van Woesik. 2003. Simultaneous time resolution of the emission spectra of fluorescent proteins and Zooxanthellar chlorophyll in reef-building corals. Photochem. Photobiol. 77:515-523.[CrossRef][ISI][Medline]
Gorbunov, M. Y., and P. G. Falkowski. 2002. Photoreceptors in the cnidarian hosts allow symbiotic corals to sense blue moonlight. Limnol. Oceanogr. 47:309-315.[ISI]
Gorbunov, M. Y., Z. S. Kolber, M. P. Lesser, and P. G. Falkowski. 2001. Photosynthesis and photoprotection in symbiotic corals. Limnol. Oceanogr. 46:75-85.[ISI]
Gorokhovatsky, A. Y., N. V. Rudenko, V. V. Marchenkov, V. S. Skosyrev, M. A. Arzhanov, N. Burkhardt, M. V. Zakharov, G. V. Semisotnov, L. M. Vinokurov, and Y. B. Alakhov. 2003. Homogeneous assay for biotin based on Aequorea victoria bioluminescence resonance energy transfer system. Anal. Bioch. 313:68-75.[CrossRef][ISI][Medline]
Gross, L. A., G. S. Baird, R. C. Hoffman, K. K. Baldridge, and R. Y. Tsien. 2000. The structure of the chromophore within DsRed, a red fluorescent protein from coral. Proc. Natl. Acad. Sci. USA 97:11990-11995.
Gurskaya, N. G., A. F. Fradkov, N. I. Pounkova, D. B. Staroverov, M. E. Bulina, Y. G. Yanushevich, Y. A. Labas, S. Lukyanov, and K. A. Lukyanov. 2003. Colourless green fluorescent protein homologue from the non-fluorescent hydromedusa Aequorea coerulescens and its fluorescent mutants. Biochem. J. 373:403-408.[CrossRef][ISI][Medline]
Gurskaya, N. G., A. F. Fradkov, A. Terskikh, M. V. Matz, Y. A. Labas, V. I. Martynov, Y. G. Yanushevich, K. A. Lukyanov, and S. A. Lukyanov. 2001a. GFP-like chromoproteins as a source of far-red fluorescent proteins. FEBS Lett. 507:16-20.[CrossRef][ISI][Medline]
Gurskaya, N. G., A. P. Savitsky, Y. G. Yanushevich, S. A. Lukyanov, and K. A. Lukyanov. 2001b. Color transitions in coral's fluorescent proteins by site-directed mutagenesis. BMC Biochem. 2:6.[CrossRef][Medline]
Herring, P. J. 1978. Bioluminescence of invertebrates other than insects. Pp. 199240 in P. J. Herring ed. Bioluminescence in action. Academic Press, London.
Herring, P. J. 1988. Copepod luminescence. Hydrobiologia 167/168:183-195.
Hopf, M., W. Gohring, A. Ries, R. Timpl, and E. Hohenester. 2001. Crystal structure and mutational analysis of a perlecan-binding fragment of nidogen-1. Nat. Struct. Biol. 8:634-640.[CrossRef][ISI][Medline]
Huelsenbeck, J. P., and F. Ronquist. 2001. MrBayes: Bayesian inference of phylogenetic trees. Bioinformatics 17:754-755.
Johnson, F. H., L. C. Gershman, J. R. Waters, G. T. Reynolds, Y. Saiga, and O. Shimomura. 1962. Quantum efficiency of Cypridina luminescence, with a note that of Aequorea. J. Cell. Comp. Physiol. 60:85-104.[ISI]
Karan, M., F. Brugliera, J. Mason, E. L. Jones, S. G. Dove, O. Hoegh-Guldberg, and M. Prescott. 2002. Cell visual characteristic-modifying sequences. In patent: WO 02070703-A 179. Nufarm Australia Limited, The University of Queensland.
Kawaguti, S. 1944. On the physiology of reef corals. VI. Study of the pigments. Palao. Trop. Biol. Stn. Stud. 2:617-674.
Kelmanson, I., and M. Matz. 2003. Molecular basis and evolutionary origins of color diversity in great star coral Montastraea cavernosa (Scleractinia: Faviida). Mol. Biol. Evol. 20:1125-1133.
Kishino, H., and M. Hasegawa. 1989. Evaluation of the maximum likelihood estimate of the evolutionary tree topologies from DNA-sequence data, and the branching order in Hominoidea. J. Mol. Evol. 29:170-179.[ISI][Medline]
Knoll, A. H., and S. B. Carroll. 1999. Early animal evolution: emerging views from comparative biology and geology. Science 284:2129-2137.
Labas, Y. A., N. G. Gurskaya, Y. G. Yanushevich, A. F. Fradkov, K. A. Lukyanov, S. A. Lukyanov, and M. V. Matz. 2002. Diversity and evolution of the green fluorescent protein family. Proc. Natl. Acad. Sci. USA 99:4256-4261.
Lippincott-Schwartz, J., and G. H. Patterson. 2003. Development and use of fluorescent protein markers in living cells. Science 300:87-91.
Lukyanov, K. A., A. F. Fradkov, and N. G. Gurskaya, et al. (12 co-authors). 2000. Natural animal coloration can be determined by a nonfluorescent green fluorescent protein homolog. J. Biol. Chem. 275:25879-25882.
Mackie, G. O. 1995. Defensive strategies in planktonic coelenterates. Mar. Fresh Behav. Physiol. 26:119-129.[ISI]
Martynov, V. I., A. P. Savitsky, N. Y. Martynova, P. A. Savitsky, K. A. Lukyanov, and S. A. Lukyanov. 2001. Alternative cyclization in GFP-like proteins family. The formation and structure of the chromophore of a purple chromoprotein from Anemonia sulcata. J. Biol. Chem. 276:21012-21016.
Matz, M. V., A. F. Fradkov, Y. A. Labas, A. P. Savitsky, A. G. Zaraisky, M. L. Markelov, and S. A. Lukyanov. 1999. Fluorescent proteins from nonbioluminescent Anthozoa species. Nat. Biotechnol. 17:969-973.[CrossRef][ISI][Medline]
Mazel, C. H., and E. Fuchs. 2003. Contribution of fluorescence to the spectral signature and perceived color of corals. Limnol. Oceanogr. 48:390-401.[ISI]
Mazel, C. H., M. P. Lesser, M. Y. Gorbunov, T. M. Barry, J. H. Farrell, K. D. Wyman, and P. G. Falkowski. 2003. Green-fluorescent proteins in Caribbean corals. Limnol. Oceanogr. 48:402-411.[ISI]
Mizuno, H., T. K. Mal, K. I. Tong, R. Ando, T. Furuta, M. Ikura, and A. Miyawaki. 2003. Photo-induced peptide cleavage in the green-to-red conversion of a fluorescent protein. Mol. Cell 12:1051-1058.[ISI][Medline]
Ormo, M., A. B. Cubitt, K. Kallio, L. A. Gross, R. Y. Tsien, and S. J. Remington. 1996. Crystal structure of the Aequorea victoria green fluorescent protein. Science 273:1392-1395.[Abstract]
Partridge, J. C., and M. E. Cummings. 1999. Adaptation of visual pigments to the aquatic environment. Pp. 251283 in S. N. Archer et al., eds. Adaptive mechanisms in the ecology of vision. Kluwer Academic Publishers, Dordrecht, The Netherlands.
Prasher, D. C., V. K. Eckenrode, W. W. Ward, F. G. Prendergast, and M. J. Cormier. 1992. Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111:229-233.[CrossRef][ISI][Medline]
Prescott, M., M. Ling, T. Beddoe, A. J. Oakley, S. Dove, O. Hoegh-Guldberg, R. J. Devenish, and J. Rossjohn. 2003. The 2.2 A crystal structure of a pocilloporin pigment reveals a nonplanar chromophore conformation. Structure 11:275-284.[CrossRef][ISI][Medline]
Salih, A., A. Larkum, G. Cox, M. Kuhl, and O. Hoegh-Guldberg. 2000. Fluorescent pigments in corals are photoprotective. Nature 408:850-853.[CrossRef][ISI][Medline]
Schmidt, H. A., K. Strimmer, M. Vingron, and A. von Haeseler. 2002. Tree-Puzzle: maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics 18:502-504.
Shimodaira, H. 2002. An approximately unbiased test of phylogenetic tree selection. Syst. Biol. 51:492-508.[CrossRef][ISI][Medline]
Shimodaira, H., and M. Hasegawa. 1999. Multiple comparisons of log-likelihoods with applications to phylogenetic inference. Mol. Biol. Evol. 16:1114-1116.
Shimomura, O., F. H. Johnson, and Y. Saiga. 1962. Extraction, purification, and properties of Aequorin, a bioluminescent protein from luminous Hydromedusan, Aequorea. J. Cell Comp. Physiol. 59:223-239.[ISI]
Swofford, D. L. 2002. PAUP. Phylogenetic Analysis Using Parismony (* and Other Methods). Version 4. Sinauer Associates, Sunderland, Massachusetts.
Szent-Gyorgyi, C. S., B. J. Bryan, and W. Szczepaniak. 2001. Patent US 6232107-B.
Takabayashi, M., and O. Hoegh-Guldberg. 1995. Ecological and physiological differences between two color morphs of the coral Pocillopora-Damicornis. Marine Biol. 123:705-714.[ISI]
Terskikh, A., A. Fradkov, and G. Ermakova, et al. (12 co-authors). 2000. "Fluorescent timer": protein that changes color with time. Science 290:1585-1588.
Tsien, R. Y. 1998. The green fluorescent protein. Annu. Rev. Biochem. 67:509-544.[CrossRef][ISI][Medline]
Tunggal, J., M. Wartenberg, M. Paulsson, and N. Smyth. 2003. Expression of the nidogen-binding site of the laminin gamma 1 chain disturbs basement membrane formation and maintenance in F9 embryoid bodies. J. Cell Sci. 116:803-812.
Wall, M. A., M. Socolich, and R. Ranganathan. 2000. The structural basis for red fluorescence in the tetrameric GFP homolog DsRed. Nat. Struct. Biol. 7:1133-1138.[CrossRef][ISI][Medline]
Ward, W. W. 2002. Fluorescent proteins: who's got 'em and why? Pp. 123126 in P. E. Stanley and L. J. Kricka, eds. Bioluminescence and chemiluminescence. World Scientific, Cambridge, UK.
Ward, W. W., and M. J. Cormier. 1978. Energy-transfer via protein-protein interaction in Renilla bioluminescence. Photochem. Photobiol. 27:389-396.[ISI]
Wiehler, J., J. von Hummel, and B. Steipe. 2001. Mutants of Discosoma red fluorescent protein with a GFP-like chromophore. FEBS Lett. 487:384-389.[CrossRef][ISI][Medline]
Willem, M., N. Miosge, W. Halfter, N. Smyth, I. Jannetti, E. Burghart, R. Timpl, and U. Mayer. 2002. Specific ablation of the nidogen-binding site in the laminin gamma 1 chain interferes with kidney and lung development. Development 129:2711-2722.
Won, J. H., B. J. Rho, and J. I. Song. 2001. A phylogenetic study of the Anthozoa (phylum Cnidaria) based on morphological and molecular characters. Coral Reefs 20:39-50.[CrossRef][ISI]
Xia, N. S., W. X. Luo, J. Zhang, X. Y. Xie, H. J. Yang, S. W. Li, M. Chen, and M. H. Ng. 2002. Bioluminescence of Aequorea macrodactyla, a common jellyfish species in the East China Sea. Marine Biotechnol. 4:155-162.[CrossRef][ISI]
Yang, T. T., P. Sinai, G. Green, P. A. Kitts, Y. T. Chen, L. Lybarger, R. Chervenak, G. H. Patterson, D. W. Piston, and S. R. Kain. 1998. Improved fluorescence and dual color detection with enhanced blue and green variants of the green fluorescent protein. J. Biol. Chem. 273:8212-8216.
Yarbrough, D., R. M. Wachter, K. Kallio, M. V. Matz, and S. J. Remington. 2001. Refined crystal structure of DsRed, a red fluorescent protein from coral, at 2.0-A resolution. Proc. Natl. Acad. Sci. USA 98:462-467.
Zakon, H. H. 2002. Convergent evolution on the molecular level. Brain Behav. Evol. 59:250-261.[CrossRef][ISI][Medline]