Color transitions in coral's fluorescent proteins by site-directed mutagenesis.
BACKGROUND: Green Fluorescent Protein (GFP) cloned from jellyfish Aequorea victoria and its homologs from corals Anthozoa have a great practical significance as in vivo markers of gene expression. Also, they are an interesting puzzle of protein science due to an unusual mechanism of chromophore formation and diversity of fluorescent colors. Fluorescent proteins can be subdivided into cyan (approximately 485 nm), green (approximately 505 nm), yellow (approximately 540 nm), and red (>580 nm) emitters. RESULTS: Here we applied site-directed mutagenesis in order to investigate the structural background of color variety and possibility of shifting between different types of fluorescence. First, a blue-shifted mutant of cyan amFP486 was generated. Second, it was established that cyan and green emitters can be modified so as to produce an intermediate spectrum of fluorescence. Third, the relationship between green and yellow fluorescence was inspected on closely homologous green zFP506 and yellow zFP538 proteins. The following transitions of colors were performed: yellow to green; yellow to dual color (green and yellow); and green to yellow. Fourth, we generated a mutant of cyan emitter dsFP483 that demonstrated dual color (cyan and red) fluorescence. CONCLUSIONS: Several amino acid substitutions were found to strongly affect fluorescence maxima. Some positions primarily found by sequence comparison were proved to be crucial for fluorescence of particular color. These results are the first step towards predicting the color of natural GFP-like proteins corresponding to newly identified cDNAs from corals. (+info)
Interconversion of Anthozoa GFP-like fluorescent and non-fluorescent proteins by mutagenesis.
BACKGROUND: Within the family of green fluorescent protein (GFP) homologs, one can mark two main groups, specifically, fluorescent proteins (FPs) and non-fluorescent or chromoproteins (CPs). Structural background of differences between FPs and CPs are poorly understood to date. RESULTS: Here, we applied site-directed and random mutagenesis in order to to transform CP into FP and vice versa. A purple chromoprotein asCP (asFP595) from Anemonia sulcata and a red fluorescent protein DsRed from Discosoma sp. were selected as representatives of CPs and FPs, respectively. For asCP, some substitutions at positions 148 and 165 (numbering in accordance to GFP) were found to dramatically increase quantum yield of red fluorescence. For DsRed, substitutions at positions 148, 165, 167, and 203 significantly decreased fluorescence intensity, so that the spectral characteristics of these mutants became more close to those of CPs. Finally, a practically non-fluorescent mutant DsRed-NF was generated. This mutant carried four amino acid substitutions, specifically, S148C, I165N, K167M, and S203A. DsRed-NF possessed a high extinction coefficient and an extremely low quantum yield (< 0.001). These spectral characteristics allow one to regard DsRed-NF as a true chromoprotein. CONCLUSIONS: We located a novel point in asCP sequence (position 165) mutations at which can result in red fluorescence appearance. Probably, this finding could be applied onto other CPs to generate red and far-red fluorescent mutants. A possibility to transform an FP into CP was demonstrated. Key role of residues adjacent to chromophore's phenolic ring in fluorescent/non-fluorescent states determination was revealed. (+info)
Additional cytotoxic diacetylenes from the stony coral Montipora sp.
Three new diacetylenes (1, 4, 6) have been isolated as cytotoxic constituents from the methanolic extract of the stony coral Montipora sp. The structures have been elucidated on the basis of spectroscopic evidence. The compounds were evaluated for cytotoxicity against a small panel of human tumor cell lines and showed moderate to significant activity. (+info)
Sympatric populations of the highly cross-fertile coral species Acropora hyacinthus and Acropora cytherea are genetically distinct.
High cross-fertilization rates in vitro and non-monophyletic patterns in molecular phylogenies challenge the taxonomic status of species in the coral genus Acropora. We present data from eight polymorphic allozyme loci that indicate small, but significant, differentiation between sympatric populations of Acropora cytherea and Acropora hyacinthus (F(ST) = 0.025-0.068, p < 0.05), a pair of acroporid corals with very high interspecific fertilization rates in vitro. Although no fixed allelic differences were found between these species, the absence of genetic differentiation between widely allopatric populations suggests that allele frequency differences between A. cytherea and A. hyacinthus in sympatry are biologically significant. By contrast, populations of Acropora tenuis, a species which spawns 2-3 hours earlier and shows low cross-fertilization rates with congeners in vitro, were clearly distinct from A. cytherea and A. hyacinthus (F(ST) = 0.427-0.465, p < 0.05). Moreover, allopatric populations of A. tenuis differed significantly, possibly as a consequence of its relatively short period of larval competency. Our results effectively rule out the possibility that A. hyacinthus and A. cytherea are morphotypes within a single species, and indicate that hybridization occurs relatively infrequently between these taxa in nature. (+info)
Effect of increased calcium concentration in sea water on calcification and photosynthesis in the scleractinian coral Galaxea fascicularis.
The relationship between calcification and photosynthesis in coral was investigated using standard sea water with enhanced calcium concentration. In standard sea water at 23 degrees C with the calcium concentration increased by 2.5 mmol l(-1), incorporation of calcium into the skeleton increased by 30-61 %, depending on the method of data normalisation, and photosynthesis, measured as (14)C incorporation into the tissues, also increased by 87 %. At 29 degrees C, calcium incorporation into the skeleton increased by 54-84 % and (14)C incorporation increased by 32 % when sea water calcium concentration was increased by 5 mmol l(-1). However, photosynthesis measured as net photosynthetic oxygen production did not increase. Similarly there was no change in respiration rate when coral polyps were incubated in high-calcium sea water. It is conjectured that an increase in photorespiration may be responsible for the latter observations. Bisphosphonate has been considered to inhibit calcification but not photosynthesis in corals. We show that bisphosphonate may not inhibit formation of amorphous calcium carbonate and that the inhibition of calcification is possibly illusory. The data are consistent with the trans-calcification model, which suggests that calcification is a source of CO(2) for photosynthesis in corals. (+info)
Coral development: from classical embryology to molecular control.
The phylum Cnidaria is the closest outgroup to the triploblastic metazoans and as such offers unique insights into evolutionary questions at several levels. In the post-genomic era, a knowledge of the gene complement of representative cnidarians will be important for understanding the relationship between the expansion of gene families and the evolution of morphological complexity among more highly evolved metazoans. Studies of cnidarian development and its molecular control will provide information about the origins of the major bilaterian body axes, the origin of the third tissue layer, the mesoderm, and the evolution of nervous system patterning. We are studying the cnidarian Acropora millepora, a reef building scleractinian coral, and a member of the basal cnidarian class, the Anthozoa. We review ourwork on descriptive embryology and studies of selected transcription factor gene families, where our knowledge from Acropora is particularly advanced relative to other cnidarians. We also describe a recent preliminary whole genome initiative, a coral EST database. (+info)
An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein.
We have cloned a gene encoding a fluorescent protein from a stony coral, Trachyphyllia geoffroyi, which emits green, yellow, and red light. The protein, named Kaede, includes a tripeptide, His-Tyr-Gly, that acts as a green chromophore that can be converted to red. The red fluorescence is comparable in intensity to the green and is stable under usual aerobic conditions. We found that the green-red conversion is highly sensitive to irradiation with UV or violet light (350-400 nm), which excites the protonated form of the chromophore. The excitation lights used to elicit red and green fluorescence do not induce photoconversion. Under a conventional epifluorescence microscope, Kaede protein expressed in HeLa cells turned red in a graded fashion in response to UV illumination; maximal illumination resulted in a 2,000-fold increase in the ratio of red-to-green signal. These color-changing properties provide a simple and powerful technique for regional optical marking. A focused UV pulse creates an instantaneous plane source of red Kaede within the cytosol. The red spot spreads rapidly throughout the cytosol, indicating its free diffusibility in the compartment. The extensive diffusion allows us to delineate a single neuron in a dense culture, where processes originating from many different somata are present. Illumination of a focused UV pulse onto the soma of a Kaede-expressing neuron resulted in filling of all processes with red fluorescence, allowing visualization of contact sites between the red and green neurons of interest. (+info)
Low temperature X-ray microanalysis of calcium in a scleractinian coral: evidence of active transport mechanisms.
Element concentrations were measured by X-ray microanalysis in seawater (SW) compartments and mucocytes in bulk, frozen-hydrated preparations of the scleractinian coral Galaxea fascicularis. Quantitative X-ray microanalysis of polyps sampled in the daytime revealed that concentrations of the elements Na, S, K and Ca were all significantly higher in a thin (10-20 micro m) external SW layer adjacent to the oral ectoderm (P<0.05, <0.05, <0.0001 and <0.01, respectively) than in standard SW. In polyps sampled during night-time, concentrations of Ca and S in this external SW layer were significantly reduced (P<0.05). Ca concentration in the coelenteron and extrathecal coelenteron was significantly higher (P<0.001) than in the external SW layer, regardless of time of sampling, suggesting that Ca(2+) transport across the oral epithelium occurs via an active, transcellular route. X-ray microanalyses of mucocytes revealed that the concentration of S was high and did not vary between epithelial layers, while that of Ca increased in an inward gradient toward the skeleton. We suggest that throughout the day, secreted mucus behaves as a Donnan matrix at the oral ectoderm-SW interface, facilitating intracellular Ca(2+) uptake. The accumulation within internal SW compartments of high concentrations of Ca relative to standard SW levels, however, appears to be independent of mucus secretion and is likely to be a consequence of active transport processes. (+info)