The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Konstantin A Lukyanov - One of the best experts on this subject based on the ideXlab platform.
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fluorescent proteins as a toolkit for in vivo imaging
Trends in Biotechnology, 2005Co-Authors: Dmitriy M Chudakov, Sergey Lukyanov, Konstantin A LukyanovAbstract:Green fluorescent protein (GFP) from the jellyfish Aequorea victoria, and its mutant variants, are the only fully genetically encoded fluorescent probes available and they have proved to be excellent tools for labeling living specimens. Since 1999, numerous GFP homologues have been discovered in Anthozoa, Hydrozoa and Copepoda species, demonstrating the broad evolutionary and spectral diversity of this protein family. Mutagenic studies gave rise to diversified and optimized variants of fluorescent proteins, which have never been encountered in nature. This article gives an overview of the GFP-like proteins developed to date and their most common applications to study living specimens using fluorescence microscopy.
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the molecular properties and applications of Anthozoa fluorescent proteins and chromoproteins
Nature Biotechnology, 2004Co-Authors: Vladislav V Verkhusha, Konstantin A LukyanovAbstract:The green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its fluorescent homologs from Anthozoa corals have become invaluable tools for in vivo imaging of cells and tissues. Despite spectral and chromophore diversity, about 100 cloned members of the GFP-like protein family possess common structural, biochemical and photophysical features. Anthozoa GFP-like proteins are available in colors and properties unlike those of A. victoria GFP variants and thus provide powerful new fluorophores for molecular labeling and intracellular detection. Although Anthozoa GFP-like proteins provide some advantages over GFP, they also have certain drawbacks, such as obligate oligomerization and slow or incomplete fluorescence maturation. In the past few years, effective approaches for eliminating some of these limitations have been described. In addition, several Anthozoa GFP-like proteins have been developed into novel imaging agents, such as monomeric red and dimeric far-red fluorescent proteins, fluorescent timers and photoconvertible fluorescent labels. Future studies on the structure of this diverse set of proteins will further enhance their use in animal tissues and as intracellular biosensors.
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far red fluorescent tag for protein labelling
Biochemical Journal, 2002Co-Authors: Arkady F Fradkov, Sergey Lukyanov, Vladislav V Verkhusha, Dmitry B Staroverov, Maria E Bulina, Yurii G Yanushevich, Vladimir I Martynov, Konstantin A LukyanovAbstract:Practical applications of green fluorescent protein ('GFP')-like fluorescent proteins (FPs) from species of the class Anthozoa (sea anemones, corals and sea pens) are strongly restricted owing to their oligomeric nature. Here we suggest a strategy to overcome this problem by the use of two covalently linked identical red FPs as non-oligomerizing fusion tags. We have applied this approach to the dimeric far-red fluorescent protein HcRed1 and have demonstrated superiority of the tandem tag in the in vivo labelling of fine cytoskeletal structures and tiny nucleoli. In addition, a possibility of effective fluorescence resonance energy transfer ('FRET') between enhanced yellow FP mutant ('EYFP') and tandem HcRed1 was demonstrated in a protease assay.
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diversity and evolution of the green fluorescent protein family
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Yulii Aleksandrovich Labas, Arkady F Fradkov, Nadya G. Gurskaya, Yurii G Yanushevich, Konstantin A Lukyanov, S A Lukyanov, Mikhail V MatzAbstract:The family of proteins homologous to the green fluorescent protein (GFP) from Aequorea victoria exhibits striking diversity of features, including several different types of autocatalytically synthesized chromophores. Here we report 11 new members of the family, among which there are 3 red-emitters possessing unusual features, and discuss the similarity relationships within the family in structural, spectroscopic, and evolutionary terms. Phylogenetic analysis has shown that GFP-like proteins from representatives of subclass Zoantharia fall into at least four distinct clades, each clade containing proteins of more than one emission color. This topology suggests multiple recent events of color conversion. Combining this result with previous mutagenesis and structural data, we propose that (i) different chromophore structures are alternative products synthesized within a similar autocatalytic environment, and (ii) the phylogenetic pattern and color diversity in reef Anthozoa is a result of a balance between selection for GFP-like proteins of particular colors and mutation pressure driving the color conversions.
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gfp like chromoproteins as a source of far red fluorescent proteins
FEBS Letters, 2001Co-Authors: Nadya G. Gurskaya, Arkady F Fradkov, Yulii Aleksandrovich Labas, Mikhail V Matz, Yurii G Yanushevich, Vladimir I Martynov, Konstantin A Lukyanov, Alexey V Terskikh, Sergey LukyanovAbstract:Abstract We have employed a new approach to generate novel fluorescent proteins (FPs) from red absorbing chromoproteins. An identical single amino acid substitution converted novel chromoproteins from the species Anthozoa (Heteractis crispa, Condylactis gigantea, and Goniopora tenuidens) into far-red FPs (emission λmax=615–640 nm). Moreover, coupled site-directed and random mutagenesis of the chromoprotein from H. crispa resulted in a unique far-red FP (HcRed) that exhibited bright emission at 645 nm. A clear red shift in fluorescence of HcRed, compared to drFP583 (by more than 60 nm), makes it an ideal additional color for multi-color labeling. Importantly, HcRed is excitable by 600 nm dye laser, thus promoting new detection channels for multi-color flow cytometry applications. In addition, we generated a dimeric mutant with similar maturation and spectral properties to tetrameric HcRed.
Sergey Lukyanov - One of the best experts on this subject based on the ideXlab platform.
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fluorescent proteins as a toolkit for in vivo imaging
Trends in Biotechnology, 2005Co-Authors: Dmitriy M Chudakov, Sergey Lukyanov, Konstantin A LukyanovAbstract:Green fluorescent protein (GFP) from the jellyfish Aequorea victoria, and its mutant variants, are the only fully genetically encoded fluorescent probes available and they have proved to be excellent tools for labeling living specimens. Since 1999, numerous GFP homologues have been discovered in Anthozoa, Hydrozoa and Copepoda species, demonstrating the broad evolutionary and spectral diversity of this protein family. Mutagenic studies gave rise to diversified and optimized variants of fluorescent proteins, which have never been encountered in nature. This article gives an overview of the GFP-like proteins developed to date and their most common applications to study living specimens using fluorescence microscopy.
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far red fluorescent tag for protein labelling
Biochemical Journal, 2002Co-Authors: Arkady F Fradkov, Sergey Lukyanov, Vladislav V Verkhusha, Dmitry B Staroverov, Maria E Bulina, Yurii G Yanushevich, Vladimir I Martynov, Konstantin A LukyanovAbstract:Practical applications of green fluorescent protein ('GFP')-like fluorescent proteins (FPs) from species of the class Anthozoa (sea anemones, corals and sea pens) are strongly restricted owing to their oligomeric nature. Here we suggest a strategy to overcome this problem by the use of two covalently linked identical red FPs as non-oligomerizing fusion tags. We have applied this approach to the dimeric far-red fluorescent protein HcRed1 and have demonstrated superiority of the tandem tag in the in vivo labelling of fine cytoskeletal structures and tiny nucleoli. In addition, a possibility of effective fluorescence resonance energy transfer ('FRET') between enhanced yellow FP mutant ('EYFP') and tandem HcRed1 was demonstrated in a protease assay.
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gfp like chromoproteins as a source of far red fluorescent proteins
FEBS Letters, 2001Co-Authors: Nadya G. Gurskaya, Arkady F Fradkov, Yulii Aleksandrovich Labas, Mikhail V Matz, Yurii G Yanushevich, Vladimir I Martynov, Konstantin A Lukyanov, Alexey V Terskikh, Sergey LukyanovAbstract:Abstract We have employed a new approach to generate novel fluorescent proteins (FPs) from red absorbing chromoproteins. An identical single amino acid substitution converted novel chromoproteins from the species Anthozoa (Heteractis crispa, Condylactis gigantea, and Goniopora tenuidens) into far-red FPs (emission λmax=615–640 nm). Moreover, coupled site-directed and random mutagenesis of the chromoprotein from H. crispa resulted in a unique far-red FP (HcRed) that exhibited bright emission at 645 nm. A clear red shift in fluorescence of HcRed, compared to drFP583 (by more than 60 nm), makes it an ideal additional color for multi-color labeling. Importantly, HcRed is excitable by 600 nm dye laser, thus promoting new detection channels for multi-color flow cytometry applications. In addition, we generated a dimeric mutant with similar maturation and spectral properties to tetrameric HcRed.
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alternative cyclization in gfp like proteins family the formation and structure of the chromophore of a purple chromoprotein from anemonia sulcata
Journal of Biological Chemistry, 2001Co-Authors: Vladimir I Martynov, Alexander P. Savitsky, Konstantin A Lukyanov, Natalya Y. Martynova, Pavel A. Savitsky, Sergey LukyanovAbstract:Anemonia sulcata purple protein (asFP595) belongs to a family of green fluorescent protein (GFP)-like proteins from the Anthozoa species. Similar to GFP, asFP595 apparently forms its chromophore by modifying amino acids within its polypeptide chain. Until now, the GFP-like proteins from Anthozoa were thought to contain chromophores with the same imidazolidinone core as GFP. Mass spectral analysis of a chromophore-containing tryptic pentapeptide from asFP595 demonstrates that chromophore formation in asFP595 is stoichiometrically the same as that in GFP: one H(2)O and two H(+) are released while a Schiff base and dehydrotyrosine are formed. However, structural studies of this asFP595 chromopeptide show that in contrast to GFP, the other peptide bond nitrogen and carbonyl carbon are required for chromophore cyclization, a reaction that yields the six-membered heterocycle 2-(4-hydroxybenzylidene)-6-hydroxy-2,5-dihydropyrazine. Spectrophotometric titration reveals three pH-dependent forms of the asFP595 chromopeptide: yellow (absorption maximum = 430 nm) at pH 3.0; red (absorption maximum = 535 nm) at pH 8.0; and colorless (absorption maximum = 380 nm) at pH 14.0. The pK(a) values for these spectral transitions (6.8 and 10.9) are consistent with the ionization of the phenolic group of dehydrotyrosine and deprotonation of the amidinium cation in the chromophore heterocycle, respectively. The amidinium group in asFP595 accounts for the unique absorption spectrum of the protein, which is substantially red-shifted relative to that of GFP. When the asFP595 chromophore cyclizes, the Cys-Met bond adjacent to the chromophore hydrolyzes, splitting the chromoprotein into 8- and 20-kDa fragments. High performance liquid chromatography analysis of a tryptic digest of denatured asFP595 shows that a pentapeptide with the cleaved Cys-Met bond is the only fragment associated with the red-shifted absorbance. These results imply that fragmentation of asFP595 is a critical step in protein maturation.
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THE FORMATION AND STRUCTURE OF THE CHROMOPHORE OF A PURPLE CHROMOPROTEIN FROM ANEMONIA SULCATA
2001Co-Authors: Vladimir I Martynov, Alexander P. Savitsky, Konstantin A Lukyanov, Natalya Y. Martynova, Pavel A. Savitsky, Sergey LukyanovAbstract:Anemonia sulcata purple protein (asFP595) belongs to a family of green fluorescent protein (GFP)-like proteins from the Anthozoa species. Similar to GFP, asFP595 apparently forms its chromophore by modifying amino acids within its polypeptide chain. Until now, the GFP-like proteins from Anthozoa were thought to contain chromophores with the same imidazolidinone core as GFP. Mass spectral analysis of a chromophore-containing tryptic pentapeptide from asFP595 demonstrates that chromophore formation in asFP595 is stoichiometrically the same as that in GFP: one H2O and two H 1 are released while a Schiff base and dehydrotyrosine are formed. However, structural studies of this asFP595 chromopeptide show that in contrast to GFP, the other peptide bond nitrogen and carbonyl carbon are required for chromophore cyclization, a reaction that yields the six-membered heterocycle 2-(4-hydroxybenzylidene)-6-hydroxy-2,5-dihydropyrazine. Spectrophotometric titration reveals three pH-dependent forms of the asFP595 chromopeptide: yellow (absorption maximum 5 430 nm) at pH 3.0; red (absorption maximum 5 535 nm) at pH 8.0; and colorless (absorption maximum 5 380 nm) at pH 14.0. The pKa values for these spectral transitions (6.8 and 10.9) are consistent with the ionization of the phenolic group of dehydrotyrosine and deprotonation of the amidinium cation in the chromophore heterocycle, respectively. The amidinium group in asFP595 accounts for the unique absorption spectrum of the protein, which is substantially red-shifted relative to that of GFP. When the asFP595 chromophore cyclizes, the Cys‐Met bond adjacent to the chromophore hydrolyzes, splitting the chromoprotein into 8- and 20-kDa fragments. High performance liquid chromatography analysis of a tryptic digest of denatured asFP595 shows that a pentapeptide with the cleaved Cys‐Met bond is the only fragment associated with the red-shifted absorbance. These results imply that fragmentation of asFP595 is a critical step in protein maturation.
Ulrich Technau - One of the best experts on this subject based on the ideXlab platform.
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sox genes in the coral acropora millepora divergent expression patterns reflect differences in developmental mechanisms within the Anthozoa
BMC Evolutionary Biology, 2008Co-Authors: David C Hayward, Ulrich Technau, Chuya Shinzato, Akira Iguchi, Eldon E Ball, David J. MillerAbstract:Sox genes encode transcription factors that function in a wide range of developmental processes across the animal kingdom. To better understand both the evolution of the Sox family and the roles of these genes in cnidarians, we are studying the Sox gene complement of the coral, Acropora millepora (Class Anthozoa). Based on overall domain structures and HMG box sequences, the Acropora Sox genes considered here clearly fall into four of the five major Sox classes. AmSoxC is expressed in the ectoderm during development, in cells whose morphology is consistent with their assignment as sensory neurons. The expression pattern of the Nematostella ortholog of this gene is broadly similar to that of AmSoxC, but there are subtle differences – for example, expression begins significantly earlier in Acropora than in Nematostella. During gastrulation, AmSoxBb and AmSoxB1 transcripts are detected only in the presumptive ectoderm while AmSoxE1 transcription is restricted to the presumptive endoderm, suggesting that these Sox genes might play roles in germ layer specification. A third type B Sox gene, AmSoxBa, and a Sox F gene AmSoxF also have complex and specific expression patterns during early development. Each of these genes has a clear Nematostella ortholog, but in several cases the expression pattern observed in Acropora differs significantly from that reported in Nematostella. These differences in expression patterns between Acropora and Nematostella largely reflect fundamental differences in developmental processes, underscoring the diversity of mechanisms within the Anthozoan Sub-Class Hexacorallia (Zoantharia).
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The ancestral role of Brachyury: expression of NemBra1 in the basal cnidarian Nematostella vectensis (Anthozoa).
Development genes and evolution, 2002Co-Authors: Corinna B. Scholz, Ulrich TechnauAbstract:The T-Box transcription factor Brachyury plays important roles in the development of all bilateral animals examined so far. In order to understand the ancestral function of Brachyury we cloned NemBra1, a Brachyury homolog from the Anthozoan sea anemone Nematostella vectensis. Anthozoa are considered the basal group among the Cnidaria. First NemBra1 expression could be detected at the blastula/gastrula transition and gene activity persists until adulthood of the animals. In situ hybridization shows that NemBra1 expression in gastrulae and early planula larvae is restricted to a circle around the blastopore. When the larvae begin to metamorphose into primary polyps, the expression zone extends into the developing mesenteries. In adult polyps Brachyury expression persists in the mesenteries, but is excluded from the septal filament and the differentiated retractor muscles, which also develop from the mesenteries. We conclude that the ancestral function of Brachyury was in specifying the blastopore and its endodermal derivatives.
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Induction of gametogenesis in the basal cnidarian Nematostella vectensis(Anthozoa).
Development genes and evolution, 2002Co-Authors: Jens H. Fritzenwanker, Ulrich TechnauAbstract:A protocol was established to reproducibly induce spawning in the basal cnidarian Nematostella vectensis (Anthozoa). We found that a combination of feeding regime, dark-light cycle and temperature shift synergistically induced gametogenesis in adult polyps. Females lay between 100-600 eggs. This procedure led reproducibly to the production of thousands of eggs over the course of more than 1 year in weekly cycles. Gametes are released in a time window of about 2 h resulting in predictable and fairly synchronized development. We also present a method for in vitro fertilization allowing manipulation of early embryos. These methods as well as the simple culture conditions could provide important prerequisites for the use of Nematostella as a model system for the development of a basal Metazoa.
David J. Miller - One of the best experts on this subject based on the ideXlab platform.
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the organizer in evolution gastrulation and organizer gene expression highlight the importance of brachyury during development of the coral acropora millepora
Developmental Biology, 2015Co-Authors: David C Hayward, Robert Saint, Lauretta C. Grasso, David J. MillerAbstract:Organizer activity, once thought to be restricted to vertebrates, has ancient origins. However, among non-bilaterians, it has only been subjected to detailed investigation during embryonic development of the sea anemone, Nematostella vectensis. As a step toward establishing the extent to which findings in Nematostella can be generalized across the large and diverse phylum Cnidaria, we examined the expression of some key organizer and gastrulation genes during the embryonic development of the coral Acropora millepora. Although anemones and corals both belong to the cnidarian class Anthozoa, the two lineages diverged during the Cambrian and the morphological development of Acropora differs in several important respects from that of Nematostella. While the expression patterns of the key genes brachyury, bmp2/4, chordin, goosecoid and forkhead are broadly similar, developmental differences between the two species enable novel observations, and new interpretations of their significance. Specifically, brachyury expression during the flattened prawnchip stage before gastrulation, a developmental peculiarity of Acropora, leads us to suggest that it is the key gene demarcating ectoderm from endoderm in Acropora, and by implication in other cnidarians, whereas previous studies in Nematostella proposed that forkhead plays this role. Other novel observations include the transient expression of Acropora forkhead in scattered ectodermal cells shortly after gastrulation, and in the developing mesenterial filaments, with no corresponding expression reported in Nematostella. In addition, the expression patterns of goosecoid and bmp2/4 confirm the fundamental bilaterality of the Anthozoa.
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sox genes in the coral acropora millepora divergent expression patterns reflect differences in developmental mechanisms within the Anthozoa
BMC Evolutionary Biology, 2008Co-Authors: David C Hayward, Ulrich Technau, Chuya Shinzato, Akira Iguchi, Eldon E Ball, David J. MillerAbstract:Sox genes encode transcription factors that function in a wide range of developmental processes across the animal kingdom. To better understand both the evolution of the Sox family and the roles of these genes in cnidarians, we are studying the Sox gene complement of the coral, Acropora millepora (Class Anthozoa). Based on overall domain structures and HMG box sequences, the Acropora Sox genes considered here clearly fall into four of the five major Sox classes. AmSoxC is expressed in the ectoderm during development, in cells whose morphology is consistent with their assignment as sensory neurons. The expression pattern of the Nematostella ortholog of this gene is broadly similar to that of AmSoxC, but there are subtle differences – for example, expression begins significantly earlier in Acropora than in Nematostella. During gastrulation, AmSoxBb and AmSoxB1 transcripts are detected only in the presumptive ectoderm while AmSoxE1 transcription is restricted to the presumptive endoderm, suggesting that these Sox genes might play roles in germ layer specification. A third type B Sox gene, AmSoxBa, and a Sox F gene AmSoxF also have complex and specific expression patterns during early development. Each of these genes has a clear Nematostella ortholog, but in several cases the expression pattern observed in Acropora differs significantly from that reported in Nematostella. These differences in expression patterns between Acropora and Nematostella largely reflect fundamental differences in developmental processes, underscoring the diversity of mechanisms within the Anthozoan Sub-Class Hexacorallia (Zoantharia).
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diversity and evolution of coral fluorescent proteins
PLOS ONE, 2008Co-Authors: Naila O Alieva, David J. Miller, Karen A Konzen, Steven F Field, Ella A Meleshkevitch, Marguerite E Hunt, Victor Beltranramirez, Jorg Wiedenmann, Anya SalihAbstract:GFP-like fluorescent proteins (FPs) are the key color determinants in reef-building corals (class Anthozoa, order Scleractinia) and are of considerable interest as potential genetically encoded fluorescent labels. Here we report 40 additional members of the GFP family from corals. There are three major paralogous lineages of coral FPs. One of them is retained in all sampled coral families and is responsible for the non-fluorescent purple-blue color, while each of the other two evolved a full complement of typical coral fluorescent colors (cyan, green, and red) and underwent sorting between coral groups. Among the newly cloned proteins are a “chromo-red” color type from Echinopora forskaliana (family Faviidae) and pink chromoprotein from Stylophora pistillata (Pocilloporidae), both evolving independently from the rest of coral chromoproteins. There are several cyan FPs that possess a novel kind of excitation spectrum indicating a neutral chromophore ground state, for which the residue E167 is responsible (numeration according to GFP from A. victoria). The chromoprotein from Acropora millepora is an unusual blue instead of purple, which is due to two mutations: S64C and S183T. We applied a novel probabilistic sampling approach to recreate the common ancestor of all coral FPs as well as the more derived common ancestor of three main fluorescent colors of the Faviina suborder. Both proteins were green such as found elsewhere outside class Anthozoa. Interestingly, a substantial fraction of the all-coral ancestral protein had a chromohore apparently locked in a non-fluorescent neutral state, which may reflect the transitional stage that enabled rapid color diversification early in the history of coral FPs. Our results highlight the extent of convergent or parallel evolution of the color diversity in corals, provide the foundation for experimental studies of evolutionary processes that led to color diversification, and enable a comparative analysis of structural determinants of different colors.
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Pax gene diversity in the basal cnidarian Acropora millepora (Cnidaria, Anthozoa): Implications for the evolution of the Pax gene family
Proceedings of the National Academy of Sciences, 2000Co-Authors: David J. Miller, David C Hayward, Julian Catmull, John S. Reece-hoyes, Ingo Scholten, Walter J. Gehring, Patrick Callaerts, Jill E. LarsenAbstract:Pax genes encode a family of transcription factors, many of which play key roles in animal embryonic development but whose evolutionary relationships and ancestral functions are unclear. To address these issues, we are characterizing the Pax gene complement of the coral Acropora millepora, an Anthozoan cnidarian. As the simplest animals at the tissue level of organization, cnidarians occupy a key position in animal evolution, and the Anthozoa are the basal class within this diverse phylum. We have identified four Pax genes in Acropora: two (Pax-Aam and Pax-Bam) are orthologs of genes identified in other cnidarians; the others (Pax-Cam and Pax-Dam) are unique to Acropora. Pax-Aam may be orthologous with Drosophila Pox neuro, and Pax-Bam clearly belongs to the Pax-2/5/8 class. The Pax-Bam Paired domain binds specifically and preferentially to Pax-2/5/8 binding sites. The recently identified Acropora gene Pax-Dam belongs to the Pax-3/7 class. Clearly, substantial diversification of the Pax family occurred before the Cnidaria/higher Metazoa split. The fourth Acropora Pax gene, Pax-Cam, may correspond to the ancestral vertebrate Pax gene and most closely resembles Pax-6. The expression pattern of Pax-Cam, in putative neurons, is consistent with an ancestral role of the Pax family in neural differentiation and patterning. We have determined the genomic structure of each Acropora Pax gene and show that some splice sites are shared both between the coral genes and between these and Pax genes in triploblastic metazoans. Together, these data support the monophyly of the Pax family and indicate ancient origins of several introns.
Mikhail V Matz - One of the best experts on this subject based on the ideXlab platform.
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evolutionary origins of germline segregation in metazoa evidence for a germ stem cell lineage in the coral orbicella faveolata cnidaria Anthozoa
Proceedings of The Royal Society B: Biological Sciences, 2016Co-Authors: Sarah Barfield, Galina V Aglyamova, Mikhail V MatzAbstract:The ability to segregate a committed germ stem cell (GSC) lineage distinct from somatic cell lineages is a characteristic of bilaterian Metazoans. However, the occurrence of GSC lineage specification in basally branching Metazoan phyla, such as Cnidaria, is uncertain. Without an independently segregated GSC lineage, germ cells and their precursors must be specified throughout adulthood from continuously dividing somatic stem cells, generating the risk of propagating somatic mutations within the individual and its gametes. To address the potential for existence of a GSC lineage in Anthozoa, the sister-group to all remaining Cnidaria, we identified moderate- to high-frequency somatic mutations and their potential for gametic transfer in the long-lived coral Orbicella faveolata (Anthozoa, Cnidaria) using a 2b-RAD sequencing approach. Our results demonstrate that somatic mutations can drift to high frequencies (up to 50%) and can also generate substantial intracolonial genetic diversity. However, these somatic mutations are not transferable to gametes, signifying the potential for an independently segregated GSC lineage in O. faveolata. In conjunction with previous research on germ cell development in other basally branching Metazoan species, our results suggest that the GSC system may be a Eumetazoan characteristic that evolved in association with the emergence of greater complexity in animal body plan organization and greater specificity of stem cell functions.
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diversity and evolution of the green fluorescent protein family
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Yulii Aleksandrovich Labas, Arkady F Fradkov, Nadya G. Gurskaya, Yurii G Yanushevich, Konstantin A Lukyanov, S A Lukyanov, Mikhail V MatzAbstract:The family of proteins homologous to the green fluorescent protein (GFP) from Aequorea victoria exhibits striking diversity of features, including several different types of autocatalytically synthesized chromophores. Here we report 11 new members of the family, among which there are 3 red-emitters possessing unusual features, and discuss the similarity relationships within the family in structural, spectroscopic, and evolutionary terms. Phylogenetic analysis has shown that GFP-like proteins from representatives of subclass Zoantharia fall into at least four distinct clades, each clade containing proteins of more than one emission color. This topology suggests multiple recent events of color conversion. Combining this result with previous mutagenesis and structural data, we propose that (i) different chromophore structures are alternative products synthesized within a similar autocatalytic environment, and (ii) the phylogenetic pattern and color diversity in reef Anthozoa is a result of a balance between selection for GFP-like proteins of particular colors and mutation pressure driving the color conversions.
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gfp like chromoproteins as a source of far red fluorescent proteins
FEBS Letters, 2001Co-Authors: Nadya G. Gurskaya, Arkady F Fradkov, Yulii Aleksandrovich Labas, Mikhail V Matz, Yurii G Yanushevich, Vladimir I Martynov, Konstantin A Lukyanov, Alexey V Terskikh, Sergey LukyanovAbstract:Abstract We have employed a new approach to generate novel fluorescent proteins (FPs) from red absorbing chromoproteins. An identical single amino acid substitution converted novel chromoproteins from the species Anthozoa (Heteractis crispa, Condylactis gigantea, and Goniopora tenuidens) into far-red FPs (emission λmax=615–640 nm). Moreover, coupled site-directed and random mutagenesis of the chromoprotein from H. crispa resulted in a unique far-red FP (HcRed) that exhibited bright emission at 645 nm. A clear red shift in fluorescence of HcRed, compared to drFP583 (by more than 60 nm), makes it an ideal additional color for multi-color labeling. Importantly, HcRed is excitable by 600 nm dye laser, thus promoting new detection channels for multi-color flow cytometry applications. In addition, we generated a dimeric mutant with similar maturation and spectral properties to tetrameric HcRed.
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fluorescent proteins from nonbioluminescent Anthozoa species
Nature Biotechnology, 1999Co-Authors: Mikhail V Matz, Alexander P. Savitsky, Arkady F Fradkov, Yulii Aleksandrovich Labas, Mikhail L Markelov, Andrey G. Zaraisky, Sergey LukyanovAbstract:We have cloned six fluorescent proteins homologous to the green fluorescent protein (GFP) from Aequorea victoria. Two of these have spectral characteristics dramatically different from GFP, emitting at yellow and red wavelengths. All the proteins were isolated from nonbioluminescent reef corals, demonstrating that GFP-like proteins are not always functionally linked to bioluminescence. The new proteins share the same beta-can fold first observed in GFP, and this provided a basis for the comparative analysis of structural features important for fluorescence. The usefulness of the new proteins for in vivo labeling was demonstrated by expressing them in mammalian cell culture and in mRNA microinjection assays in Xenopus embryos.