Mikhail V. Matz

Fluorescent proteins from nonbioluminescent Anthozoa species.

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 β-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.

Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues

Green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its homologs from diverse marine animals are widely used as universal genetically encoded fluorescent labels. Many laboratories have focused their efforts on identification and development of fluorescent proteins with novel characteristics and enhanced properties, resulting in a powerful toolkit for visualization of structural organization and dynamic processes in living cells and organisms. The diversity of currently available fluorescent proteins covers nearly the entire visible spectrum, providing numerous alternative possibilities for multicolor labeling and studies of protein interactions. Photoactivatable fluorescent proteins enable tracking of photolabeled molecules and cells in space and time and can also be used for super-resolution imaging. Genetically encoded sensors make it possible to monitor the activity of enzymes and the concentrations of various analytes. Fast-maturing fluorescent proteins, cell clocks, and timers further expand the options for real time studies in living tissues. Here we focus on the structure, evolution, and function of GFP-like proteins and their numerous applications for in vivo imaging, with particular attention to recent techniques.

Sequencing and de novo analysis of a coral larval transcriptome using 454 GSFlx

BackgroundNew methods are needed for genomic-scale analysis of emerging model organisms that exemplify important biological questions but lack fully sequenced genomes. For example, there is an urgent need to understand the potential for corals to adapt to climate change, but few molecular resources are available for studying these processes in reef-building corals. To facilitate genomics studies in corals and other non-model systems, we describe methods for transcriptome sequencing using 454, as well as strategies for assembling a useful catalog of genes from the output. We have applied these methods to sequence the transcriptome of planulae larvae from the coral Acropora millepora.ResultsMore than 600,000 reads produced in a single 454 sequencing run were assembled into ~40,000 contigs with five-fold average sequencing coverage. Based on sequence similarity with known proteins, these analyses identified ~11,000 different genes expressed in a range of conditions including thermal stress and settlement induction. Assembled sequences were annotated with gene names, conserved domains, and Gene Ontology terms. Targeted searches using these annotations identified the majority of genes associated with essential metabolic pathways and conserved signaling pathways, as well as novel candidate genes for stress-related processes. Comparisons with the genome of the anemone Nematostella vectensis revealed ~8,500 pairs of orthologs and ~100 candidate coral-specific genes. More than 30,000 SNPs were detected in the coral sequences, and a subset of these validated by re-sequencing.ConclusionThe methods described here for deep sequencing of the transcriptome should be widely applicable to generate catalogs of genes and genetic markers in emerging model organisms. Our data provide the most comprehensive sequence resource currently available for reef-building corals, and include an extensive collection of potential genetic markers for association and population connectivity studies. The characterization of the larval transcriptome for this widely-studied coral will enable research into the biological processes underlying stress responses in corals and evolutionary adaptation to global climate change.

A ubiquitous family of putative gap junction molecules.

References 1. Artavanis-Tsakonas S, Rand MD, Lake RJ: Notch signaling: cell fate control and signal integration in development. Science 1999, 284:770-776. 2. Artavanis-Tsakonas S, Matsuno K, Fortini ME: Notch signaling. Science 1995, 268:225-232. 3. Petcherski AG, Kimble J: LAG-3 is a putative transcriptional activator in the C. elegans Notch pathway. Nature 2000, 405:364-368. 4. Lehmann R, Jimenez F, Dietrich U, Campos-Ortega J: On the phenotype and development of mutants of early neurogenesis in Drosophila melanogaster. Wilhelm Roux’s Archiv Dev Biol 1983, 192:62-74. 5. Xu T, Rebay I, Fleming RJ, Scottgale TN, Artavanis-Tsakonas S: The Notch locus and the genetic circuitry involved in early Drosophila neurogenesis. Genes Dev 1990, 4:464-475. 6. Kopan R, Nye JS, Weintraub H: The intracellular domain of mouse Notch: a constitutively activated repressor of myogenesis directed at the basic helix-loop-helix region of MyoD. Development 1994, 120:2385-2396. 7. Smoller D, Friedel C, Schmid A, Bettler D, Lam L, Yedvobnick B: The Drosophila neurogenic locus mastermind encodes a nuclear protein unusually rich in amino acid homopolymers. Genes Dev 1990, 4:1688-1700. 8. Bettler D, Pearson S, Yedvobnick B: The nuclear protein encoded by the Drosophila neurogenic gene mastermind is widely expressed and associates with specific chromosomal regions. Genetics 1996, 143:859-875. 9. Schuldt AJ, Brand AH: Mastermind acts downstream of Notch to specify neuronal cell fates in the Drosophila central nervous system. Dev Biol 1999, 205:287-295. 10. Mitchell PJ, Tjian R: Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science 1989, 245:371-378. 11. Kodoyianni V, Maine EM, Kimble J: Molecular basis of loss-of-function mutations in the glp-1 gene of Caenorhabditis elegans. Mol Biol Cell 1992, 3:1199-1213. 12. Jarriault S, Brou C, Logeat F, Schroeter EH, Kopan R, Israël A: Signaling downstream of activated mammalian Notch. Nature 1995, 377:355-358. 13. Kato H, Taniguchi Y, Kurooka H, Minoguchi S, Sakai T, Nomura-Okazaki S, Tamura K, Honjo T: Involvement of RBP-J in biological functions of mouse Notch1 and its derivatives. Development 1997, 124:4133-4141.

Diversity and evolution of the green fluorescent protein family

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.

Diversity and evolution of coral fluorescent proteins.

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.

GFP-like chromoproteins as a source of far-red fluorescent proteins.

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 λ max=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.

2b-RAD: a simple and flexible method for genome-wide genotyping

We describe 2b-RAD, a streamlined restriction site–associated DNA (RAD) genotyping method based on sequencing the uniform fragments produced by type IIB restriction endonucleases. Well-studied accessions of Arabidopsis thaliana were genotyped to validate the methods accuracy and to demonstrate fine-tuning of marker density as needed. The simplicity of the 2b-RAD protocol makes it particularly suitable for high-throughput genotyping as required for linkage mapping and profiling genetic variation in natural populations.

Novel fluorescent protein from Discosoma coral and its mutants possesses a unique far-red fluorescence

A novel gene for advanced red‐shifted protein with an emission maximum at 593 nm was cloned from Discosoma coral. The protein, named dsFP593, is highly homologous to the recently described GFP‐like protein drFP583 with an emission maximum at 583 nm. Using the remarkable similarity of the drFP583 and dsFP593 genes, we performed a ‘shuffling’ procedure to generate a pool of mutants consisting of various combinations of parts of both genes. One ‘hybrid gene’ was chosen for subsequent random mutagenesis, which resulted in a mutant variant with a uniquely red‐shifted emission maximum at 616 nm.

Profiling gene expression responses of coral larvae (Acropora millepora) to elevated temperature and settlement inducers using a novel RNA‐Seq procedure

Elevated temperatures resulting from climate change pose a clear threat to reef‐building corals; however, the traits that might influence corals’ survival and dispersal during climate change remain poorly understood. Global gene expression profiling is a powerful hypothesis‐forming tool that can help elucidate these traits. Here, we applied a novel RNA‐Seq protocol to study molecular responses to heat and settlement inducers in aposymbiotic larvae of the reef‐building coral Acropora millepora. This analysis of a single full‐sibling family revealed contrasting responses between short‐ (4‐h) and long‐term (5‐day) exposures to elevated temperatures. Heat shock proteins were up‐regulated only in the short‐term treatment, while the long‐term treatment induced the down‐regulation of ribosomal proteins and up‐regulation of genes associated with ion transport and metabolism (Ca2+ and CO32−). We also profiled responses to settlement cues using a natural cue (crustose coralline algae, CCA) and a synthetic neuropeptide (GLW‐amide). Both cues resulted in metamorphosis, accompanied by differential expression of genes with known developmental roles. Some genes were regulated only by the natural cue, which may correspond to the recruitment‐associated behaviour and morphology changes that precede metamorphosis under CCA treatment, but are bypassed under GLW‐amide treatment. Validation of these expression profiles using qPCR confirmed the quantitative accuracy of our RNA‐Seq approach. Importantly, qPCR analysis of different larval families revealed extensive variation in these responses depending on genetic background, including qualitative differences (i.e. up‐regulation in one family and down‐regulation in another). Future studies of gene expression in corals will have to address this genetic variation, which could have important adaptive consequences for corals during global climate change.