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Fogel and Hastings first hypothesized the existence of voltage-gated proton channels in 1972 in bioluminescent dinoflagellates, where they were thought to trigger the flash by activating luciferase. Proton channel genes were subsequently identified in human, mouse, and Ciona intestinalis, but their existence in dinoflagellates remained unconfirmed. We identified a candidate proton channel gene from a Karlodinium veneficum cDNA library based on homology with known proton channel genes. K. veneficum is a predatory, nonbioluminescent dinoflagellate that produces toxins responsible for fish kills worldwide. Patch clamp studies on the heterologously expressed gene confirm that it codes for a genuine voltage-gated proton channel, kHV1: it is proton-specific and activated by depolarization, its gH-V relationship shifts with changes in external or internal pH, and mutation of the selectivity filter (which we identify as Asp51) results in loss of proton-specific conduction. Indirect evidence suggests that kHV1 is monomeric, unlike other proton channels. Furthermore, kHV1 differs from all known proton channels in activating well negative to the Nernst potential for protons, EH. This unique voltage dependence makes the dinoflagellate proton channel ideally suited to mediate the proton influx postulated to trigger bioluminescence. In contrast to vertebrate proton channels, whose main function is acid extrusion, we propose that proton channels in dinoflagellates have fundamentally different functions of signaling and excitability. [ABSTRACT FROM AUTHOR]

The article discusses the two different domains of the luciferase gene in the heterotrophic dinoflagellate Noctiluca scintillans occur as two separate genes in photosynthetic species. It describes the Noctiluca scintillans as a heterotrophic unarmored unicellular bioluminescent dinoflagellate. It explains the molecular cloning of the full-length Noctiluca luciferase gene and the two-domain protein.

The article profiles Lawrence Bogorad, Maria Moors Cabot professor of biology emeritus at Harvard University in Cambridge, Massachusetts. He died of stroke while he and his family is vacationing in Puerto Vallarta, Mexico on December 28, 2003. The organization of influential symposia has been helped by Bogorad. He was survived by his wife Rosalyn.

Tandemly arranged luciferase genes were previously reported in two dinoflagellates species, but their intergenic regions were strikingly different and no canonical promoter sequences were found. Here, we examined the intergenic regions of the luciferase genes of five other dinoflagellate species along with those of the earlier two. In all cases, the genes exist in multiple copies and are arranged tandemly, coding for proteins of similar sizes and sequences. However, the 5′ untranslated region, 3′ untranslated region, and intergenic regions of the seven genes differ greatly in length and sequence, except for two stretches that are conserved in the intergenic regions of two pairs of phylogenetically close species. Microsatellites and minisatellites were detected in the intergenic sequences of four species, Alexandrium affine (H. Inoue & Y. Fukuyo) E. Balech, A. tamarense (Lebour) E. Balech, Protoceratium reticulatum (Claparède & Lachmann) Butschli, and Pyrocystis lunula (Schütt) Schütt, the first three of which have unusually high percentages of particular sets of dinucleotides. Most remarkably, the P. reticulatum intergenic region is almost exclusively made up of 19 nearly identical repeats of an 11-nucleotide sequence. Dinoflagellate luciferase intergenic regions bear similarities to ribosomal genes and to some protein-encoding genes in trypanosomes, both of which are transcribed by RNA polymerase I. It is possible that the transcription of the dinoflagellate genes are catalyzed by an RNA polymerase with novel properties. [ABSTRACT FROM AUTHOR]

In many dinoflagellate species, the plastid genome has been proposed to exist as a limited number of single-gene minicircles, and many genes normally found in the plastid genome are nuclear- encoded. Unlike the nuclear-encoded plastid-directed gene products whose expression is often regulated by the circadian clock, little is known about expression of minicircle genes. Furthermore, even the plastid location of the minicircies has recently been challenged. We have examined the incorporation in vivo of (35S]methionine into the proteins of purified plastids, and we find that several plastid proteins are labeled in the presence of cycloheximide but not chloramphenicol. One of these proteins, labeled in two different dinoflagellate species, was identified as psbA by Western blot analysis. Furthermore, this psbA has the expected physiological characteristics, because both synthesis and degradation are induced by light. We find no evidence for circadian control over either synthesis or degradation of psbA, unlike the several nuclear-encoded plastid-directed proteins studied. Finally, we find that levels of psbA protein or RNA do not change over a 24-h light-dark cycle, suggesting that this protein may not be involved in mediating the circadian rhythm in oxygen evolution rates. This demonstration is the first, to our knowledge, that minidrcle genes encoding plastid proteins are translated in dinoflagellate plastids, and it suggests that a proteomic approach to characterizing the dinoflagellate plastid genome is feasible. [ABSTRACT FROM AUTHOR]

The luciferase of Lingulodinium polyedrum, a marine bioluminescent dinoflagellate, consists of three similar but not identical domains in a single polypeptide. Each encodes an active luciferase that catalyzes the oxidation of a chlorophyll-derived open tetra- pyrrole (dinoflagellate luciferin) to produce blue light. These domains share no sequence similarity with any other in the GenBank database and no structural or motif similarity with any other luciferase. We report here the 1.8-Å crystal structure of the third domain, D3, at pH 8, and a mechanism for its activity regulation by pH. D3 consists of two major structural elements: a β-barrel pocket putatively for substrate binding and catalysis and a regulatory three-helix bundle. N-terminal histidine residues previously shown to regulate activity by pH are at the interface of the helices in the bundle. Molecular dynamics calculations indicate that in response to changes in pH, these histidines could trigger a large molecular motion of the bundle, thereby exposing the active site to the substrate. [ABSTRACT FROM AUTHOR]

Enzymes with multiple catalytic sites are rare, and their evolutionary significance remains to be established. This study of luciferases from seven dinoflagellate species examines the previously undescribed evolution of such proteins. All these enzymes have the same unique structure: three homologous domains, each with catalytic activity, preceded by an N-terminal region of unknown function. Both pairwise comparison and phylogenetic inference indicate that the similarity of the corresponding individual domains between species is greater than that between the three different domains of each polypeptide. Trees constructed from each of the three individual domains are congruent with the tree of the full-length coding sequence. Luciferase and ribosomal DNA trees both indicate that the Lingulodinium polyedrum luciferase diverged `early from the other six. In all species, the amino acid sequence in the central regions of the three domains is strongly conserved, suggesting it as the catalytic site. Synonymous substitution rates also are greatly reduced in the central regions of two species but not in the other five. This lineage-specific difference in synonymous substitution rates in the central region of the domains correlates inversely with the content of GC³, which can be accounted for by the biased usage toward C-ending codons at the degenerate sites. RNA modeling of the central region of the L. polyedrum luciferase domain suggests a function of the constrained synonymous substitutions in the circadian-controlled protein synthesis. [ABSTRACT FROM AUTHOR]

Our objective was to identify cell surface proteins in the dinoflagellate Lingulodinium polyedrum. Proteins on the surface of living cells that had regions exposed to the external medium were labeled with Na125I. After partial purification of membrane proteins and analysis by two-dimensional gel electrophoresis and autoradiography, a protein of roughly 43 kDa was found to have incorporated the radiolabel. This protein was cloned using a combination of protein microsequencing and PCR amplification. The derived protein sequence in the cDNA has a signal peptide at the N-terminal end of the protein and thus represents the first plasma membrane protein ever reported for a dinoflagellate. The function of the protein is unknown, but its cloning provides a proof of principle for the general use of vectorial labeling in identifying cell surface proteins of marine algae. [ABSTRACT FROM AUTHOR]

Indicative of the importance of protein phosphorylation in the core circadian clock mechanism, chronically applied inhibitors of both protein kinases and phosphoprotein phosphatases have significant effects on the period, phase, and light-dependent regulation of circadian rhythms in the dinoflagellate Lingulodinium polyedrum. This study was aimed at identifying the presence of the affected phosphatase(s). Dephosphorylation of a PP1/PP2A-specific substrate by L. polyedrum extracts was inhibited by okadaic acid only at concentrations greater than 100nM, as in vivo, by mammalian inhibitor-2 (I-2), and by an endogenous inhibitor with properties similar to I-2, indicating that a type-1 protein phosphatase (PP1) was predominant. AcDNA encoding a highly conserved PP1 was isolated, the 1st such signaling molecule identified in dinoflagellates. Antisera specific for this type of phosphatase recognized a 34 kDa protein in L. polyedrum extract, this being the same size as the PP1 encoded by the isolated cDNA. These findings are consistent with the suggestion that the L. polyedrum PP1 may be a part of the clock mechanism in this species. [ABSTRACT FROM AUTHOR]

In a genome-wide study of circadian gene expression, we constructed microarrays containing 3500 cDNAs from the dinoflagellate Pyrocystis lunula Schütt and compared the abundance of transcripts at circadian times separated by 12 h. About 3% of the unique genes screened were identified as circadian controlled, more than 50% of which could be identified with diverse known genes. Most were preferentially expressed in either early subjective day or late subjective night. Light exposures at times expected to induce phase shifts in the rhythm revealed 30 differentially expressed genes, including some potentially participating in photic entrainment and others in pathways connecting a central oscillator to output rhythms. Those that appeared in both screens were considered as possible core clock genes, but there were no similarities with such genes from other organisms. This is the first report of circadian regulation of transcript levels in a dinoflagellate. [ABSTRACT FROM AUTHOR]

Lingulodinium polyedrum luciferase is a bioluminescent protein found in the marine dinoflagellate formerly known as Gonyaulax. It is located in organelles called scintillons that emit brief and bright flashes of light that are regulated by an endogenous circadian clock. The complete luciferase molecule has a molecular mass of 136 994 Da and contains three homologous domains, each of which is a separately active luciferase. Two of these domains, D2-LCF and D3-LCF, have been cloned, expressed and crystallized. Crystals of D2-LCF were obtained from PEG 10 000 in space group P2[sub 1]2[sub 1]2[sub 1], with unit-cell parameters a = 49.1, b = 104.7, c = 180.3 Å. They diffract to 2.9 Å on a rotating anode. Crystals of D3-LCF were grown from PEG 2000 in space group P2[sub 1]2[sub 1]2[sub 1], with unit-cell parameters a = 58.86, b = 63.98, c = 95.76 Å. They diffract to 2.3 Å on a rotating anode. [ABSTRACT FROM AUTHOR]

Although nucleosomes and histones are lacking in dinoflagellate nuclei, small basic histone-like proteins have been reported, but their function(s) is unknown. In this study we cloned and sequenced a gene for a histone-like protein from the dinoflagellate Lingulodinium polyedrum (Stein) Dodge (HLp) (formerly Gonyaulax polyedra Stein) and investigated its post-translational modification and DNA-binding activities. HLp appears to be acetylated in L. polyedrum , and we identified several L. polyedrum proteins that possess histone acetyltransferase activity and may be responsible for this modification. HLp binds weakly to L. polyedrum DNA but to certain specific sequences with higher affinity, consistent with its having a regulatory function. [ABSTRACT FROM AUTHOR]

Presents a lecture on biological rhythms. People involved in studies on circadian biology; Works on the culture of the marine dinoflagellate Gonyaulax polyedra; Expedition on the research vessel Alpha Helix.

Presents information on a study which characterizes the unusual action of the protein kinase inhibitor staurosporine on the Gonyaulax circadian system. Materials and methods; Results; Discussion.

Bioluminescence has evolved independently many times; thus the responsible genes are unrelated in bacteria, unicellular algae, coelenterates, beetles, fishes, and others. Chemically, all involve exergonic reactions of molecular oxygen with different substrates (luciferins) and enzymes (luciferases), resulting in photons of visible light (approx. 50 kcal). In addition to the structure of luciferan, several factors determine the color of the emissions, such as the amino acid sequence of the luciferase (as in beetles, for example) or the presence of accessory proteins, notably GFP, discovered in coelenterates and now used as a reporter of gene expression and a cellular marker. The mechanisms used to control the intensity and kinetics of luminescence, often emitted as flashes, also vary. Bioluminescence is credited with the discovery of how some bacteria, luminous or not, sense their density and regulate specific genes by chemical communication, as in the fascinating example of symbiosis between luminous bacteria and squid. [ABSTRACT FROM AUTHOR]

Compares the biochemistry and circadian regulation of luminescence in two Pyrocystis species P. lunula Hulburt and P. noctiluca Murray et Haeckel, with a well-studied species, Gonyaulax polyedra Stein. Lack of a second protein by the Pyrocystis involved in the reaction in Gonyaulax, the luciferin binding protein; Difference in the circadian regulation of luminescence which in the Gonyaulax involves the daily synthesis and destruction of the two proteins.

Radiolabeled RNA transcripts from a cDNA clone of Gonyaulax polyedra Stein luciferin-binding protein (a protein involved in bioluminescence and which increases intracellularly at night) were synthesized in an in vitro transcription assay and hybridized to fixed, thin sections of Gonyaulax cells in both day and night phases. When visualized under dark-field microscopy, probes, which were complementary in sequence (anti-sense probes) to the luciferin-binding protein mRNA, bound to cell sections and revealed a dense, even distribution of photographic silver grains over the cytoplasm, with more intense labeling in night phase cells. Control probes, consisting of the identical sequence as the cellular mRNA (sense probe), revealed little specific labeling in day and night cells. This suggests that cellular levels of luciferin-binding protein mRNA are higher in night phase cells, which is in contrast to recent biochemical findings that the mRNA levels are constant throughout the day and night. This new data is compared with the previous work, and the utility and limitations of the in situ hybridization technique in analyzing message distribution and abundance is discussed. [ABSTRACT FROM AUTHOR]

Bioluminescence in the unicellular dinoflagellate Gonyaulax polyedra represents an excellent model for studying a circadian controlled process at the biochemical and molecular levels. There are three key components involved in the bioluminescence reaction: the enzyme, luciferase, its substrate, luciferin, and a luciferin-binding protein (LBP), which sequesters the substrate at pH 7.5 and thus prevents it from reacting with the enzyme. All components are tightly packed together in organelles, designated scintillons. The entire bioluminescent system is under circadian control with maximum amounts in the night. For both proteins circadian control is exerted at the translational level. In case of lbp mRNA a small interval in its 3′ untranslated region serves as a cis-acting element to which a trans-factor binds in a circadian manner. The binding activity of this factor decreases at the beginning of the night phase, when synthesis of LBP starts, and it increases at the end of the night, when synthesis of LBP stops indicating that it functions as a clock-controlled repressor. [ABSTRACT FROM AUTHOR]

Three of ten cultures of Gonyaulax excavata (Braarud) Balech isolated from the 1972 New England red tide are nonluminescent. Biochemical components of dinoflagellate bioluminescent. Biochemical components of dinoflagellate bioluminescence were not detected in the extracts from these three isolates. Cells of the nonluminescent cultures were identical to those of iuminescent cultures as compared by light microscopy, major body plate tabulation, cell size and growth. Both luminescent and nonluminescent cells were toxic as determined by using the mouse bioassay for paralytic shellfish poisoning. All the 122 clones made from on of the luminescent isolates were luminescent suggesting this feature is a stable trait. Wer conclude that these isolates represent luminescent and nonluminescent strains of G. exacavata. This is the first intraspecfic investigation of in vitro bioluminescent components between nonluminescent and luminescent strains of a dinoflagellate. [ABSTRACT FROM AUTHOR]

Describes that phase shifts in response to light pulses differ for two different rhythms in the unicellular alga Gonyaulax polyhedra. Dinoflagellates; Circadian oscillator; Phase response curve; Spectral effects; Photoperiod.

Constant red light (RR) influences the Gonyaulax clock in several ways: (1) Phase resetting by white or blue light pulses is stronger under background RR than in constant white light (WW); (2) frequency of the rhythm is less in RR than in WW; and (3) the amplitude of the spontaneous flashing rhythm is greater in RR than in WW. The phase response curve (PRC) to 4-hr white or blue light pulses is of high amplitude (Type 0) for cells in RR, but is of lower amplitude (Type 1) for cells in WW. In all cases, the PRC is highly asymmetrical: The magnitude of advance phase resetting is far higher than that of delay resetting. Consistent with this PRC, Gonyaulax cells in RR (free-running period greater than 24 hr) will entrain to T cycles of between 21 and 26.5 hr. The bioluminescence rhythms exhibit "masking" by blue light pulses while entrained to these T cycles. The fluence response of phase resetting to light-pulse intensity is not linear or logarithmic—rather, it is discontinuous. This feature is consistent with a limit cycle interpretation of Type 0 resetting of circadian clocks. Light pulses that cause large phase shifts also shorten the subsequent free-running period. The phase angle difference between the clock and the previous LD cycle is within 2 hr of the same phase between 16°C and 25°C, as determined from the light PRCs at various temperatures. Several drugs that inhibit mitochondria and/or electron transport will partially inhibit the phase shift by light. [ABSTRACT FROM PUBLISHER]

Populations of Paramecium bursaria cells display a circadian rhythm of photoac cumulation. Both Chlorella-containing cells and Chlorella-free cells exhibit this rhythm. Sev eral other species of Paramecium do not express rhythmic photoaccumulation when tested under the same conditions. In P. bursaria, photoaccumulation rhythms persist in continuous conditions (constant temperature and either continuous light or continuous darkness). The period of this rhythm is "temperature-compensated," with a Q10 of 1.10-1.12. The rhythm can be reset by pulses of light or darkness in a phase-dependent manner. Therefore, an endoge nous circadian oscillator controls photoaccumulation behavior in P. bursaria. [ABSTRACT FROM PUBLISHER]

Like other flagellates, Gonyaulax polyedra exhibits diurnal vertical migration and pattern formation. Shape and size of the aggregations depend on container type, light inten sity, and cell density. In Petri dishes, cells form oval "swarms"; within these, cells move downward in the highly dense center and rise up at the periphery. We have investigated the daily rhythm of this swarming activity in Petri dishes illuminated from the side, using time- lapse video recordings. At night, a "lawn" of cells forms at the bottom of the dish toward the light source (independent of light intensity). Before dawn, cells rise toward the surface and aggregate in swarms. The daily vertical migration occurs independent of light direction and intensity. The diurnal swarms, however, form every day at the same location within the dish, at a distance from the light that depends on light intensity, indicating a self-selection of light intensity. In constant light and temperature and with negligible vertical nutrient differences, all aspects of the rhythm continue to oscillate for up to 3 weeks, when the rhythm of the population becomes desynchronized.Under cycles of bright white-dim red light (WR), cell entrain to WR 10: 10 but free run in WR 8:8 and shorter cycles, showing relative coordination (von Holst, 1939) to the driving light cycle. They also entrain to the 24-hr multiple of WR 6:6. Under nonentrained conditions, swarming activity is still influenced by light changes, and in spite of the apparent free run, the phasing of the averaged activity varies systematically with different T-cycle frequencies. [ABSTRACT FROM PUBLISHER]

A size filtration method to synchronize cultures of the dinoflagellate Gonyaulax polyedra to the beginning of the G1 phase has been developed. This technique selects newly born cells by two sequential filtrations, based on the fact that cell division is restricted to the beginning of the day, so that a decrease in cell volume occurs at this time. The fraction of synchronized cells immediately after the second filtration is about 90%; the procedures do not alter the free-running period or phase of glow rhythm, and the selected cells divide again in a few days. Applying this method, we have found that the generation times of this species in a light-dark cycle (LD 12:12) are indeed quantized to multiples of 24 hr, but are variable from generation to generation. [ABSTRACT FROM PUBLISHER]

We describe a method whereby the effect of protein synthesis inhibitors upon protein synthesis in Gonyaulax cultures may be reliably measured. Using this method, we found that protein synthesis inhibition and clock resetting were correlated, but that the correlation was not as close as has been reported in other systems. The effect of the inhibitors anisomycin and cycloheximide upon phase shifting of the circadian clock was a function of the illumination and temperature conditions to which the cells were subjected, but these factors did not appear to influence the inhibition of protein synthesis by these drugs. Cellular protein synthesis did not recover immediately from the inhibitors' effects; depending upon the previous concentration of the inhibitor, translational recovery from the drugs may require hours. This observation has important implications for the analysis of any phase response curve when the stimulus is a chemical. [ABSTRACT FROM PUBLISHER]

During measurements of the circadian (approximately 24-hr) rhythms of spontaneous bioluminescence in the marine dinoflagellate Gonyaulax polyedra, the individual cultures in vials were shielded from otherwise constant dim light for 1-3 min every 20-60 min by a photomultiplier housing that was moved from vial to vial. The high-frequency dark pulses caused a small but consistent shortening of the free-running circadian period, but there was no indication that the dark pulses caused entrainment. Hardware and software components of the microcomputer-controlled data collection system are described. A microcomputer controlled the movement of the photomultiplier and acquired the data via an analog-to-digital converter. The algorithms distinguished and separately recorded background glow, intermittent flashes, and total light from populations ranging in number from 103 to 105 cells in volumes from 1 to 10 ml. Fast video display techniques allowed continuous on-line viewing of incoming data, together with a display of the data recorded over the preceding day or two. Detection of mechanical and software errors coupled with recovery systems maintained high reliability of data collection. [ABSTRACT FROM PUBLISHER]

The reaction of reduced 1-d-FMN with oxygen and decanal results in bioluminescence with kinetic and spectral properties similar to those of the reaction with FMNH2, even though the spectral (absorbance, fluorescence) and chemical properties of the oxidized forms differ greatly. This emission, which is about 10-15% as efficient as with FMNH2, is postulated to involve the intermediacy of the corresponding 4a-hydroperoxide, the fluorescence of which occurred transiently. The N(1) protonated species had been proposed as the emitter in the reaction with FMNH2, but the 1-deaza analog cannot be protonated at the corresponding position, thus excluding this possibility. [ABSTRACT FROM AUTHOR]

A luciferase-bound neutral flavin semiquinone radical can be formed upon the oxidation of the luciferase- FMNH2 complex by molecular oxygen. This species can also be formed anaerobically by comproportionation of FMN and FMNH2 in the presence of luciferase. The radical is kinetically stable (t1/2 ≈ 20 h at 0 °C in air: the Arrhenius ΔHdecay= being about 170 kJ/mol) and can be prepared in pure form by Sephadex G-25 chromatography at 0-4 C. The pure enzyme-bound radical is inactive for light emission either with or without aldehyde, and is not in (relevantly rapid) equilibrium with the luciferase 4a-peroxyflavin, the active intermediate in the bioluminescent reaction. [ABSTRACT FROM AUTHOR]