Permanent expression of a cyclin B homologue in the cell cycle of the dinoflagellate Karenia brevis

The eukaryotic cell cycle is driven by a set of cyclin-dependent kinases associated with their regulatory partners, the cyclins, which confer activity, substrate specificities and proper localization of the kinase activity. We describe the cell cycle of Karenia brevis and provide evidence for the presence of a cyclin B homologue in this dinoflagellate using two antibodies with different specificities. This cyclin B homologue has an unusual behavior, since its expression is permanent and it has a cytoplasmic location throughout the cell cycle. There is no evidence for translocation to the nucleus during mitosis. However, it appears also to be specifically bound to the nucleolus throughout the cell cycle. The permanent expression and the cytoplasmic localization during mitosis of this cyclin B homologue is similar to p56, a cyclin B homologue previously described in a different species of dinoflagellate, Crypthecodinium cohnii. Here we discuss this unusual behavior of the cyclin B homologue in dinoflagellates, its relationship to the unusual characteristics of dinomitosis, and its potential implications regarding the evolution of cell cycle regulation among eukaryotes.     

Microsatellite DNA markers for population genetic studies in the dinoflagellate Karenia brevis

Nine nuclear‐encoded microsatellites from an enriched genomic DNA library of the HAB (harmful algal bloom) dinoflagellate Karenia brevis were isolated and characterized. The microsatellites include five perfect (three dinucleotide and two trinucleotide) and four imperfect (two dinucleotide and two trinucleotide) repeat motifs. Gene (haplotype) diversity ranged from 0.153 to 0.750 among a sample of 13 isolates; the number of alleles among the isolates ranged from two to six and pairwise tests of genotypic disequilibria were nonsignificant. The microsatellites developed in this study will provide insight into the genetic diversity of this HAB species and tools that may prove useful in predicting source populations and physiological parameters of individual K. brevis blooms.     

Chimeric plastid proteome in the Florida "red tide" dinoflagellate Karenia brevis

Current understanding of the plastid proteome comes almost exclusively from studies of plants and red algae. The proteome in these taxa has a relatively simple origin via integration of proteins from a single cyanobacterial primary endosymbiont and the host. However, the most successful algae in marine environments are the chlorophyll c-containing chromalveolates such as diatoms and dinoflagellates that contain a plastid of red algal origin derived via secondary or tertiary endosymbiosis. Virtually nothing is known about the plastid proteome in these taxa. We analyzed expressed sequence tag data from the toxic "Florida red tide" dinoflagellate Karenia brevis that has undergone a tertiary plastid endosymbiosis. Comparative analyses identified 30 nuclear-encoded plastid-targeted proteins in this chromalveolate that originated via endosymbiotic or horizontal gene transfer (HGT) from multiple different sources. We identify a fundamental divide between plant/red algal and chromalveolate plastid proteomes that reflects a history of mixotrophy in the latter group resulting in a highly chimeric proteome. Loss of phagocytosis in the "red" and "green" clades effectively froze their proteomes, whereas chromalveolate lineages retain the ability to engulf prey allowing them to continually recruit new, potentially adaptive genes through subsequent endosymbioses and HGT. One of these genes is an electron transfer protein (plastocyanin) of green algal origin in K. brevis that likely allows this species to thrive under conditions of iron depletion.     

Sterol biosynthesis in the harmful marine dinoflagellate,Karenia brevis: Identification of biosynthetic intermediates produced during exposure to the fungicide fenpropidine

Karenia brevis is a harmful marine dinoflagellate that forms yearly blooms in the Gulf of Mexico. Under normal growth conditions, K. brevis forms two predominant sterols (24R)‐4α‐methyl‐5α‐ergosta‐8(14),22‐dien‐3β‐ol (gymnodinosterol) and its 27‐nor isomer (brevesterol). At the current time, there are no published studies concerning the biosynthesis of these two sterols. We have therefore undertaken experiments in which K. brevis was exposed to the fungicide fenpropidine, an inhibitor of the Δ¹⁴‐reductase and the Δ^8→7‐isomerase that operate in sterol biosynthesis in both fungal and plant systems. Such exposure to fenpropidine has produced two, tri‐unsaturated intermediates. The identifications of these two K. brevis sterol biosynthesis intermediates, via gas chromatography/mass spectrometry and nuclear magnetic resonance spectroscopy techniques, were 4α‐methyl‐5α‐ergosta‐8,14,22‐trien‐3β‐ol and 5α‐ergosta‐8,14,22‐trien‐3β‐ol.     

Localization of polyketide synthase encoding genes to the toxic dinoflagellate Karenia brevis

Karenia brevis is a toxic marine dinoflagellate endemic to the Gulf of Mexico. Blooms of this harmful alga cause fish kills, marine mammal mortalities and neurotoxic shellfish poisonings. These harmful effects are attributed to a suite of polyketide secondary metabolites known as the brevetoxins. The carbon framework of all polyketides is assembled by a polyketide synthase (PKS). Previously, PKS encoding genes were amplified from K. brevis culture and their similarity to a PKS gene from the closely related protist, Cryptosporidium parvum, suggested that these genes originate from the dinoflagellate. However, K. brevis has not been grown axenically. The associated bacteria might be the source of the toxins or the PKS genes. Herein we report the localization of PKS encoding genes by a combination of flow cytometry/PCR and fluorescence in situ hybridization (FISH). Two genes localized exclusively to K. brevis cells while a third localized to both K. brevis and associated bacteria. While these genes have not yet been linked to toxin production, the work describes the first definitive evidence of resident PKS genes in any dinoflagellate.     

Molecular detection of the brevetoxin‐producing dinoflagellate Karenia brevis and closely related species using rRNA‐targeted probes and a semiautomated sandwich hybridization assay1

Brevetoxins produced by the marine dinoflagellate Karenia brevis (C. C. Davis) G. Hansen et Moestrup cause neurotoxic shellfish poisoning (NSP) in human consumers and also endanger a variety of coastal wildlife. In the eastern Gulf of Mexico the presence and abundance of this species have traditionally been monitored using light microscopy (LM) observations of whole water samples. Various molecular probe methods now enable detection of multiple species from a single sample, allowing rapid sample analysis. We describe the development of sandwich hybridization assays (SHAs) for Karenia brevis, K. selliformis Haywood, Steid. et L. MacK., K. mikimotoi (Miyake et Kominami ex M. Oda) G. Hansen et Moestrup, K. papilionacea Haywood et Steid., the Karlotoxin‐producer Karlodinium veneficum (D. Ballant.) J. Larsen (=K. micrum), and Gymnodinium aureolum (Hulburt) G. Hansen, comb. nov. The assays require no nucleic acid purification and use LSU rRNA‐targeted probes and a semiautomated, 96‐well plate format. Probes tested in matrix format were specific relative to rRNAs of all nontarget species used. The response of the SHA for a constant number of K. brevis cells per unit volume of homogenate depended on the growth status of a culture, decreasing for senescent cells relative to actively growing cells. The results of preliminary field tests of the K. brevis SHA indicated that cells collected from natural populations tended to return a lower signal than those harvested from laboratory cultures, but these results are nonetheless very encouraging. These preliminary field studies show that robust standards are required for cell identification and enumeration, with which new methods can be compared.     

Severe Karenia brevis red tides influence juvenile bottlenose dolphin (Tursiops truncatus) behavior in Sarasota Bay,Florida

Harmful algal blooms (HABs) are natural stressors in the coastal environment that may be increasing in frequency and severity. This study investigates whether severe red tide blooms, caused by Karenia brevis, affect the behavior of resident coastal bottlenose dolphins in Sarasota Bay, Florida through changes to juvenile dolphin activity budgets, ranging patterns, and social associations. Behavioral observations were conducted on free‐ranging juvenile dolphins during the summer months of 2005–2007, and behavior during red tide blooms was compared to periods of background K. brevis abundance. We also utilized dolphin group sighting data from 2004 to 2007 to obtain comparison information from before the most severe recent red tide of 2005 and incorporate social association information from adults in the study area. We found that coastal dolphins displayed a suite of behavioral changes associated with red tide blooms, including significantly altered activity budgets, increased sociality, and expanded ranging behavior. At present, we do not fully understand the mechanism behind these red tide‐associated behavioral effects, but they are most likely linked to underlying changes in resource availability and distribution. These behavioral changes have implications for more widespread population impacts, including increased susceptibility to disease outbreaks, which may contribute to unusual mortality events during HABs.     

Uptake and localization of fluorescently‐labeled Karenia brevis metabolites in non‐toxic marine microbial taxa

Brevetoxin (PbTx) is a neurotoxic secondary metabolite of the dinoflagellate Karenia brevis. We used a novel, fluorescent BODIPY‐labeled conjugate of brevetoxin congener PbTx‐2 (B‐PbTx) to track absorption of the metabolite into a variety of marine microbes. The labeled toxin was taken up and brightly fluoresced in lipid‐rich regions of several marine microbes including diatoms and coccolithophores. The microzooplankton (20–200 μm) tintinnid ciliate Favella sp. and the rotifer Brachionus rotundiformis also took up B‐PbTx. Uptake and intracellular fluorescence of B‐PbTx was weak or undetectable in phytoplankton species representative of dinoflagellates, cryptophytes, and cyanobacteria over the same (4 h) time course. The cellular fate of two additional BODIPY‐conjugated K. brevis associated secondary metabolites, brevenal (B‐Bn) and brevisin (B‐Bs), were examined in all the species tested. All taxa exhibited minimal or undetectable fluorescence when exposed to the former conjugate, while most brightly fluoresced when treated with the latter. This is the first study to observe the uptake of fluorescently‐tagged brevetoxin conjugates in non‐toxic phytoplankton and zooplankton taxa, demonstrating their potential in investigating whether marine microbes can serve as a significant biological sink for algal toxins. The highly variable uptake of B‐PbTx observed among taxa suggests some may play a more significant role than others in vectoring lipophilic toxins in the marine environment.     

GFP-PCNA as an S-phase marker in embryos during the first and subsequent cell cycles

BACKGROUND INFORMATION: Proliferating cell nuclear antigen (PCNA) is a key component of the DNA replication machinery involved in the process of DNA elongation, recombination, methylation and repair. We have used PCNA fused with green fluorescent protein (GFP-PCNA) as a convenient tool to show the progress of S-phase in single embryos in vivo. Here we make a comparison between Hoechst 33342 and GFP-PCNA as in vivo event markers for DNA synthesis. Hoechst 33342 and DAPI (4,6-diamidino-2-phenylindole) have been used as a simple and rapid method for assessing membrane permeability and staining DNA in mammalian cells. However, it is difficult to use these dyes in living embryos during cell cycle progression studies over long periods of time as they are phototoxic. Moreover, though Hoechst staining reveals nuclear morphology, it gives no information about the progress of S-phase. RESULTS: We have microinjected or expressed a GFP-PCNA chimera to develop a method which enables visualization of S-phase in sea urchin and Caenorhabditis elegans embryos during the first and subsequent embryonic cell cycles and in Drosophila stage 4 embryos during syncytial nuclear divisions. We find that nuclear accumulation of GFP-PCNA correlates with S-phase onset. Loss of the chimera from the nucleus occurs when the nuclear envelope breaks down at mitosis. CONCLUSIONS: GFP-PCNA is a accurate and non-toxic marker of S-phase in embryos during early development.     

转基因植物转录后基因沉默的特点、机理及应用

自在转基因植物中发现转基因沉默现象以来 ,很多学者对该现象进行了广泛的研究 ,认为其作用机制有三种 :位置依赖性基因沉默、转录水平基因沉默、转录后水平基因沉默。目前主要集中于转录后水平基因沉默的研究 ,通过研究发现它具有广泛性、可传导性及特异性的特点 ,并对其机制提出了一些假说。最近 ,人们还发现转录后水平基因沉默与植物抗病毒能力有联系。本文对转基因植物转录后基因沉默的特点、机理及应用方面的进展作了综述     

Effects of the red tide dinoflagellate, Karenia brevis, on grazing and fecundity in the copepod Acartia tonsa

Among studies of copepod grazers fed harmful algae, decreasedgrazing and fecundity are the most common results. The causesof decreased grazing (physiological incapacitation, behavioralavoidance or lack of stimulation) and decreased fecundity (toxicversus nutritional effect) vary among studies. This study useda series of controlled laboratory experiments to investigatethe cause of decreased grazing and fecundity in the copepodAcartia tonsa fed sole and mixed diets of the harmful alga,Karenia brevis. Copepods fed K. brevis mixed with the nutritionallyviable dinoflagellate Peridinium foliaceum had higher ingestionrates and offspring production than copepods fed a sole dietof K. brevis (even when K. brevis was virtually nontoxic). Copepodsfed mixtures did not discriminate between P. foliaceum and K.brevis while feeding. The results of this study suggest thatK. brevis is not toxic to A. tonsa but lacks some chemical componentresponsible for stimulating a grazing response in A. tonsa aswell as the nutritional requirements for normal offspring production.