The copy number of chloroplast gene minicircles changes dramatically with growth phase in the dinoflagellate Amphidinium operculatum

The chloroplast genome of algae and plants typically comprises a circular DNA molecule of 100-200kb, which harbours approximately 120 genes, and is present in 50-100 copies per chloroplast. However, in peridinin dinoflagellates, an ecologically important group of unicellular algae, the chloroplast genome is fragmented into plasmid-like 'minicircles', each of 2-3kb. Furthermore, the chloroplast gene content of dinoflagellates is dramatically reduced. Only 14 genes have been found on dinoflagellate minicircles, and recent evidence from EST studies suggests that most of the genes typically located in the chloroplast in other algae and plants are located in the nucleus. In this study, Southern blot analysis was used to estimate the copy number per cell of a variety of minicircles during different growth stages in the dinoflagellate Amphidinium operculatum. It was found that minicircle copy number is low during the exponential growth stage but increases during the later growth phase to resemble the situation seen in other plants and algae. The control of minicircle replication is discussed in the light of these findings.     

Mortality in cultures of the dinoflagellate Amphidinium carterae during culture senescence and darkness

The study of cell death in higher plants and animals has revealed the existence of an active ('programmed') process in most types of cell, and similarities in cell death between plants, animals, yeast and bacteria suggest an evolutionarily ancient origin of programmed cell death (PCD). Despite their global importance in primary production, information on algal cell death is limited. Algal cell death could have similarities with metazoan cell death. One morphotype of metazoan PCD, apoptosis, can be induced by light deprivation in the unicellular chlorophyte Dunaliella tertiolecta. The situation in other algal taxa is less clear. We used a model dinoflagellate (Amphidinium carterae) to test whether mortality during darkness and culture senescence showed apoptotic characteristics. Using transmission electron microscopy, fluorescent biomarkers, chlorophyll fluorescence and particulate carbon analysis we analysed the process of cell mortality and found that light deprivation caused mass mortality. By contrast, fewer dead cells (5-20% of the population) were found in late-phase cultures, while a similar degenerate cell morphology (shrunken, chlorotic) was observed. On morphological grounds, our observations suggest that the apoptotic cell death described in D. tertiolecta does not occur in A. carterae. Greater similarity was found with paraptosis, a recently proposed alternative morphotype of PCD. A paraptotic conclusion is supported by inconclusive DNA fragmentation results. We emphasize the care that must be taken in transferring fundamental paradigms between phylogenetically diverse cell types and we argue for a greater consistency in the burden of proof needed to assign causality to cell death processes.     

Amphidinol C,a major polyketide from an Irish strain of the dinoflagellate Amphidinium carterae

An Irish strain of the dinoflagellate Amphidinium carterae was previously shown to produce antibacterial amphidinol derivatives of unknown masses. Inspection of the major metabolites present in a bulk culture of this strain led to the isolation and structure elucidation of a new amphidinol derivative named amphidinol C featuring an unprecedented tetrahydropyran ring between the positions C-7 and C-11. The structure was determined using extensive analyses of NMR and MS data and comparison with data of analogues. The new ring was proposed to stem from a nucleophilic substitution of the sulphate present on the side chain of Amphidinol B. The major metabolites isolated were tested for their antibacterial and antifungal activities and Amphidinol C showed moderate fungicidal activity against yeast and filamentous fungi at 8–16 µg mL⁻¹.     

Flow cytometric analysis of chronic and acute toxicity of copper(II) on the marine dinoflagellate Amphidinium carterae.

BACKGROUND: Copper(II) is a heavy metal whose levels have increased in some marine ecosystems to polluting levels. Dinoflagellates, an important phytoplankton group, are at the base of aquatic food chains and bioaccumulation of copper by these microorganisms can result in complex ecosystem alterations, so we investigated how copper disturbs those cells. METHODS: Cytotoxic effects of sublethal and lethal copper concentrations ranging from 4.2 nM (control condition) to 3.13 microM estimated labile copper were studied in batch cultures of Amphidinium carterae. Cell morphology, motility, autofluorescence, and fluorescein diacetate (FDA)-dependent fluorescence generation were evaluated by flow cytometry (FCM) and microscopy. RESULTS: Exposure of A. carterae to toxic levels of copper impaired cell mobility, delayed cell proliferation, led to increased green autofluorescence, and at 3.13 microM labile copper also induced encystment and death. Chlorophyll fluorescence, however, was not affected. Kinetic FCM assay of FDA-dependent fluorescence generation showed a dose-dependent enhancement of fluorescein fluorescence immediately after copper addition and in cultures with sustained exposure to this toxicant. CONCLUSIONS: Our data suggest that copper toxicity occurs quickly at the membrane level in relation to oxidative stress generation. Based on fluorescence kinetic studies, the Na(+)/H(+) antiporter seemed to be affected by copper, thereby affecting intracellular pH.     

Evidence for cAMP-dependent protein kinase in the dinoflagellate,Amphidinium operculatum

A cAMP dependent protein kinase (PKA) was identified in the dinoflagellate Amphidinium operculum. In vitro kinase activity towards kemptide, a PKA-specific substrate, was not detectable in crude lysates. However, fractionation of dinoflagellate extracts by gel filtration chromatography showed PKA-like activity toward kemptide at approximately 66 kDa. These findings suggest that possible low molecular mass inhibitors in crude lysates were removed by the gel filtration chromatography. Pre-incubation of extracts with cAMP prior to chromatography resulted in an apparent molecular mass shift in the in vitro kinase assay to 40 kDa. An in-gel kinase assay reflected activity of the free catalytic subunit at approximately 40 kDa. Furthermore, western blotting with an antibody to the human PKA catalytic subunit confirmed a catalytic subunit with a mass of approximately 40 kDa. Results from this study indicate that the PKA in A. operculatum has a catalytic subunit of similar size to that in higher eukaryotes, but with a holoenzyme of a size suggesting a dimeric, rather than tetrameric structure.     

'Empty' minicircles and petB/atpA and psbD/psbE (cytb559 alpha) genes in tandem in Amphidinium carterae plastid DNA

Amphidinium carterae minicircle chloroplast DNA was separated from total DNA by centrifugation through a sucrose/NaCl gradient. Sequences of minicircles with psbA and 23S rRNA contained a common region of 67 bp. Primers designed from this generated numerous polymerase chain reaction products of 1.5-2.6 kb. These contained psaA and psaB as one gene/circle, and petB/atpA and psbD/psbE as two genes/circle. 'Empty' minicircles of 1.7-2.5 kb containing no identifiable genes or parts of genes were more abundant than gene-containing circles. From 15 minicircles a minimum common region of 48 bp was identified, with little identity to that from other dinoflagellate minicircles.     

Integrated analysis of gene copy number, copy neutral LOH, and microRNA profiles in adult acute lymphoblastic leukemia

We adopted an integrated analysis of gene copy number alterations (CNAs), copy number neutral loss of heterozygosity (CNN LOH), and microRNA (miRNA) profiling in 21 adult acute lymphoblastic leukemia (ALL) patients. This study revealed the most frequent CNAs to be at chromosomes 9p, 7, and 17 and recurrent CNN LOH at 5p, 9p, and Xq. As for the most differentially expressed miRNAs, they included 8 upregulated and 14 downregulated miRNAs, of which miR-148a at 7p15.2, miR-22 at 17p13.3, miR-223 at Xq12, as well as miR-101-2 at 9p24.1 exhibited recurrent CNAs or CNN LOH. miR-101-2 was recurrently downregulated, and although the related CNN LOH was detected only in BCR-ABL1 negative cases (2/14), deletions of miR-101-2 were observed solely in BCR-ABL1 positive cases (4/7). Finally, BCR-ABL1 positive cases, in contrast to negative ones, were characterized by slightly, but still significantly, higher expression levels of miR-29b.     

Energy transfer in the peridinin chlorophyll-a protein of Amphidinium carterae studied by polarized transient absorption and target analysis

The peridinin chlorophyll-a protein (PCP) of dinoflagellates differs from the well-studied light-harvesting complexes of purple bacteria and green plants in its large (4:1) carotenoid to chlorophyll ratio and the unusual properties of its primary pigment, the carotenoid peridinin. We utilized ultrafast polarized transient absorption spectroscopy to examine the flow of energy in PCP after initial excitation into the strongly allowed peridinin S2 state. Global and target analysis of the isotropic and anisotropic decays reveals that significant excitation (25-50%) is transferred to chlorophyll-a directly from the peridinin S2 state. Because of overlapping positive and negative features, this pathway was unseen in earlier single-wavelength experiments. In addition, the anisotropy remains constant and high in the peridinin population, indicating that energy transfer from peridinin to peridinin represents a minor or negligible pathway. The carotenoids are also coupled directly to chlorophyll-a via a low-lying singlet state S1 or the recently identified SCT. We model this energy transfer time scale as 2.3 +/- 0.2 ps, driven by a coupling of approximately 47 cm(-1). This coupling strength allows us to estimate that the peridinin S1/SCT donor state transition moment is approximately 3 D.     

Investigations on gene copy number, introns and chromosomal arrangement of genes encoding the fucoxanthin chlorophyll a/c-binding proteins of the centric diatom Cyclotella cryptica

The gene arrangement, existence of introns and the number of gene copies of genes (fcps) encoding fucoxanthin chlorophyll a/c-binding proteins (Fcps) of the centric diatom Cyclotella cryptica were investigated by polymerase chain reaction (PCR), Southern blotting and denaturing gradient gel electrophoresis (DGGE) experiments. PCR-mediated amplification of the fcp genes using chromosomal DNA as template demonstrated the absence of introns within the amplified regions. Clustering of genes could not be demonstrated in these experiments. Digestion of chromosomal DNA of Cy. cryptica followed by Southern blotting and hybridization with specific fcp probes revealed minimum and maximum values of 12 and 20, respectively, for the gene copies. In addition, the DGGE technique confirmed and strengthened the results obtained from Southern blotting experiments as amplification of gene fragments from genomic DNA with different sets of specific primers revealed values of 21 and 23, for the minimum and maximum gene copy number, respectively.