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1.
Eighty-seven axenic clones of the colorless inshore dinoflagellate Crypthecodinium cohnii were found by mating experiments to fall into 52 sibling species, seven wide ranging (two possibly global)—called major sibling species—and 45 found only once—minor sibling species. Electrophoretic analysis of three soluble enzymes from these strains revealed the following: 1) Despite some polymorphism most members of major sibling species closely resemble one another electrophoretically. 2) Major sibling species and most minor ones are electrophoretically distinct. 3) Sharing of electromorphs is sufficiently extensive, however, that no major sibling species is totally unrelated to all others. 4) Some minor sibling species are electrophoretically indistinguishable from a member of a major sibling species or from one another, suggesting recent origin by sexual isolation in situ. 5) Other minor sibling species differ from majors by one, two, or all three of the enzymes studied. A “model” of sexual isolation and diversification is offered.  相似文献   

2.
ABSTRACT. Sixty-five members of the Ctypthecodinium cohnii species complex were analyzed for sequence differences within the D2 region of the 23S ribosomal RNA molecule. On the basis of 46 sequence differences the strains fell into 19 distinct ribosets (strains of identical sequence), some with many members. Members of four of the seven major sibling species (widespread breeding groups) were each found within single ribosets. Members of three other major sibling species were each, however, divided into two ribosets by a single sequence difference correlated with geographic separation and with previously reported electrophoretic polymorphisms of soluble enzymes within the sibling species. In addition to members of major sibling species, some ribosets include many minor sibling species (each represented by only one strain). Of 38 minor sibling species, 22 shared sequence with a major sibling species. Of these 22, 14 were identical in soluble enzymes to their related major sibling species or differed by only one of three enzymes. Other minor sibling species appear to have diverged extensively from any others in both rRNA sequence and electrophoretic profile. As a group, major sibling species differ markedly in the number of minor sibling species associated with them, suggesting differences in frequency of sexually isolating events in their past histories. These findings are discussed in the context of the previously proposed model of sympatric speciation.  相似文献   

3.
P J Rizzo  R L Morris 《Bio Systems》1983,16(3-4):211-216
The histone-like protein from Crypthecodinium cohnii (HCc) was examined in regard to its amino acid composition and the peptide pattern resulting from protease digestion. A revised amino acid composition indicated a higher lysine and arginine content and a lower glycine content than that determined previously. Comparative peptide mapping of HCc with HTa, a histone-like protein from Thermoplasma acidophilum, and with a histone-like protein from the dinoflagellate Gyrodinium dorsum showed significant differences in the peptide patterns produced.  相似文献   

4.
Poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase have been detected in chromatin extracts from the dinoflagellate Crypthecodinium cohnii. Poly(ADP-ribose) glycohydrolase was detected by the liberation of ADP-ribose from poly(ADP-ribose). Poly(ADP-ribose) polymerase was proved by (a) demonstration of phosphoribosyl-AMP in the phosphodiesterase digest of the reaction product, (b) demonstration of ADP-ribose oligomers by fractionation of the reaction product on DEAE-Sephadex. The (ADP-ribose)-protein transfer is dependent on DNA; it is inhibited by nicotinamide, thymidine, theophylline and benzamide. The protein-(ADP-ribose bond is susceptible to 0.1 M NaOH (70%) and 0.4 M NH2OH (33%). Dinoflagellates, nucleated protists, are unique in that their chromatin lacks histones and shows a conformation like bacterial chromatin [Loeblich, A. R., III (1976) J. Protozool. 23, 13--28]; poly(ADP-ribose) polymerase, however, has been found only in eucaryotes. Thus our results suggest that histones were not relevant to the establishment of poly(ADP-ribose) during evolution.  相似文献   

5.
SYNOPSIS.
The carotenoid compositions of 15 nitrosoguanidine-induced mutants of Crypthecodinium cohnii , a heterotrophic dinoflagellate, were determined by chromatographic and mass spectral analyses. Wild-type C. cohnii grown with irradiation of 250 W/cm2 visible light at 27 C synthesizes β-carotene (33%) and γ-carotene (67%) amounting to 0.083 mg/g dry wt. There are 4 types of carotenoid-deficient mutants: (I) albinos which synthesize no C40-carotonoids: (II) albinos blocked at the level of phytoene desaturation; (III) cream-colored cells which accumulate mainly §–carotene, with phytoene and/or β-zeacarotene also present; and (IV) light-orange strains which synthesize reduced amounts of β-carotene and γ-carotene.
Dark-grown wild-type cells produced 35% as much carotenoids as light-grown cells. Inhibition studies revealed that diphenylamine (3 γ) caused phytoene accumulation; nicotine at 0.9 mM blocked the final cyclization, to cause γ-carotene to accumulate in wild-type cells. Inhibition by adenine and guanine (1.5 mM) of carotenogenesis was demonstrated for the first time in any system. The effect of these purines was similar to that of diphenylamine addition: phytoene desaturation was largely inhibited.
The carotenogenic system in this dinoflagellate is similar to that of green algae and higher plants, and is under nuclear genetic control.  相似文献   

6.
Dinoflagellates are a major group of organisms with an extranuclear spindle. As the purpose of the spindle checkpoint is to ensure proper alignment of the chromosomes on the spindle, dinoflagellate cell cycle control may be compromised to accomodate the extranuclear spindle. In the present study, we demonstrated that nocodazole reversibly prolonged the G2 + M phase of the dinoflagellate cell cycle, in both metaphase and anaphase. The regulation of the spindle checkpoint involves the activation and inhibition of the anaphase promoting complex (APC), which in turn degrades specific cell cycle regulators in the metaphase to anaphase transition. In Crypthecodinium cohnii, nocodazole was also able to induce a prolongation of the degradation of mitotic cyclins and a delay in the inactivation of p13(suc1)-associated histone kinase activities. In addition, cell extracts prepared from C. cohnii in G1 phase and G2/M phase (or nocodazole treated) were able to activate and inhibit, respectively, the degradation of exogenous human cyclin B1 in vitro. The present study thus demonstrated the presence of the spindle checkpoint and APC-mediated cyclin degradation in dinoflagellates. This is discussed in relation to a possible role of the nuclear membrane in mitosis in dinoflagellates.  相似文献   

7.
The valuable polyunsaturated fatty acid, docosahexaenoic acid, can be produced by cultivation of the heterotrophic microalga, Crypthecodinium cohnii. During batch growth of C. cohnii on glucose, sea salt and yeast extract for 5 days, so far unreported extracellular polysaccharides were produced. These caused an increased viscosity and a strong drop in the maximum oxygen transfer. The viscosity increased most markedly as cells entered the stationary phase. The polysaccharides varied in size (from 6 kDa to >1,660 kDa) and monomer distribution. A high molecular mass fraction (from 100 kDa to >1,660 kDa) and a medium molecular mass fraction (6-48 kDa) were prepared. The high molecular mass fraction contained (on a molar basis) 71.7% glucose, 13.1% galactose and 3.8% mannose, whereas the medium molecular mass fraction contained 37.7% glucose, 19.8% galactose and 28.1% mannose. Other monomers present in both fractions were fucose, uronic acid and xylose. Monomers were coupled mainly via alpha-(1-3) links. Increased viscosity due to polysaccharide production complicates the development of commercial, high cell-density processes for the production of docosahexaenoic acid.  相似文献   

8.
Atypical eukaryotic RNA polymerase activity was demonstrated in nuclei of Crypthecodinium cohnii, a eukaryote devoid of histones. Nuclei were isolated from growing cultures of this dinoflagellate and assayed for endogenous RNA polymerase (EC 2.7.7.6) activity. There was a biphasic response to Mg2+ with optima at ? 0.01 and 0.02 M MgCl2, but in contrast to other eukaryotic RNA polymerases, this enzyme activity was inhibited by low MnCl2 concentrations. In the presence of 0.01 M MgCl2 the optimum (NH4)2SO4 concentration was 0.025 M, a concentration at which the nuclei were lysed. Incorporation of [3H]UMP into RNA was inhibited by actinomycin D and dependent on the presence of undegraded DNA, and the reaction product was sensitive to ribonuclease and KOH digestion. Omission of one or more ribonucleoside triphosphates greatly reduced the incorporation. Only a slight enhancement of RNA polymerase activity resulted from the addition of various amounts of native and denatured calf thymus DNA. Spermine caused a marked inhibition while spermidine had little effect on RNA synthesis in the nuclei. Under the optimum conditions described in the present paper the nuclei incorporated ? 3 pmoles of [3H]UMP/muml; DNA at 25 C for 15 min, and ? 80% of this activity was inhibited by the eukaryotic RNA polymerase II inhibitor, α-amanitin (20 m?/ml). A unique situation therefore exists in C. cohnii nuclei, in which absence of histones (a prokaryotic trait) is combined with α-amanitin-sensitive RNA polymerase activity (a eukaryotic trait).  相似文献   

9.
The distribution of four enzymes within the endomembrane system of the protist Crypthecodinium cohnii has been determined using cytochemical localizations with lead as a capture agent. Nucleoside diphosphatase (NDPase) activity, using inosine diphosphate (IDP) and thiamine pyrophosphate (TPP) as substrates, was observed in the Golgi apparatus, with a gradient of increasing reaction product noted in some cells from the cis to trans cisternae. Tubules and vesicles associated with the trans cisternae also contained reaction product. The endoplasmic reticulum exhibited a high activity of glucose-6-phosphatase [with glucose-6-phosphate (G-6-P) as substrate]. Traces of reaction product were also observed in the cis-most and trans-most cisternae of the dictyosomes. Activity of acid phosphatase (AcPase) was observed in Golgi cisternae as well as in associated cytoplasmic vesicles. Heaviest deposition was localized in medial and trans dictyosome cisternae. The cytoplasmic system of flattened vesicles subtending the surface membranes in these cells did not exhibit reactivity with any of the substrates used. The distribution of these enzymes in this algal cell appears similar to that observed in animal cells and suggests that these enzymes may represent markers for algal cell endomembrane compartments.  相似文献   

10.
Mutant strains were chemically induced by treatment with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and UV irradiation. UV and NTG mutation rates were obtained that were both consistent with the organism being haploid. Three types of mutants were produced: (a) strains deficient in both beta- and gamma-carotene, the only carotenoids found in the wild type; phenotypes include albinos (translucent, dull white, "snow white") and cream-colored on agar as compared to the yellow-orange color of wild type colonies; (b) strains requiring adenine, guanine or cytosine in addition to the minimal medium for growth; (c) mutants that grow at a rate less than 40% of the wild type in minimal medium.  相似文献   

11.
Atypical eukaryotic RNA polymerase activity was demonstrated in nuclei of Crypthecodinium cohnii, a eukaryote devoid of histones. Nuclei were isolated from growing cultures of this dinoflagellate and assayed for endogenous RNA polymerase (EC 2.7.7.6) activity. There was a biphasic response to Mg2+ with optima at approximately 0.01 and 0.02 M MgCl2, but in contrast to other eukaryotic RNA polymerases, this enzyme activity was inhibited by low MnCl2 concentrations. In the presence of 0.01 M MgCL2 the optimum (NH4)2SO4 concentration was 0.025 M, a concentration at which the nuclei were lysed. Incorporation of [3H]UMP into RNA was inhibited by actinomycin D and dependent on the presence of undergraded DNA, and the reaction product was sensitive to ribonuclease and KOH digestion. Omission of one or more ribonucleoside triphosphates greatly reduced the incorporation. Only a slight enhancement of RNA polymerase activity resulted from the addition of various amounts of native and denatured calf thymus DNA. Spermine caused a marked inhibition while spermidine had little effect on RNA synthesis in the nuclei. Under the optimum conditions described in the present paper the nuclei incorporated approximately 3 pmoles of [3H]UMP/microgram DNA at 25 C for 15 min, and approximately 80% of this activity was inhibited by the eukaryotic RNA polymerase II inhibitor, alpha-amanitin (20 micrograms/ml). A unique situation therefore exists in C. cohnii nuclei, in which absence of histones (a prokaryotic trait) is combined with alpha-amanitin-sensitive RNA polymerase activity (a eukaryotic trait).  相似文献   

12.
The heterotrophic marine microalga Crypthecodinium cohnii produces docosahexaenoic acid (DHA), a polyunsaturated fatty acid with food and pharmaceutical applications. So far, DHA production has been studied with glucose and acetic acid as carbon sources. This study investigates the potential of ethanol as an alternative carbon source for DHA production by C. cohnii. In shake-flask cultures, the alga was able to grow on ethanol. The specific growth rate was optimal with 5 g l(-1) ethanol and growth did not occur at 0 g l(-1) and above 15 g l(-1). By contrast, in fed-batch cultivations with a controlled feed of pure ethanol, cumulative ethanol addition could be much higher than 15 g l(-1), thus enabling a high final cell density and DHA production. In a representative fed-batch cultivation of C. cohnii with pure ethanol as feed, 83 g dry biomass l(-1), 35 g total lipid l(-1) and 11.7 g DHA l(-1) were produced in 220 h. The overall volumetric productivity of DHA was 53 mg l(-1 )h(-1), which is the highest value reported so far for this alga.  相似文献   

13.
The potential use of n-dodecane as an oxygen vector for enhancement of Crypthecodinium cohnii growth and docosahexaenoic acid (DHA) production was studied. The volumetric fraction of oxygen vector influenced the gas–liquid volumetric mass transfer coefficient k L a positively. The k L a increased almost linearly with the increase of volumetric fraction of n-dodecane up to 1%. The stirring rate showed a higher influence on the k L a than the aeration rate. The effects of this hydrocarbon on C. cohnii growth and DHA production were then investigated. A control batch fermentation without n-dodecane addition (CF) and a batch fermentation where n-dodecane 1% (v/v) was added (DF) were carried out simultaneously under the same experimental conditions. It was found that, before 86.7 h of fermentation, the biomass concentration, the specific growth rate, the DHA, and total fatty acids (TFA) production were higher in the CF. After this fermentation time, the biomass concentration, the DHA and TFA production were higher in the DF. The highest DHA content of biomass (6.14%), DHA percentage of TFA (51%), and DHA production volumetric rate r DHA (9.75 mg l−1 h−1) were obtained at the end of the fermentation with n-dodecane (135.2 h). The dissolved oxygen tension (DOT) was always higher in the DF, indicating a better oxygen transfer due to the oxygen vector presence. However, since the other C. cohnii unsaturated fatty acids percentages did not increase with the oxygen availability increase due to the n-dodecane presence, a desaturase oxygen-dependent mechanism involved in the C. cohnii DHA biosynthesis was not considered to explain the DHA production increase. A selective extraction through the n-dodecane was suggested.  相似文献   

14.
Calcium plays several important roles in the signal transduction pathways of dinoflagellates. We describe here the development of calcium orange-AM as an intracellular calcium reporter for the heterotrophic dinoflagellate Crypthecodinium cohnii. We demonstrated with confocal microscopy that by restricting the incubation period to 30-45 min, no compartmentalization of the dye occurs in the mitochondria or endoplasmic reticulum. The dye fluorescence responded well to the effects of calcium ionophores and calcium chelators. By calibrating the dye with known calcium concentrations, we determined the intracellular calcium concentration of C. cohnii to be 158 +/- 56 nM, which rose to about 550 nM upon mechanical stimulation.  相似文献   

15.
Alexandrium catenella (Whedon et Kof.) Balech, A. tamarense (M. Lebour) Balech, and A. fundyense Balech comprise the A. tamarense complex, dinoflagellates responsible for paralytic shellfish poisoning worldwide. The relationships among these morphologically defined species are poorly understood, as are the reasons for increases in range and bloom occurrence observed over several decades. This study combines existing data with new ribosomal DNA sequences from strains originating from the six temperate continents to reconstruct the biogeography of the complex and explore the origins of new populations. The morphospecies are examined under the criteria of phylogenetic, biological, and morphological species concepts and do not to satisfy the requirements of any definition. It is recommended that use of the morphospecies appellations within this complex be discontinued as they imply erroneous relationships among morphological variants. Instead, five groups (probably cryptic species) are identified within the complex that are supported on the basis of large genetic distances, 100% bootstrap values, toxicity, and mating compatibility. Every isolate of three of the groups that has been tested is nontoxic, whereas every isolate of the remaining two groups is toxic. These phylogenetic groups were previously identified within the A. tamarense complex and given geographic designations that reflected the origins of known isolates. For at least two groups, the geographically based names are not indicative of the range occupied by members of each group. Therefore, we recommend a simple group‐numbering scheme for use until the taxonomy of this group is reevaluated and new species are proposed.  相似文献   

16.
Details of the general DNA sequence organization in the dinoflagellate Crypthecodinium cohnii have been obtained by using hydroxylapatite binding experiments, S1 nuclease digestion .and electron microscopy of reassociated DNA. It has been found that roughly half of the genome is made up of unique sequences interspersed with repeated sequence elements with a period of approximately 600 nucleotides. This class represents roughly 95% of the total number of interspersed unique elements in the genome. The remaining 5% are uninterrupted by repeated sequences for at least 4000 nucleotide pairs. The interspersed repeated elements are narrowly distributed in length with 80% under 300 nucleotide pairs in length. About half of the repeated DNA (20-30% of the genome) is not interspersed among unique sequences. The close spacing of the short repeats interspersed throughout much of the genome is consistent with the occurrence of the huge network structures observed in the electron microscope for low Cot reassociation of moderately long fragments. An unusual class of heteroduplexes was detected in the electron microscope which is believed to derive from the reassociation of repeated sequences from different families which are frequently found adjacent to one another in different locations in the genome. The occurrence of this novel arrangement of repeated sequences may reflect the unusual organization of the dinoflagellate nucleus. However, in most respects the sequence arrangement in this unicellular alga is very typical of higher plants and animals.  相似文献   

17.
Dinolflagellate is one of the primitive eukaryotes,whose nucleus may represent one of the transition stages from prokaryotic nucleoid to typical eukaryotic nucleus,Using selective extraction together with embeddment-free section and whole mount electron microscopy,a delicate nuclear matrix filament network was shown,for the first time,in dinoflagellate Crypthecodinium cohnii nucleus,Chromosome residues are connected with nuclear matrix filaments to form a complete network spreading over the nucleus,Moreover,we demonstrated that the dinoflagellate chromosome retains a protein scafflod after the depletion of DNA and soluble proteins.This scaffold preserves the characterstic morphology of the chromosome.Two dimensional electrophoreses indicated that the nuclear matrix and chromosome scaffold are mainly composed of acidic proteins.Our results demonstrated that a framework similar th the nuclear matrix and chromosome scaffold in mammalian cells appears in this primitive eukaryote,suggesting that these structures may have been originated from the early stages of eukaryote evolution.  相似文献   

18.
ABSTRACT By adding the protein synthesis inhibitor, emetine (10-4 M) to a highly synchronized population of Crypthecodinium cohnii Biecheler 1938 at different phases of its cycle, we were able to determine: 1. The existence and the lengthening of the G2-Phase (30 min) in the first cycle (cycle with swimming G1 phase). 2. The time of the second cell cycle phases (cycle in the cyst): G1, 30 min; S, 1.5 h; G2, 2 h and M, 2 h. These results, together with the estimation of the cell volume of the two and four swimming daughter cells emerging from the cysts, allowed us to state the existence of two transition points: G1/S and G2/M, which are necessary for completion of mitosis. We completed this refined approach of the cell cycle in studying the activities of the histone H1 kinase either in dividing or in non-dividing Crypthecodinium cohnii cells with either total soluble proteins or the isolated mitotic kinase complex. The H1 kinase activity of this purified complex is noticeably higher (twice as high) in the dividing cells than in the non-dividing ones. These data are discussed in the light of the basic characteristics of the dinokaryon, and also compared with recent biochemical observations on the same organism and studies on other higher eukaryotic protists and metazoa.  相似文献   

19.
Microalgae biomass can be a feasible source of ω‐3 fatty acids due to its stable and reliable composition. In the present study, the Crypthecodinium cohnii growth and docosahexaenoic acid (DHA, 22:6ω3) production in a 100 L glucose‐fed batch fermentation was evaluated. The lipid compounds were extracted by supercritical carbon dioxide (SC‐CO2) from C. cohnii CCMP 316 biomas, was and their fatty acid composition was analysed. Supercritical fluid extraction runs were performed at temperatures of 313 and 323 K and pressures of 20.0, 25.0 and 30.0 MPa. The optimum extraction conditions were found to be 30.0 MPa and 323 K. Under those conditions, almost 50% of the total oil contained in the raw material was extracted after 3 h and the DHA composition attained 72% w/w of total fatty acids. The high DHA percentage of total fatty acids obtained by SC‐CO2 suggested that this extraction method may be suitable for the production of C. cohnii value added products directed towards pharmaceutical purposes. Furthermore, the fatty acid composition of the remaining lipid fraction from the residual biomass with lower content in polyunsaturated fatty acids could be adequate for further uses as feedstock for biodiesel, contributing to the economy of the overall process suggesting an integrated biorefinery approach.  相似文献   

20.
Unlike typical eukaryotes, the Dinoflagellate Crypthecodinium cohnii does not contain histones but six major basic, low molecular weight nuclear proteins which represent only 10% of the DNA mass and differ from histones in their electrophoretic and DNA-binding properties. These proteins are resolved in two-dimensional electrophoresis (AUT-PAGE x SDS-PAGE). Three proteins with an apparent molecular mass of 16, 16.5 and 17 kDa (p16, p16.5 and p17) are present in addition to the major 14 kDa basic nuclear component (HCc). HCc itself is resolved in three proteins (alpha, beta and gamma). When the proteins are not reduced with 2-mercaptoethanol before 2D-PAGE, the migration of HCc alpha, beta and gamma is modified in a way which suggests the formation of both inter- and intramolecular disulfide bridges and thus, the presence of at least two cysteines. The amino-acid analysis of HCc proteins resolved in 2D gels confirms that they are lysine-rich. HCc alpha, beta and gamma as well as p16, p16.5 and p17 are removed from isolated chromatin with 0.6 M NaCl, indicating that their affinity for DNA in vivo is lower than that of core histones. Furthermore, in vitro, they bind more tightly to single-stranded than to double-stranded DNA.  相似文献   

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