CHARACTERIZATION AND MOLECULAR PHYLOGENYOF A PROTEIN KINASE cDNA FROM THE DINOFLAGELLATE GONYAULAX (DINOPHYCEAE)1

Degenerate primers corresponding to conserved protein kinase motifs were used to amplify potential kinase DNA fragments from a Gonyaulax polyedra Stein cDNA library using PCR. One PCR fragment, potentially encoding a CAMP-dependent protein kinase, was used as a probe to isolate a near full-length cDNA from the library. The nucleic acid sequence of the entire cDNA clone had a high homology to the catalytic subunit of cAMP-dependent protein kinase (cAPK subfamily and affiliated members. Northern blot analysis showed that the corresponding mRNA had a size (about 1.4 kb) and a relative high abundance consistent with a cAPK homologue. Southern blot analysis showed that while there are roughly 30 copies of the kinase gene per genome, the pattern of restriction fragments is inconsistent with the hypothesis of a large gene family. Phylogenetic analyses comparing the deduced amino acid sequence from the Gonyaulax cDNA with other cAPK sequences place Gonyaulax close to the slime mold Dictyostelium discoideum. This is the first phylogenetic analysis of dinoflagellates based on protein sequence, and the results are in agreement with similar analyses based on rRNA sequences.     

MICROSATELLITE GENOTYPING OF SINGLE CELLS OF THE DINOFLAGELLATE SPECIES LINGULODINIUM POLYEDRUM (DINOPHYCEAE): A NOVEL APPROACH FOR MARINE MICROBIAL POPULATION GENETIC STUDIES1

In recent years, two new approaches have been introduced in genetic studies of phytoplankton species. One is the application of highly polymorphic microsatellite markers, which allow detailed population genetic studies; the other is the development of methods that enable the direct genetic characterization of single cells as an alternative to clonal cultures. The aim of this study was to combine these two approaches in a method that would allow microsatellite genotyping of single phytoplankton cells, providing a novel tool for high‐resolution population genetic studies. The dinoflagellate species Lingulodinium polyedrum (F. Stein) J. D. Dodge was selected as a model organism to develop this novel approach. The method we describe here is based on several key developments: (i) a simple and efficient DNA extraction method for single cells, (ii) the characterization of microsatellite markers for L. polyedrum, (iii) a protocol for the species identification of single cells through the analysis of partial rRNA gene sequences, and (iv) a two‐step multiplex PCR protocol for the simultaneous amplification of microsatellite markers and partial rRNA gene sequences from single cells. Our protocol allowed the amplification of up to six microsatellite loci together with either the complete ITS1‐5.8S‐ITS2 region or a partial 18S region of the ribosomal gene of L. polyedrum from single motile cells and resting cysts. This article describes and evaluates the developed approach and discusses its significance for population genetic studies of L. polyedrum and other phytoplankton species.     

BACTERIA‐INDUCED MOTILITY REDUCTION IN LINGULODINIUM POLYEDRUM (DINOPHYCEAE)1

Biotic factors that affect phytoplankton physiology and behavior are not well characterized but probably play a crucial role in regulating their population dynamics in nature. We document evidence that some marine bacteria can decrease the swimming speed of motile phytoplankton through the release of putative protease(s). Using the dinoflagellate Lingulodinium polyedrum (F. Stein) J. D. Dodge as a model system, we showed that the motility‐reducing components of bacterial‐algal cocultures were mostly heat labile, were of high molecular weight (>50 kDa), and could be partially neutralized by incubations with protease inhibitors. We further showed that additions of the purified protease pronase E decreased dinoflagellate swimming speed in a concentration‐dependent manner. We propose that motility can be used as a marker for dinoflagellate stress or general unhealthy status due to proteolytic bacteria, among other factors.     

DESCRIPTION OF TYRANNODINIUM GEN. NOV., A FRESHWATER DINOFLAGELLATE CLOSELY RELATED TO THE MARINE PFIESTERIA‐LIKE SPECIES1

On the basis of morphological (light and electron microscopy) as well molecular data, we show that the widely distributed freshwater dinoflagellate presently known as Peridiniopsis berolinensis is a member of the family Pfiesteriaceae, an otherwise marine and estuarine family of dinoflagellates. P. berolinensis is a close relative of the marine species, which it resembles in morphology, mode of swimming, food‐uptake mechanism, and partial LSU rRNA sequences. It differs from all known genera of the family in plate tabulation. P. berolinensis is only distantly related to the type species of Peridiniopsis, P. borgei, and is therefore transferred to the new genus Tyrannodinium as T. berolinense comb. nov. T. berolinense is a very common freshwater flagellate that feeds vigorously on other protists and is able to consume injured metazoans much larger than itself. Production of toxins has not been reported.     

CLONING,EXPRESSION PATTERNS,AND PRELIMINARY CHARACTERIZATION OF AccCPR24, A NOVEL RR‐1 TYPE CUTICLE PROTEIN GENE FROM Apis cerana cerana

Cuticular proteins (CPs) are key components of insect cuticle, a structure that plays a pivotal role in insect development and defense. In this study, we cloned the full‐length cDNA of a CP gene from Apis cerana cerana (AccCPR24). An amino acid sequence alignment indicated that AccCPR24 contains the conserved Rebers and Riddiford consensus sequence and shares high similarity with the genes from other hymenopteran insects. We then isolated the genomic DNA and found that the first intron, which is present in other CP genes, is absent in AccCPR24. Real‐time quantitative polymerase chain reaction (qPCR) analysis revealed that AccCPR24 is highly expressed in the late pupal stage and midgut. Expression was inhibited by an exogenous ecdysteroid in vitro but was enhanced by this hormone in vivo; environmental stressors, such as heavy metals and pesticides, also influenced gene expression. In addition, a disc diffusion assay showed that AccCPR24 enhanced the ability of bacterial cells to resist multiple stresses. We infer from our results that AccCPR24 acts in honeybee development and in protecting these insects from abiotic stresses.     

NOVEL STEROLS OF THE TOXIC DINOFLAGELLATE KARENIA BREVIS (DINOPHYCEAE): A DEFENSIVE FUNCTION FOR UNUSUAL MARINE STEROLS?1

The “red tide” organism Karenia brevis (Davis) Hansen & Moestrup (=Gymnodinium breve Davis) produces a mixture of brevetoxins, potent neurotoxins responsible for neurotoxic shellfish poisoning in humans and massive fish kills in the Gulf of Mexico and the southern Atlantic coast of the United States. The sterol composition of K. brevis was found to be a mixture of six novel and rare Δ⁸⁽¹⁴⁾ sterols. The two predominant sterols, (24R)‐4α‐methylergosta‐8(14), 22‐dienol and (24R)‐4α‐methyl‐27‐norergosta‐8(14), 22‐dienol, were named gymnodinosterol and brevesterol and represent potentially useful biomarkers for K. brevis. A possible function for such unusual marine sterols is proposed whereby structural modifications render the sterols non‐nutritious to marine invertebrates, reducing predation and thereby enhancing the ability of the dinoflagellates to form massive blooms.     

OCCURRENCE AND CHARACTERIZATION OF ARABINOGALACTAN‐LIKE PROTEINS AND HEMICELLULOSES IN MICRASTERIAS (STREPTOPHYTA)1

The cell wall of the green alga Micrasterias denticulata Bréb. ex Ralfs (Desmidiaceae, Zygnematophyceae, Streptophyta) was investigated to obtain information on the composition of component polysaccharides and proteoglycans to allow comparison with higher plants and to understand cell wall functions during development. Various epitopes currently assigned to arabinogalactan‐proteins (AGPs) of higher plants could be detected in Micrasterias by immuno TEM and immunofluorescence methods, but the walls did not bind the β‐d ‐glycosyl‐Yariv (β‐GlcY) reagent. Secretory vesicles and the primary wall were labeled by antibodies against AGPs (JIM8, JIM13, JIM14). Dot and Western blot experiments indicated a proteoglycan nature of the epitopes recognized, which consisted of galactose and xylose as major sugars by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC‐PAD). Epitopes of alkali‐soluble polysaccharides assigned to noncellulosic polysaccharides in higher plants could be detected and located in the wall during its formation. The polyclonal anti‐xyloglucan (anti‐XG) antibody labeled primary and secondary wall of Micrasterias, whereas the monoclonal antibody CCRC‐M1, directed against the fucose/galactose side chain of xyloglucan (XyG), did not recognize any structures. Labeling by anti‐XG antibody at the trans‐sites of the dictyosomes and at wall material containing vesicles indicated that secretion of the epitopes occurred similar to higher plants. The presence of (1→3, 1→4)‐β‐glucan (mixed linked glucan) in the secondary cell wall but not in the primary cell wall of Micrasterias could be demonstrated by an antibody recognizing this glucan type, whereas (1→3)‐β‐glucan (callose) could not be detected. The analytical results revealed that alkali‐soluble polysaccharides in the secondary wall of Micrasterias consist mostly of (1→3, 1→4)‐β‐d ‐glucan.     

MORPHOLOGY AND ECOLOGY OF THE MARINE BENTHIC DINOFLAGELLATE SCRIPPSIELLA SUBSALSA (DINOPHYCEAE)1

The thecal surface morphology of Scrippsiella subsalsa (Ostenfeld) Steidinger et Balech was examined using the scanning electron microscope. This species is distinguished by a number of morphological characteristics. Apical plate 1′ is wide, asymmetric, and pentagonal, and it ends at the anterior margin of the cingulum. Intercalary plates 2a and 3a are separated by apical plate 3′. The apical pore complex includes a large P_o plate with a raised dome at the center and a deep canal plate with thickened margins at plates 2′, 3′, and 4′. The intercalary bands are wide and deeply striated. The cingulum is deep, formed by six cingular plates; its surface is transversely striated and aligned with a row of minute pores. The cingular list continues around postcingular plate 1′” to form a sulcal list. The sulcal list is a flexible ribbon with a rounded tip that protrudes posteriorly, partially covering the sulcal plates. The hypotheca is lobed, and the antapical plates are irregularly shaped and wide in antapical view. The thecal surface is vermiculate to reticulate. A comparison in morphology and ecology is presented between S. subsalsa and other known Scrippsiella species.     

CHARACTERIZATION OF GYMNODINIUM MIKIMOTOI (DINOPHYCEAE) NUCLEI AND IDENTIFICATION OF THE MAJOR HISTONE-LIKE PROTEIN, HGm

A simple and rapid method for isolation of nuclei from Gymnodinium mikimotoi Miyake et Kominami ex Oda is described along with chemical characterization of the nuclei. The isolated nuclei were completely free of whole cells, 99.96% free of cytoplasmic contamination, and were collected with a yield of 40% from harvested whole cells. Each nucleus contained 47 pg of DNA and the ratio of DNA to acid-soluble proteins to acid-insoluble proteins was 1:0.25:1.21, respectively. SDS electrophoresis of acid-extracted proteins showed one histone-like protein, which we termed HGm, with an apparent molecular mass of 12 kDa. V8 protease digestion analysis of HGm, the histone-like protein from Crypthecodinium cohnii (HCc), and two histone-like proteins from Gymnodinium dorsum , showed that the HGm digestion pattern was more similar to that of HCc than to that of either of the G. dorsum histone-like proteins.     

THE RECLASSIFICATION OF PFIESTERIA SHUMWAYAE (DINOPHYCEAE): PSEUDOPFIESTERIA,GEN. NOV.1

Pfiesteria shumwayae Glasgow et Burkholder is assigned to a new genus Pseudopfiesteria gen. nov. Plate tabulation differences between Pfiesteria and Pseudopfiesteria gen. nov. as well as a maximum likelihood phylogenetic analysis based on rDNA sequence data warrant creation of this new genus. The Kofoidian thecal plate formula for the new genus is Po, cp, X, 4′, 1a, 6′′, 6c, PC, 5+s, 5′′′, 0p, 2′′′′. In addition to having six precingular plates, P. shumwayae comb. nov. also has a distinctive diamond or rectangular‐shaped anterior intercalary plate. Both Pfiesteria and Pseudopfiesteria gen. nov. are reassigned to the order Peridiniales based on an apical pore complex (APC) with a canal (X) plate that contacts a symmetrical 1′, four to five sulcal plates, and the conservative hypothecal tabulation of 5′′′, 0p, and 2′′′′. These morphological characters and the life histories of Pfiesteria and Pseudopfiesteria are consistent with placement of both genera in the Peridiniales. Based on the plate tabulations for P. shumwayae, P. piscicida, and the closely related “cryptoperidiniopsoid” and “lucy” groups, the family Pfiesteriaceae is amended to include species with the following tabulation: 4‐5′, 0‐2a, 5‐6′′, 6c, PC, 5+s, 5′′′, 0p, and 2′′′′ as well as an APC containing a pore plate (Po), a closing plate (cp), and an X plate; the tabulation is expanded to increase the number of sulcal plates and to include a new plate, the peduncle cover (PC) plate. Members of the family have typical dinoflagellate life cycles characterized by a biflagellated free‐living motile stage, a varying number of cyst stages, and the absence of multiple amoeboid stages.     

酵母菌类金属硫蛋白的分离、纯化及性质鉴定*

经过对酵母菌的金属抗性试验和培养条件试验,确定了高产类金属硫蛋白的酿酒酵母菌株Cu-21（Saccharomyces cerevisiae Cu-21）及其最佳培养条件。对该菌株的菌体蛋白进行SephadexG-100、DEAE-52结合的两次柱分离纯化,获得了类金属硫蛋白的三个亚型,蛋白性质鉴定结果表明含三个亚型、分子量小、富含半胱氨酸和金属元素,具有典型的巯基吸收特性。     

MOLECULAR CHARACTERIZATION OF A PHOSPHATE‐REGULATED CELL‐SURFACE PROTEIN FROM THE COCCOLITHOPHORID,EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE)1

Emiliania huxleyi (Lohmann) Hay et Mohler is a cosmopolitan coccolithophorid that is known to be an excellent competitor for phosphate. A previous survey of cell‐surface proteins induced by phosphorus limitation in strain CCMP 374 yielded three abundant proteins. Using CCMP 1516, the strain chosen for genome sequence determination, we report the cDNA, genomic, and amino acid sequence of one cell‐surface phosphorus‐limitation induced protein and evidence that a second protein is highly similar. The introns within the genomic DNA encoding this cell‐surface protein as well as those defined by other phosphate‐regulated expressed sequence tags are analyzed. As these proteins are the most abundant cell‐surface proteins present under phosphorus limitation, they likely have a role in the ability of this organism to compete for phosphate.     

酵母菌类金属硫蛋白的分离、纯化及性质鉴定

经过对酵母菌的金属抗性试验和培养条件试验,确定了高产类金属硫蛋白的酿酒酵母菌株Cu-21（Saccharomyces cerevisiae Cu-21）及其最佳培养条件。对该菌株的菌体蛋白进行SephadexG-100、DEAE-52结合的两次柱分离纯化,获得了类金属硫蛋白的三个亚型,蛋白性质鉴定结果表明含三个亚型、分子量小、富含半胱氨酸和金属元素,具有典型的巯基吸收特性。