Identification of the nuclear matrix and chromosome scaffold in dinoflagellate Crypthecodinium cohnii~1

Dinoflagellate is one of the primitive eukaryotes,whosenucleus may represent one of the transition stages fromprokaryotic nucleoid to typical eukaryotic nucleus.Usingselective extraction together with embeddment-free sectionand whole mount electron microscopy,a delicate nuclearmatrix filament network was shown,for the first time,indinoflagellate Crypthecodinium cohnii nucleus.Chromosomeresidues are connected with nuclear matrix filaments to forma complete network spreading over the nucleus.Moreover,we demonstrated that the dinoflagellate chromosome retainsa protein scaffold after the depletion of DNA and solubleproteins.This scaffold preserves the characteristic mor-phology of the chromosome.Two dimensional elec-trophoreses indicated that the nuclear matrix and chromo- some scaffold are mainly composed of acidic proteins.Ourresults demonstrated that a framework similar to the nuclearmatrix and chromosome scaffold in mammalian cells appearsin this primitive eukaryote,suggesting that these structuresmay have been originated from the early stages of eukaryoteevolution.     

涡鞭毛虫（甲藻）着丝粒／动粒蛋白的检查

利用ＡＣＡ血清、抗人着丝粒蛋白Ｂ的单抗和多抗、抗ＣＨＯ细胞动粒蛋白的单抗,对典型涡鞭毛虫隐沟虫（隐甲藻）（Ｃｒｙｐｔｈｅｃｏｄｉｎｉｕｍｃｏｈｎｉｉ）和特殊涡鞭毛虫尖尾虫（尖尾藻）（Ｏｘｙｒｒｈｉｓｍａｒｉｎａ）的着丝粒／动粒蛋白进行了检查。用ＡＣＡ血清作的荧光观察表明,隐沟虫的这些蛋白虽结合在核骨架上,但在间期时并不形成点状的前着丝粒。免疫印迹检查表明两种涡鞭毛虫的着丝粒蛋白Ｂ彼此一致,而且与四膜虫和眼虫的也高度一致。但用ＡＣＡ血清作免疫印迹检查时,尖尾虫的蛋白虽与四膜虫和眼虫的相近,与隐沟虫的却有极大的差异。以抗动粒蛋白的单抗作此种检查时,尖尾虫与眼虫的反应带相同,而隐沟虫则与源真核生物（Ａｒｃｈｅｚｏａ）贾第虫（Ｇｉａｒｄｉａｌａｍｂｌｉａ）的相同;而且隐沟虫和贾第虫都与几种原细菌有两条相同的反应带,其中５０ｋＤ的一条是尖尾虫和眼虫都没有的。上述发现不仅从一个新的方面支持了认为应把尖尾虫从典型涡鞭毛虫分出来独立为一个门的主张（李靖炎,１９９０）,而且指出典型涡鞭毛虫在后真核生物（Ｍｅｔａｋａｒｙｏｔａ）中间是非常原始的。     

Further Studies of Breeding Restrictions among Crypthecodinium cohnii-like Dinoflagellates. Evidence of a New Interbreeding Group

SYNOPSIS. Studies on sexual compatibility among populations of different origins of axenically cultivated, heterotrophic, homothallic dinoflagellates resembling Crypthecodinium cohnii have been undertaken using motility mutants and complementation testing. We showed previously that one interbreeding group or biologic species can be found on the East and West coasts of the United States, thus far associated only with Fucus. In addition, we found several strains of these organisms each of which, though fertile in intrastrain crosses, appeared infertile with all other isolates. A continuation of these studies has revealed another biologic species of wide geographic distribution (Taiwan, California, Florida, New York) and apparently associated with sea weeds other than Fucus.     

A Detailed Study of the Complex Cell Cycle of the Dinoflagellate Crypthecodinium cohnii Biecheler and Evidence for Variation in Histone H1 Kinase Activity

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.     

Sexual Isolation and Genetic Diversification Among Some Strains of Crypthecodinium cohnii-like Dinoflagellates Evidence of Speciation

SYNOPSIS. Nine new isolates of Crypthecodinium-like dinoflagellates from diverse geographic locations, together with 3 established strains from Woods Hole and Puerto Rico, were analyzed for sexual compatibility by means of a complementation test using motility mutants. The results indicate that 5 of the 12 are mutually compatible and thereby represent one species. Five others are clearly reproductively isolated from this group and from each other and therefore may belong to separate species. The position of 2 other isolates remains uncertain. Only one, MC-5, is markedly distinct morphologically from all the others. It is also sympatric with one of the others, strain G, and its separation from Crypthecodinium cohnii seems therefore more fully justified than that of the other sexually isolated strains. G and MC-5 would be considered "good" species by an evolutionary biologist. The others are from widely separate geographic origins and. though still poorly characterized, all superficially resemble C. cohnii . Comparison of many characters (DNA profile, radiation response, drug sensitivity, macroalgal association, etc.) of the incompatible strains show significant disparities which are discussed in considering speciation.     

伊朗卡尚地区始新世沟鞭藻类

文章简要报道了伊朗卡尚地区库姆组首次发现的沟鞭藻类。计6属6种。根据沟鞭藻化石组合面貌,并结合介形虫和钙质超微化石组合。探讨了含沟鞭藻化石的地层时代．推测其沉积环境为正常浅海大陆架环境。     

GLYCOPROTEIN MOIETY IN THE CELL WALL OF THE RED MICROALGA PORPHYRIDIUM SP. (RHODOPHYTA) AS THE BIORECOGNITION SITE FOR THE CRYPTHECODINIUM COHNII-LIKE DINOFLAGELLATE

The Crypthecodinium cohnii -like heterotrophic dinoflagellate preys on the cells of the red microalga Porphyridium sp. UTEX 637, and not on other microalgae. The dinoflagellate contains enzymes that degrade the cell wall complex of this species of alga and not that of other red microalgae. The cells of the red microalgae are encapsulated within a cell wall complex composed of about 10 sugars, sulfate, and proteins. We previously hypothesized that the dinoflagellate recognizes the cell wall of this alga. In this study, we have shown that the biorecognition site is the 66-kDa glycoprotein in the algal cell wall complex. The methodology used in this study was based on changing the algal cell wall composition and examining the prey and chemosensory response of the dinoflagellate. The dinoflagellate was not attracted to the cell wall of other red microalgae, which are similar to that of Porphyridium sp., or to sugars composing its cell wall. However, the dinoflagellate preyed on and was attracted to Porphyridium sp. mutants (DCB resistant) having modified cell wall polysaccharide composition, probably because the 66-kDa cell wall glycoprotein was not changed. The dinoflagellate did not respond chemotactically to enzymatically degraded cell wall complex. Treatment of the cell wall complex with antiserum to the 66-kDa glycoprotein or with the lectin concanavalin A (con A), which binds specifically to α-d-mannosyl and α-d-glucosyl residues, did not affect the chemotactic attraction. However, prey by the dinoflagellate was prevented when the algal cells were blocked with antiserum specific to the 66-kDa glycoprotein or with con A. These latter results provide direct proof that the 66-kDa cell wall glycoprotein isthe recognition site and prey-prevention results from the blocking of this site on the cell wall.     

cAMP IN THE CELL CYCLE OF THE DINOFLAGELLATE CRYPTHECODINIUM COHNII (DINOPHYTA)

The second messenger cAMP is a key regulator of growth in many cells. Previous studies showed that cAMP could reverse the growth inhibition of indoleamines in the dinoflagellate Crypthecodinium cohnii Biecheler. In the present study, we measured the level of intracellular cAMP during the cell cycle of C. cohnii . cAMP peaked during the G₁ phase and decreased to a minimum during S phase. Similarly, cAMP-dependent protein kinase activities peaked at both G₁ and G₂+M phases of the cell cycle, decreasing to a minimum at S phase. Addition of N6, O₂'-dibutyryl (Bt₂)-cAMP directly stimulated the growth of C. cohnii . Flow cytometric analysis of synchronized C. cohnii cells suggested that 1 mM cAMP shortened the cell cycle, probably at the exit from mitosis. The size of Bt₂-cAMP treated cells at G₁ was also larger than the control cells. The present study demonstrated a regulatory role of cAMP in the cell cycle progression in dinoflagellates.     

Effects of Visible and Ultraviolet Light on Carotene-deficient Mutants of Crypthecodinium cohnii

Synopsis.
The ability of carotenoids to protect a heterotrophic dinoflagellate, Crypthecodinium cohnii , from photodynamic damage by sunlight in the presence of an exogenous dye was demonstrated. Wild-type C. cohnii and 2 carotcnoid-deficient mutants were plated on agar-solidified media and exposed to natural sunlight. The wild-type strain, which synthesizes γ–carotene and β–carotene, had the lowest mortality. Mutant car 17 which accumulates mainly §–carotene had an intermediate mortality rate while the albino mutant, car 3, which contains only phytoene, lost viability most rapidly. Wild-type cells treated with diphenylaminc, a carotenogenic inhibitor, were killed at the same rate as mutant car 3. The survival rate of mutants on exposure to sunlight was dependent upon the chromophore length of the accumulated carotenoids. The 3 strains showed no difference in rate of mortality when exposed to ultraviolet light. Protection from sunlight by accumulation of carotenes may be an important ecological factor for this species whose natural habitat is tidepools.     

隐甲藻深层培养产生二十碳五烯酸的研究

二十碳五烯酸 (Ｃ2 0 :5 ,Ｅｉｃｏｓａｐｅｎｔａｅｎｏｉｃａｃｉｄ ,简称ＥＰＡ)是一种重要的ω 3系列人体必需多不饱和脂肪酸 (Ｐｏｌｙｕｎ ｓａｔｕｒａｔｅｄｆａｔｔｙａｃｉｄｓ,ＰＵＦＡ) [1～ 2 ] 。自从Ｄｙｅｒｂｅｒｇｅ等人报道了二十碳五烯酸对防止和治疗血栓、关节硬化、抗炎症、抗癌、促进脑组织发育等方面具有明显效果以来 ,人们对它的营养和医药价值及生产方法进行了广泛的研究[3～ 6] 。目前 ,二十碳五烯酸主要来源于深海鱼油 ,但其产量不稳定 ,纯化工艺复杂且含有难以消除的鱼腥味 ,难以满足市场的需求。由于二十碳五…     

寇氏隐甲藻(Crypthecodinium cohnii)细胞核骨架与中间纤维的研究

甲藻(涡鞭毛虫,dinoflagellate)的细胞核是现存真核生物中最原始的。我们采用整装细胞制样和非树脂包埋去包埋剂超薄切片电镜技术,结合选择性生化抽提方法显示在寇氏隐甲藻(Cryptheccdinium cohnii)细胞中存在一个以水不溶性纤维蛋白成份为主的,贯穿于细胞核和细胞质的纤维网架系统,即核骨架-中间纤维结构体系,而Lamina结构不明显。免疫印迹法显示,甲藻细胞中存在类角蛋白组分,分子量为63kD和67kD,哺乳动物Lamin抗体与甲藻细胞全蛋白反应阴性。实验结果表明,在原始真核细胞中已经出现了类似于哺乳动物细胞的核骨架和中间纤维,并提示核骨架-中间纤维细胞骨架体系可能起源于真核细胞起源早期。本文对Lamina与中间纤维在进化上的关系及Lamina在真核细胞进化中的功能意义作了讨论。     

CHARACTER EVOLUTION IN POLYKRIKOID DINOFLAGELLATES1

A synthesis of available data on the morphological diversity of polykrikoid dinoflagellates allowed us to formulate a hypothesis of relationships that help explain character evolution within the group. Phylogenetic analyses of new SSU rDNA sequences from Pheopolykrikos beauchampii Chatton, Polykrikos kofoidii Chatton, and Polykrikos lebourae Herdman helped refine this hypothetical framework. Our results demonstrated that “pseudocolonies” in dinoflagellates evolved convergently at least three times independently from different Gymnodinium‐like ancestors: once in haplozoans; once in Ph. beauchampii; and at least once within a lineage containing Ph. hartmannii, P. kofoidii, and P. lebourae. The Gymnodiniales sensu stricto was strongly supported by the data, and the type species for the genus, namely Gymnodinium fuscum (Ehrenb.) F. Stein, formed the nearest sister lineage to a well‐supported Polykrikos clade. The best synapomorphy for the Polykrikos clade was the presence of two nuclei irrespective of zooid number. Two unidentified Gymnodinium species formed the nearest sister clade to Ph. beauchampii, which has four nuclei and four zooids per pseudocolony. The chain‐forming dinoflagellate G. catenatum L. W. Graham branched closely to the clade containing all members of Polykrikos and Pheopolykrikos, suggesting that an ancestral capacity toward chain formation existed before the evolution of pseudocolonies in this group. Our results also clarified the phylogenetic significance of nematocysts, ocelloids, and photosynthesis in reconstructing the evolution of polykrikoids and warnowiids. The molecular phylogenies exposed taxonomic problems associated with Polykrikos, Pheopolykrikos, and Gymnodinium, and suggested that a revision for some of these genera is warranted.     

Growth inhibition of dinoflagellate algae in shake flasks: Not due to shear this time!

Large scale algae cultures present interesting challenges in that they exhibit characteristics of typical bacterial and animal cell cultures. One current commercial food additive, docosahexaenoic acid (DHA), is produced using the dinoflagellate algae, Crypthecodinium cohnii. Like animal cell culture, the perceived sensitivity of algae culture to hydrodynamic forces has potentially limited the agitation and aeration applied to these systems. However, the high density cultivation of C. cohnii required for an economically feasible process inevitably results in high oxygen demand. In this study, we demonstrated what first appeared to be a problem with shear sensitivity in shake flasks is most probably a mass transfer limitation. We subsequently demonstrated the limit of chronic and rapid energy dissipation rate, EDR, that C. cohnii cells can experience. This limit was determined using a microfluidic device connected in a recirculation loop to a stirred tank bioreactor, which has been previously used to repeatedly expose animal cells to high levels of EDR. Inhibition of cell growth was observed when C. cohnii cells were subjected to an EDR of 5.9 × 10⁶ W/m³ with an average frequency of 0.2/min or more. This level of EDR is sufficiently high that C. cohnii can withstand typically encountered hydrodynamic forces in bioprocesses. This result suggests that at least one dinoflagellate algae, C. cohnii, is quite robust with respect to hydrodynamic forces and the scale‐up of process using this type of algae should be more concerned with providing sufficient gas transfer given the relatively high oxygen demand. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

几种不同碳、氮源对隐甲藻生长及二十二碳六烯酸产量的影响

目的：找出有利于隐甲藻生长和二十二碳六烯酸（DHA）积累的碳、氮源。方法：利用不同碳、氮源培养隐甲藻,收集藻体后提取脂肪酸并甲酯化,然后利用毛细管气相色谱法进行分析。结果：最适的单一碳、氮源分别为葡萄糖、酵母粉,在此培养条件下隐甲藻培养72h后的生物量（干重）和DHA产量分别为3．90和0．642g／L。结论：葡萄糖、酵母粉分别作为碳、氮源时更有利于隐甲藻的生长和DHA的积累。     

寇氏隐甲藻(Crypthecodinium cohnii)染色体的提取及其独特的超微结构

甲藻(dinoflagellate)作为最原始的一类真核生物,在进化研究中有着重要的地位。它的种种原始特点都表明,这类生物很可能是介于原核生物和真核生物之间的中间过渡类型。例如:它有独立的细胞核,但双层核膜不连续,核膜在整个细胞周期中永远存在;染色体附着在核膜周围,但在细胞间期和分裂期无明显的结构变化,并且永远处于高度凝集状态,其染色     

SURVEY FOR KARLOTOXIN PRODUCTION IN 15 SPECIES OF GYMNODINIOID DINOFLAGELLATES (KARENIACEAE,DINOPHYTA)1

Toxin analysis of 15 species of Kareniaceae revealed the presence of karlotoxin, KmTx 2, in only a single species (Karlodinium veneficum) but with variable activity in strains from the Swan (Km^SwanTx 2‐1, 2.1 pg · cell^?1; and Km^SwanTx 2‐2, 0.53 pg · cell^?1), Huon (Km^HuonTx 2, 0.86 pg · cell^?1), and Derwent rivers (<0.001 pg · cell^?1) in Australia. A newly isolated Southern Ocean species, Karlodinium conicum, contained a novel poorly hemolytic karlotoxin analogue (Km_conicumTx, 2.8 pg · cell^?1). The hemolytic potency (HD50%) of the Australian karlotoxins were as follows: Km^SwanTx 2‐1 (65.9 ± 4.8 ng) and Km^SwanTx 2‐2 (63.4 ± 3.7 ng), Km^HuonTx 2 (343 ± 4.9 ng), and Km_conicumTx (>4,000 ng). Species from the closely related genera Takayama (T. helix, T. tasmanica, T. tuberculata), Karenia (K. asterichroma, K. brevis, K. mikimotoi, K. papilionacea, K. umbella), and Karlodinium (Ka. australe, Ka. antarcticum, Ka. ballantinum, Ka. corrugatum, Ka. decipiens) were all consistently negative for karlotoxin production. Brevetoxin (PbTx) was only detected in K. brevis, and hemolytic activity was only observed in Ka. veneficum strains.     

ENVIRONMENTAL AND ENDOGENOUS REGULATION OF CYST GERMINATION IN TWO FRESHWATER DINOFLAGELLATES

The role of excystment in relation to seasonal succession was investigated in two freshwater dinoflagellates, Ceratium hirundinella (O.F. Müller) Dujardin and Peridinium aciculiferum (Lemmermann). Field studies and laboratory experiments were performed to determine which factors regulate the timing of cyst germination. Environmental factors (temperature, light, nutrients, and anoxia) and endogenous factors (maturation period and biological clock) were investigated. Our main results indicate that temperature and internal maturation period determine when germination can occur. C. hirundinella had a maturation period of 4.5 months and germinated in the laboratory and in the field at temperatures above 6° C. P. aciculiferum had a maturation period of 2.5 months and germinated in the laboratory and in the field at temperatures below 7° C. In addition, our results indicated that both species were regulated by a biological clock. Furthermore, anoxia prevented the germination of C. hirundinella, contrary to results in earlier studies. To conclude, we could explain the appearance in plankton of the two dinoflagellate species through two main factors regulating excystment, that is, temperature and maturation period.     

甲藻染色体在非细胞体系核重建过程中核小体的组装

利用纯化的原始真核生物寇氏隐甲藻（Crypthecodinium cohnii E.）染色体,使之与非洲爪蟾（Xenopus laevis L.）S期卵提取物温育,发现甲藻染色体经历了一系列去凝集、再凝集的形态变化,最后形成类似典型高等真核生物的间期核结构。小球菌核酸酶酶切分析表明,不具备组蛋白和核小体结构的甲藻染色体在非洲爪蟾卵提取物中进行了核小体装配,此过程与DNA序列本身、核膜以及核纤层蛋白（Lamin）是否存在无关,但部分拓扑异构酶Ⅱ（TopoⅡ）参与了这个过程,说明核小体的组装并非为核重建所必需,决定染色体高级结构的因素并不在DNA本身,而可能是非细胞体系中的组蛋白和非组蛋白。     

Carotenes from Mutants of the Dinoflagellate, Crypthecodinium cohnii

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.