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。     

用碱性品红染色法显示涡鞭毛虫的染色体

涡鞭毛虫(dinoflagellate)亦称甲藻,是原核生物进化到真核生物的过渡类型。在研究涡鞭毛虫染色体时,染色往往比较困难。目前,显示涡鞭毛虫染色体多采用Fculgen法,醋酸洋红和醋酸地依红染色法(Dodgc,1966;Shyam,1978;Hanic,1979),用这些方法染色,涡鞭毛虫染色体往往颜色较浅,不便于作观察分析。我们采用改良的碱性品红染色法得到了较为满意的结果。     

源真核生物贾第虫与原细菌着丝粒／动粒蛋白的免疫印迹检查

以抗人着丝粒蛋白Ｂ的单抗和多抗以及抗ＣＨＯ细胞动粒蛋白的单抗对源真核生物（ａｒｃｈｅｚｏａ）蓝氏贾第虫（Ｇｉａｒｄｉａｌａｍｂｌｉａ）和分别代表原细菌的３个枝的３种原细菌（Ｈａｌｏｂａｃｔｅｒｉｕｍ、Ｔｈｅｒｍｏｐｌａｓｍａ、Ｓｕｌｆｏｓｐｈａｅｒｅｌｌｕｓ）作了免疫电泳检查,并以小眼虫和大肠杆菌作为对照。结果表明,３种原细菌都呈阳性反应;而且贾第虫的反应情况显然比纤毛虫、眼虫、典型涡鞭毛虫、尖尾虫（Ｏｘｙｒｒｈｉｓ）等单细胞后真核生物的更接近于原细菌的情况。这不仅从一个新的方面为真核细胞起源于古代的原细菌的学说提供了新的佐证,而且从着丝粒／动粒蛋白方面证明了源真核生物贾第虫的原始性。本工作还为认识着丝粒蛋白Ｂ和动粒蛋白的起源和演化提供了线索。     

典型涡鞭毛虫(Zooxanthella microadriatica)含有多种染色质碱性蛋白

以往的研究都表明涡鞭毛虫类不含真核细胞普遍具有的组蛋白,而仅含1—2种分子量较小、含量较低和碱性较弱的染色质碱性蛋白。但我们采用自己建立的先固定后抽提的方法从典型涡鞭毛虫Zooxanthella microadriatica获得了多种碱性蛋白成分。经SDS-PAGE分析,其中有六条带的迁移率分别十分接近地对应着小牛胸腺的五种组蛋白(H1有两条亚带)。另外迁移率在H1与H3之间的三条互相靠近的电泳带,据其分子量(17—19KDa)分析极可能来自于此细胞中含量极为丰富的叶绿体类核体的染色质,由于碱性性质相似而被一同提取出来。既然我们利用先固定后抽提的方法提取小牛胸腺组蛋白和提取涡鞭毛虫(Crypthecodinium cohnii)的染色质碱性蛋白获得了良好的提取效果和很好地重复了前人的结果,我们认为本工作首次报道了在典型涡鞭毛虫类中也有含有多种染色质碱性蛋白并且很相似于组蛋白(至少在分子量上)的情形,为揭示和澄清组蛋白的起源进化问题提供了新的实验依据。     

关于一种含两种核的涡鞭毛虫(光簿甲藻)的真核型小核的分裂

涡鞭毛虫（甲藻）类（Dinoflagellate）由于其细胞周期的任何时候核中都含有致密的染色体结构,且其化学组成上缺乏通常真核生物所含的几种组蛋白组份,因而在进化上具有独特的地位。1971年Dodge首次发现波罗底海多甲藻（Peridinium balticum）含有两种不同类型的核,其中大核为涡鞭毛虫类所固有的核,小核却属于真核类型。以后别人又陆续有所发现,但迄今含有两种类型的核的涡鞭毛虫已报道的还是不多的。除上     

典型涡鞭毛虫（Zooxanthella microadriatica）含有多种染色质碱…

以往的研究都表明涡鞭毛虫类不含真核细胞普遍具有的组蛋白,而仅含１－２种分子量较小、含量较低和碱性较弱的染色质碱性蛋白。但我们采用自己建立的先固定后抽提的方法从典型涡鞭毛虫Ｚｏｏｘａｎｔｈｅｌｌａ ｍｉｃｒｏａｄｒｉａｔｉｃａ获得了多种碱性蛋白成分。经ＳＤＳ－ＰＡＧＥ分析,其中有六条带的迁称铉分别十分接近地对应着小牛胸腺的五种组蛋白（Ｈ１有两条亚带）。另外迁移率在Ｈ１与Ｈ３之间的三条互相靠近的电泳带     

前环藻(Amphidinium carterae)(涡鞭毛虫)染色质碱性蛋白的研究

本研究用甲醇固定、细胞匀浆、0.3NHCl抽提及丙酮沉降的四步法提取了属于典型涡鞭毛虫类的前环藻(Amphidinium carterae)之染色质碱性蛋白及作为对照的小牛胸腺组蛋白,并以酸尿素系统的聚丙烯酰胺凝胶电泳,对所提取的蛋白作了对比检查。结果小牛胸腺的总组蛋白被分离成H1、H3、H2A、H2B及H4五条电泳带;前环藻的蛋白样品在组蛋白电泳区唯一出现了一条电泳带,其电泳迁移率相当于小牛胸腺组蛋白H4。根据本结果和另外一些作者对其他一些涡鞭毛虫类的生化和细胞化学研究的结果,表明以往主要根据经典的细胞化学研究之结果而认为涡鞭毛虫类的细胞核或染色体不含组蛋白或碱性蛋白作为其一重要特征,是并不全面和可靠的。包括本研究在内的几个生化研究结果也暗示了涡鞭毛虫类的染色质主要含一种电泳迁移率类似于组蛋白H4的碱性蛋白可能是一普遍现象。     

特殊涡鞭毛虫—尖尾藻的核骨架

采用分级抽提,DGD包埋-去包埋剂电镜技术在特殊涡鞭毛虫——尖尾藻的细胞核内显示出了一个纤维网络结构。此网络结构不溶于CSK液和可抽去微管与微丝的溶液,不被DNase所酶解和热三氯醋酸所抽提,因而是一个非DNA性质的纤维蛋白网络结构。它的一系列形态结构特征——整个呈网络形态,纤维的粗细为2.8—24nm,与细胞质内的中间纤维有广泛的连接,含有少量对维持其结构完整性所必需的RNA成分等,都十分相似于典型真核细胞的核骨架结构,但所显示的核纤层为一层不均匀、不连续的结构,这有别于典型真核细胞的核纤层。 本工作首次证实了特殊涡鞭毛虫——尖尾藻已进化产生了核骨架结构,且与典型真核细胞的核骨架在形态结构上已很接近。     

涡鞭毛虫(甲藻)细胞核碱性蛋白的细胞化学研究 Ⅰ.双脚多甲藻(Peridinium bipes)

用除去DNA以后以酸性染料染示碱性蛋白的方法,在一般涡鞭毛虫的染色体上得不到阳性的结果。但是这不足以排除在它们的染色体中有碱性蛋白分子分散地结合在DNA 纤维上的可能性。为此,我们在双脚多甲藻(Peridinium bipes)上,用Tramezzani 等的氨银法,在保持住染色体中的DNA 的条件下进行了检验。结果证明,虽则有些个体的细胞核是阴性反应的,表明其中不含碱性蛋白,但是在许多个体的细胞核中,致密的涡鞭毛虫染色体都是作阳性反应的,意味着其中含有碱性蛋白,而染色体外的核质则是作阴性反应的。如果事先用稀盐酸抽去碱性蛋白,则它们的染色体也就不再能作阳性反应。在除去了染色体中的DNA 以后,涡鞭毛虫的染色体就会完全消失,而只留下相应的空洞。此时再作氨银反应,就可发现有些细胞核中的核质已经变成会作弱阳性反应的了。这表明,随着染色体的破坏,有些原来位于染色体中的碱性蛋白已经被吸附到了核质上。这就可以说明Dodge和Ris 与KuBai 何以会得到涡鞭毛虫类的染色体中不含碱性蛋白,而染色体外的核质却含有它们的看法。我们曾在不除去细胞核中的DNA 的条件下,设法用固绿的弱碱性溶液(Alfert与Geschwind 的方法)、偶氮洋红G 的中性溶液(李靖炎的方法)、溴酚蓝(Bloch与Hew 的方法)来染示核质中未曾与DNA相结合的碱性蛋白,结果双脚多甲藻的细胞核完全不着色。这也证明,在染色体被破坏以前,它们的核质中是不含游离的碱性蛋白的。     

麦积山肉鞭毛虫的物种多样性

用活体观察法对迄今缺乏了解的麦积山肉鞭类原生动物的物种多样性进行了调查,共鉴定出水生肉鞭虫100种,包括2个未知种。鞭毛亚门（Mastigophora）有2纲9目15科31属59种。植鞭纲（Phytomastigophorea）有52种,其中眼虫目(Euglenida) 36种,占鞭毛虫物种总数的61.02%,为优势类群;动鞭纲（Zoomastigophorea）仅有7种,没有明显的优势类群。肉足亚门（Sarcodina）有4纲6目19科32属41种。叶足纲（Lobosea）有31种,其中变形目（Amoebida）16种,占肉足虫物种总数的39.02%,为优势类群;表壳目（Arcellinida）12种,占29.27%,为次优势类群。在麦积山风景名胜区的南坡（为嘉陵江水系）分布有71种,北坡（为渭河水系）分布有56种,分水岭分布有13种,水生肉鞭毛虫的群落组成也显著不同;与国内典型的长江水系、雅鲁藏布江水系和黄河水系相比,麦积山分布的水生肉鞭毛虫群落组成均存在显著性差异。结果表明,麦积山水生肉鞭毛虫物种十分丰富,特有和稀有物种繁多,具有鲜明的独特性。     

Description of two species of early branching dinoflagellates, Psammosa pacifica n. g., n. sp. and P. atlantica n. sp

In alveolate evolution, dinoflagellates have developed many unique features, including the cell that has epicone and hypocone, the undulating transverse flagellum. However, it remains unclear how these features evolved. The early branching dinoflagellates so far investigated such as Hematodinium, Amoebophrya and Oxyrrhis marina differ in many ways from of core dinoflagellates, or dinokaryotes. Except those handful of well studied taxa, the vast majority of early branching dinoflagellates are known only by environmental sequences, and remain enigmatic. In this study we describe two new species of the early branching dinoflagellates, Psammosa pacifica n. g., n. sp. and P. atlantica n. sp. from marine intertidal sandy beach. Molecular phylogeny of the small subunit (SSU) ribosomal RNA and Hsp90 gene places Psammosa spp. as an early branch among the dinoflagellates. Morphologically (1) they lack the typical dinoflagellate epicone-hypocone structure, and (2) undulation in either flagella. Instead they display a mosa?c of dinokaryotes traits, i.e. (3) presence of bi-partite trychocysts; Oxyrrhis marina-like traits, i.e. (4) presence of flagellar hairs, (5) presence of two-dimensional cobweb scales ornamenting both flagella (6) transversal cell division; a trait shared with some syndineansand Parvilucifera spp. i.e. (7) a nucleus with a conspicuous nucleolus and condensed chromatin distributed beneath the nuclear envelope; as well as Perkinsus marinus -like features i.e. (8) separate ventral grooves where flagella emerge and (9) lacking dinoflagellate-type undulating flagellum. Notably Psammosa retains an apical complex structure, which is shared between perkinsids, colpodellids, chromerids and apicomplexans, but is not found in dinokaryotic dinoflagellates.     

Alveolate phylogeny inferred using concatenated ribosomal proteins

Dinoflagellates and apicomplexans are a strongly supported monophyletic group in rDNA phylogenies, although this phylogeny is not without controversy, particularly between the two groups. Here we use concatenated protein-coding genes from expressed sequence tags or genomic data to construct phylogenies including "typical" dinophycean dinoflagellates, a parasitic syndinian dinoflagellate, Amoebophrya sp., and two related species, Oxyrrhis marina, and Perkinsus marinus. Seventeen genes encoding proteins associated with the ribosome were selected for phylogenetic analysis. The dataset was limited for the most part by data availability from the dinoflagellates. Forty-five taxa from four major lineages were used: the heterokont outgroup, ciliates, dinoflagellates, and apicomplexans. Amoebophrya sp. was included in this phylogeny as a sole representative of the enigmatic marine alveolate or syndinian lineage. The atypical dinoflagellate O. marina, usually excluded from rDNA analyses due to long branches, was also included. The resulting phylogenies were well supported in concatenated analyses with only a few unstable or weakly supported branches; most features were consistent when different lineages were pruned from the tree or different genes were concatenated. The least stable branches involved the placement of Cryptosporidium spp. within the Apicomplexa and the relationships between P. marinus, Amoebophrya sp., and O. marina. Both bootstrap and approximately unbiased test results confirmed that P. marinus, Amoebophrya sp., O. marina, and the remaining dinoflagellates form a monophyletic lineage to the exclusion of Apicomplexa.     

Characterization of the DNA from the dinoflagellate Crypthecodinium cohnii and implications for nuclear organization.

Although dinoflagellates are eucaryotes, they possess many bacterial nuclear traits. For this reason they are thought by some to be evolutionary intermediates. Dinoflagellates also possess some unusual nuclear traits not seen in either bacteria or higher eucaryotes, such as a very large number of identical appearing, permanently condensed chromosomes suggesting polyteny or polyploidy. We have studied the DNA of the dinoflagellate Crypthecodinium cohnii with respect to DNA per cell, chromosome counts, and renaturation kinetics. The renaturation kinetic results tend to refute extreme polyteny and polyploidy as the mode of nuclear organization. This organism contains 55-60% repeated, interspersed DNA typical of higher eucaryotes. These results, along with the fact that dinoflagellate chromatin contains practically no basic protein, indicate that dinoflagellates may be organisms with a combination of both bacterial and eucaryotic traits.     

Characterization of p80, a novel nuclear and cytoplasmic protein in dinoflagellates.

The presence of myosin in dinoflagellates was tested using an anti-Acanthamoeba castellanii myosin II polyclonal antibody on the heterotrophic dinoflagellate Crypthecodinium cohnii Seligo. Western blots revealed the presence of a unique band of 80 kDa in total protein extracts and after immunoprecipitation. Expression of this 80 kDa protein appeared constant during the different phases of the cell cycle. In protein extracts from various other dinoflagellates, this 80 kDa protein was detected only in the autotrophic species Prorocentrum micans Ehr. Screening of a C. cohnii cDNA expression library with this antibody revealed a cDNA coding for an amino acid sequence without homology in the databases. However, particular regions were detected: - a polyglutamine repeat domain in the N-terminal part of the protein, - four peptide sequences associated with GTP-binding sites, - a sequence with slight homology to the rod tail of Caenorhabditis elegans myosin II, -a sequence with homology to a human kinesin motor domain. Immunocytolocalization performed on C. cohnii thin sections with a polyclonal antibody raised against the recombinant protein showed p80 to be present both within the nucleus and in the cytoplasm. Labelling was widespread in the nucleoplasm and more concentrated at the periphery of the permanently condensed chromosomes. In the cytoplasm, labelling appeared in a punctate region close to the nucleus and in the flagellum. Potential functions of this novel protein are discussed.     

ULTRASTRUCTURAL STUDIES ON TWO KINDS OF MESOCARYOTIC DINOFLAGELLATE NUCLEI

The nucleus of the mesocaryotic dinoflagellate has unusually distinctive features as seen under the light and electron microscope. An electron microscope study of 7 species has demonstrated two kinds of dinoflagellate nuclei. One type, characteristic of Amphidinium and most other dinoflagellates and called here the dinocaryotic type, is distinguished by the presence of discrete chromosomes visible throughout the entire cell cycle. The other type, found in vegetative cells of Noctiluca and called here the nocticaryotic type, is characteristically devoid of evident chromosomes at least during interphase. Questions are raised regarding the distinction between the nucleoplasm, chromosomes, and nucleolus of dinoflagellates. As the Heliozoa and Radiolariahave typically eucaroytic nuclei, they should not be considered as part of the Mesocaryota, as has been previously suggested.     

Molecular organization of dinoflagellate ribosomal DNA: evolutionary implications of the deduced 5.8 S rRNA secondary structure

The 5.8 S rRNA gene of Prorocentrum micans, a primitive dinoflagellate, has been cloned and its 159 base pairs (bp) have been sequenced along with the two flanking internal transcribed spacers (ITS 1 and 2), respectively, 212 and 195 bp long. Nucleotide sequence homologies between several previously published 5.8 S rRNA gene sequences including those from another dinoflagellate, an ascomycetous yeast, protozoans, a higher plant and a mammal have been determined by sequence alignment. Two prokaryotic 5'-ends of the 23 S rRNA gene have been compared owing to their probable common origin with eucaryotic 5.8 S rRNA genes. Several nucleotides are distinctive for dinoflagellates when compared with either typical eucaryotes or procaryotes. This is consistent with an early divergence of the dinoflagellate lineage from the typical eucaryotes. The secondary structure of dinoflagellate 5.8 S rRNA molecules fits the model of Walker et al. (1983). Conserved nucleotides which distinguish dinoflagellate 5.8 S rRNA from that of other eucaryotes are located in specific loops which are assumed to play a structural role in the ribosome. A 5.8 S rRNA phylogenetic tree which is proposed, based on sequence data, supports our initial assumption of the dinoflagellates.     

CHEMOSENSORY CAPABILITIES IN THE PHAGOTROPHIC DINOFLAGELLATE GYMNODINIUM FUNGIFORME11

The non-photosynthetic, phagotrophic dinoflagellate, Gymnodinium fungiforme Anissimova is attracted to a variety of amino acids and other organic compounds. Glycine, taurine and serine attracted the dinoflagellates at a threshold detection level of 10^?8 M fallowed by dextrose (10^?7 M) and alanine, proline and threonine (10^?6 M). Glycine, taurine and alanine are three of the most abundant free amino acids found in invertebrates and protozoa which are major food sources of this dinoflagellate. Three additional species of cultured heterotrophic dinoflagellates were exposed to the water soluble fraction of a shrimp extract known to attract G. fungiforme. All three species responded to the extract, but one species, Oxyrrhis marina, did so only after changing its food source. It is suggested that chemosensory behavior may be suppressed or expressed depending on culture conditions.