Redescription of Trypanosoma siniperca Chang 1964 from freshwater fish of China based on morphological and molecular data

During the parasite fauna investigation within 2005, the freshwater fish trypanosome Trypanosoma siniperca Chang 1964 was isolated from the blood of Mandarin carp (Siniperca chuatsi) from Niushan Lake, Hubei Province, central China. Blood trypomastigotes were observed only, and the density of infection was low. Light microscopy examinations of this material made it possible to study in detail the morphology of this parasite and redescribe it according to current standards. T. siniperca is characterized also on the molecular level using the sequences of SSU rRNA gene. Phylogenetic analyses based on these sequences allowed clearer phylogenetic relationships to be established with other fish trypanosomes sequenced to date.

Reevaluation of the phylogenetic relationship between Mobilid and Sessilid peritrichs (Ciliophora, Oligohymenophorea) based on small subunit rRNA genes sequences

Based on morphological characters, peritrich ciliates (Class Olygohymenophorea, Subclass Peritrichia) have been subdivided into the Orders Sessilida and Mobilida. Molecular phylogenetic studies on peritrichs have been restricted to members of the Order Sessilida. In order to shed more light into the evolutionary relationships within peritrichs, the complete small subunit rRNA (SSU rRNA) sequences of four mobilid species, Trichodina nobilis, Trichodina heterodentata, Trichodina reticulata, and Trichodinella myakkae were used to construct phylogenetic trees using maximum parsimony, neighbor joining, and Bayesian analyses. Whatever phylogenetic method used, the peritrichs did not constitute a monophyletic group: mobilid and sessilid species did not cluster together. Similarity in morphology but difference in molecular data led us to suggest that the oral structures of peritrichs are the result of evolutionary convergence. In addition, Trichodina reticulata, a Trichodina species with granules in the center of the adhesive disc, branched separately from its congeners, Trichodina nobilis and Trichodina heterodentata, trichodinids without such granules. This indicates that granules in the adhesive disc might be a phylogenetic character of high importance within the Family Trichodinidae.

The giant zooxanthellae-bearing ciliate Maristentor dinoferus (Heterotrichea) is closely related to folliculinidae

The small subunit rDNA sequence of Maristentor dinoferus (Lobban, Schefter, Simpson, Pochon, Pawlowski, and Foissner, 2002) was determined and compared with sequences from other Heterotrichea and Karyorelictea. Maristentor resembles Stentor in basic morphology and had been provisionally assigned to Stentoridae. However, our phylogenetic analyses show that Maristentor is more closely related to Folliculinidae. Our results support the creation of a separate family for Maristentor, Maristentoridae n. fam., and also confirm the phylogenetic grouping of Folliculindae, Stentoridae, Blepharismidae, and Maristentoridae, which we informally call 'stentorids'. Maristentor, rather than Stentor itself, appears to be most significant in understanding the origins of folliculinids from their aloricate ancestors. Our analyses suggest continued uncertainty in the exact placement of the root of heterotrichs with this phylogenetic marker.

Phylogeny of the cuttlerishes (Mollusca : Cephalopoda) based on mitochondrial COI and 16S rRNA gene sequence data

To clarify cuttlefish phylogeny, mitochondrial cytochrome c oxidase subunit 1 (COI) gene and partial 16S rRNA gene are sequenced for 13 cephalopod species. Phylogenetic trees are constructed, with the neighbor-joining method. Coleoids are divided into two main lineages, Decabrachia and Octobrachia. The monophyly of the order Sepioidea, which includes the families Sepiidae, Sepiolidae and Idiosepiidae, is not supported. From the two families of Sepioidea examined, the Sepiolidae are polyphyletic and are excluded from the order. On the basis of 16S rRNA and amino acid of COI gene sequences data, the two genera (Sepiella and Sepia) from the Sepiidae can be distinguished, but do not have a visible boundary using COI gene sequences. The reason is explained. This suggests that the 16S rDNA of cephalopods is a precious tool to analyze taxonomic relationships at the genus level, and COI gene is fitter at a higher taxonomic level (i.e., family).

Intraspecific phylogeography of Carchesium polypinum (Peritrichia, Ciliophora) from China, inferred from 18S-ITS1-5.8S ribosomal DNA

Based on the variation of site 34, 46, 241, 305 and 322 in the 18S-ITS1 rDNA sequence, 19 Carchesium polypinum populations collected from eight provinces of China were separated into northern and southern population along the delineation between the Yangtze River and the Pearl River. This geographic distribution pattern of Carchesium polypinum maybe results from two factors: the vicariance resulting from the formation of the delineation between the Pearl River and the Yangtze River accompanied with the uplift of Qinghai-Xizang Plateau, and the different dispersal paths of C. polypinum affected by the climate.

Taxonomic revision of water-bloom-forming species of oscillatorioid cyanobacteria

A polyphasic approach was used to clarify the taxonomy of the water-bloom-forming oscillatorioid cyanobacteria. Seventy-five strains of oscillatorioid cyanobacteria were characterized by 16S rDNA sequence analysis, DNA base composition, DNA-DNA hybridization, fatty acid composition, phycobilin pigment composition, complementary chromatic adaptation, morphological characters, growth temperature and salinity tolerance. Phylogenetic analysis based on 165 rDNA sequences divided the strains into six groups, all of which were clearly separated from the type species of the genus Oscillatoria, Oscillatoria princeps Gomont NIVA CYA 150. Therefore, these strains should be classified into genera other than Oscillatoria. Groups I-III were closely related to one another and groups IV-VI were distinct from one another and from groups I to III. Group I was further divided into two subgroups, group I-pc, which includes strains containing only phycocyanin (PC), and group I-pe, which includes strains containing large amounts of phycoerythrin (PE) in addition to PC. This phenotypic distinction was supported by DNA-DNA hybridization studies. Based on the properties examined herein and data from traditional, botanical taxonomic studies, the groups and subgroups were classified into single species and we propose either emended or new taxonomic descriptions for Planktothrix agardhii (type strain NIES 204(T)), Planktothrix rubescens (type strain CCAP 1459/22(T)) Planktothrix pseudagardhii sp. nov. (type strain T1-8-4(T)), Planktothrix mougeotii (type strain TR1-5(T)), Planktothricoides raciborskii gen. nov., comb. nov. (type strain NIES 207(T)), Tychonema bourrellyi (type strain CCAP 1459/11B(T)) and Limnothrix redekei (type strain NIVA CYA 277/1(T)).

Phylogenetic relationships among six species of Epistylis inferred from 18S-ITS1 sequences

Phylogenetic relationships among six species of Epistylis (i.e. E. plicatilis, E. urceolata, E. chrysemydis, E. hentscheli, E. wenrichi, and E, galea) were investigated using sequences of the first internal transcribed spacer region (ITS-1) of ribosomal DNA (rDNA). Amplified rDNA fragment sequences consisted of 215 or 217 bases of the flanking 18S and 5.8S regions, and the entire ITS-1 region (from 145 to 155 bases). There were more than 33 variable bases between E. galea and the other five species in both the 18S region and the ITS-1 region. The affiliation of them was assessed using Neighbor-joining (NJ), maximum parsimony (MP) and maximum likelihood (ML) analyses. In all the NJ, MP and ML analyses E. galea, whose macronucleic position and shape are distinctly different from those of the other five species, was probably diverged from the ancestor of Epistylis earlier than the other five species. The topology in which E. plicatilis and E, hentscheli formed a strongly supported sister clade to E. urceolata, E. chrysemydis, and E. wenrichi was consistent with variations in the thickness of the peristomial lip. We concluded that the macronucleus and peristomial lip might be the important phylogenetic characteristics within the genus Epistylis.

Isolation and Characterization of a Methomyl-Degrading Paracoccus sp mdw-1

Methomyl, an extremely toxic pesticide, is widely used in agriculture. A strain named mdw-1 capable of degrading methomyl rapidly was successfully isolated from activated sludge in this study. It could utilize methomyl as the sole carbon or nitrogen source. The optimal temperature and medium pH for its growth and methomyl biodegradation were 30 degrees C and 7.0, respectively. It was identified as a Paracoccus sp. according to its morphological features, physiological and biochemical characteristics, and phylogenetic analysis based on the sequence of 16S rDNA. Gas chromatography-mass spectrometry (GC-MS) analysis showed that methomyl could be completely transformed to S-methyl-N-hydroxythioacetamidate in 10 h of incubation with the isolate mdw-1.

铬污染土壤修复过程中土壤细菌群落多样性的RFLP分析

对污染土壤修复过程中土壤细菌群落多样性的变化进行研究。【方法】以淹水培养后的模拟铬污染土壤为供试材料,通过直接提取土壤中总细菌DNA,利用细菌专一引物克隆细菌16S rDNA片段,分别建立克隆文库。利用PCR-RFLP技术,分析比较了土壤淹水10 d(对照,S1)、添加Cr(Ⅵ)淹水10 d(S2)、添加Cr(Ⅵ)和Fe(OH)3淹水10 d(S3)及20 d(S4)4个处理中土壤细菌群落的变化。【结果】用专一引物克隆细菌16S rDNA片段,分别建立了克隆文库;用限制性内切酶RsaⅠ进行细菌16S rDNA PCR-RFLP分析,分别得到123,120,97和69个酶切类型,库容值分别为54.92%,55.43%,65.33%和76.60%;Shannon-Wiener指数、Gini指数、物种丰富度指数(dMa)和物种均匀度指数(Jgi)均表现为S1>S2>S3>S4,以上4个指数的变异系数分别为11.51%,1.84%,23.64%和1.55%;基于细菌多样性参数的聚类分析结果,将对照S1和添加Cr(Ⅵ)处理的S2归于一类,而2个添加Fe处理的土壤S3和S4聚为一类。【结论】经过10 d淹水处理,...

氮肥缺失对旱地土壤细菌群落多样性的影响

本文旨在研究氮肥缺失对旱地土壤细菌群落多样性的影响。采用直接提取土壤微生物总DNA的方法,对不施肥(CK)、适量施肥(F1)、和缺氮施肥(F2)3种不同施肥水平土样DNA进行提取,扩增细菌16S rDNA基因片段,建立克隆文库。用限制性内切酶HhaI和RsaI进行PCR-RFLP分析,分别得到146、187、11个酶切类型。采用α多样性的测度对试验结果进行分析统计结果表明,不同处理间土壤细菌的多样性(H′、Ds和Dg)和物种丰富度(dMa、R2和E)均为F1>CK>F2;λ、dMa、E和H′指数在不同施肥处理间的变异系数达到56.96%~163.1%,尤其Simpson指数λ是非常敏感的指标,处理间的差异最大,表明氮肥缺失严重影响土壤细菌群落多样性,合理施肥有利于土壤细菌的多样性。

贾第虫的“双等核”及其保持等同机制的研究

贾第虫是一类寄生于肠道的单细胞原生动物，也被认为是目前已知的最原始的真核细胞。它的一个十分奇特和令人感兴趣的特点是：具有在形态和大小上都很相似的左右对称的两个细胞核。已有一些证据表明贾第虫的这样的两个核在其它一些方面也都很相似甚至完全相同，如两核的DNA含量相等，两核都含有rDNA和至少一套染色体，并且在DNA复制、转录和核分裂等功能活动方面也基本都是同步的。但是，这两个核在基因的组成方面是否完全一致，甚至两核之间的对应基因（等位基因）的序列是否完全相同呢？若是，贾第虫为什么需要这样两个完全“等价”的细胞核（“双等核”）？这两个核又是如何长期保持一致而不会因两个核内发生不同的基因变异而产生差异呢？此外，目前普遍认为贾第虫至少是四倍体，即每个核至少为二倍体。那么同一个核内的等位基因是否序列也一致？保持其一致的机制又是什么？这些问题不仅饶有趣味，而且对于揭示贾第虫特殊的遗传机制乃至真核细胞基因组倍性的起源进化等问题具有重要意义。 本文首先对其两个核内是否有相同的基因组成进行了检验。我们选择了三个执行不同功能蛋白的基因为代表以检验贾第虫的两个细胞核是否都含有这些基因。这三个基因分别是：DNA拓扑异构酶II基因（topII）, 核仁蛋白KRR1基因（krr1）和目前贾第虫中报道的极少数含有内含子的基因之一的铁硫蛋白基因（fes）。利用荧光原位杂交(FISH)技术在两个核中进行了定位分析。结果表明这几个代表性的基因在贾第虫的两个细胞核中都同时存在。这提示着贾第虫的两核中具有相同的基因组成，也表明贾第虫的这两个核可能在功能方面也是“等价”的。 其次，我们对其两个核中的等位基因是否具有一致的序列进行了检验，并对其两核之间以及同一核内的等位基因保持一致的机制进行了研究。选择前人文献报道的存在多态位点（即这些位点可能是易变位点）的两个基因：fen1和pdi为研究对象，在自己建立的一个贾第虫克隆培养系上进行单细胞PCR和测序的跟踪分析。跟踪分析了18个月（约分裂1600代）后，检测到如下结果：尽管在fen1基因上尚未发现位点变异的规律，但在pdi基因上发现存在几个位点会间歇出现套峰。据此我们推测套峰的出现可能是由于其中一个等位基因发生了突变，而后套峰消失则可能是突变被恢复。这种等位基因的修复机制很可能是基因转换（gene conversion）。进而我们在贾第虫基因组中找到了参与基因转换的很多酶基因的同源基因，RT-PCR的结果也表明这些基因在贾第虫中是活跃转录的。这进一步提示了贾第虫中发生基因转换的可能性。因此以上结果不仅表明贾第虫的两核之间和同一核内的等位基因的序列都是一致的，同时还说明基因转换可能是同一核内等位基因保持一致的机制。至于两个核之间保持等位基因序列一致的机制，我们采用了两种染核的方法对大量的贾第虫细胞进行了观察，以期能从中找到两核保持等同的证据。结果发现在大量细胞群体中存在一定比例的单核细胞和一侧含有两个核的细胞。据此我们推测：贾第虫可能通过发生一侧细胞核的丢失或萎缩，然后由另外一侧的核进行复制，经过核的重排恢复正常的左右核的状态。这可能就是贾第虫消除两核基因出现的差异，保持两核之间等位基因一致乃至两个核完全等同的机制。

核仁蛋白基因组的原核起源与核仁蛋白网络进化组装分析

核仁是真核细胞间期核中最明显的结构，其主要功能是作为rDNA 的转录加 工和核糖体亚基合成、组装的场所。这一重要功能是包括原核生物和真核生物在 内的所有细胞生物都具有的，但核仁这一结构却只存在于真核细胞中。那么在从 原核到真核的进化过程中，核仁是如何进化而来，核仁的蛋白成分又是通过怎样 的方式从简单到复杂地组装起来，这些问题到目前为止都还没有得到深入的了 解。随着 “组学”的发展，核仁蛋白基因组的研究逐渐开展起来。越来越多的 核仁蛋白基因组数据为我们从“组学”的角度来探讨核仁的起源及其蛋白网络的 进化组装等问题奠定了基础。 1）本文首先通过比较目前仅有的三种真核生物（人，拟南芥，芽殖酵母） 的核仁蛋白基因组数据库，将三个核仁蛋白基因组共享的直系同源蛋白簇提取出 来构建出真核生物基本核仁蛋白基因组I (EBNP I)。由于拟南芥和芽殖酵母核仁 蛋白数据库明显偏小，为避免它们可能不全而带来的影响，进一步将人的核仁蛋 白基因组与拟南芥和芽殖酵母的全蛋白基因组共享的直系同源蛋白簇提取出来 构建出真核生物基本核仁蛋白基因组II（EBNP II）。EBNP 中的人核仁蛋白质序 列用作搜索序列去BLASTP 原核基因组，并构建同源关系矩阵进行聚类分析。 结果发现核仁蛋白基因组原核起源部分具有复合起源的特性，一部分来源于古细 菌，一部分来源于真细菌。并对EBNP II 中人核仁蛋白进行功能划分和对不同的 功能类群进行了起源分析，结果发现‘与核糖体有关’这一功能类群起源于古细 菌，‘与mRNA 有关’和‘与翻译有关’功能类群可能起源于古细菌，并在真核 阶段招募了大量蛋白成分，‘与染色质有关’这一功能类群可能起源于真细菌， 随后在真核阶段此功能类群招募了大量蛋白成分。 2）本文利用芽殖酵母的蛋白质相互作用数据，通过在原核生物基因组和原 生生物基因组的同源搜索，将芽殖酵母核仁蛋白分成不同进化时期产生的三个部 分：原核起源蛋白，原生生物起源蛋白，酵母特异蛋白。通过分析各部分蛋白质 的GO 注释，结果发现，原核起源蛋白主要行使与核糖体亚基合成与组装等核仁最基本的功能。通过比较各个部分之间以及各部分内蛋白质相互连接情况，结果 发现，芽殖酵母核仁蛋白网络按照不对称原则将招募的新蛋白组装入网络，整个 芽殖酵母核仁蛋白网络以原核起源蛋白所构建的框架为基础，原核起源蛋白起着 核心的作用。

麂属动物的DNA进化

本文分析了麂属动物及其近缘种的线粒体DNA（Mitochondrial DNA, mtDNA)和核塘体DNA（Ribosomal DNA, rDNA）限制性片段长度多态性（RFLP），建立了麂属动物mtDNA和rDNA的限制性内切酶间谱。据此计算出各个物种的种内及种间的遗传距离，构建了麂属动物的种内及种间的分子聚类图。结果表明，在现生麂类中，黑麂和贡山麂之间的关缘关系最近，其次是费氏麂；印度麂是一个特化的特种，它和黑麂支系（包括费氏麂、贡山麂和黑麂）可能是从小麂的祖先类群中独立分化出来的。在近缘物种中，毛冠鹿与麂属动物的亲缘关系较近。结合前人有关的工作，计算19种鹿科动物之间的遗传距离并绘制它们的分子聚类图。

寇氏隐甲藻(Cryp the codinium cohnii 涡鞭毛虫类)核仁的分裂

对涡鞭毛虫寇氏隐甲藻（Cryp thecodinium cohnii）细胞周期中的核仁进行了电镜连续超薄切片三维重组观察，并进行了Ag-NOR银染的光镜和电镜观察。在整个细胞周期中，寇氏隐甲藻的核仁内都有由核仁染色体发出的染色质索，又由染色质索发出细胞染色质丝进入核仁纤维区中。细胞进入分裂期后，核仁内的染色质索逐渐变粗。在核仁拉长前，核仁内大部分染色质索凝集成为“核仁内染色体”。Ag-NOR染色结果表明，有少量的rDNA也收回到核仁内染色体上，但绝大部分rDNA始终分散地分布在核仁内。核仁染色体象其它染色体一样，也是以一端结合在贯穿细胞核的细胞质管道的壁上，看来也会以同样的方式移到两极。核仁染色体向二极移动，而与核仁染色体相连的rDNA的绝大部分又始终分散地分布在核仁纤维区中，这显然是甲藻核仁拉长并分裂为二的根本原因。核仁内始终存在分散的rDNA也是它们的核仁不瓦解的根本原因。我们观察到，寇氏隐甲藻的染色体不仅连接于细胞质管道壁上，而且还与管道中的微管形成结构上的联系，从而可以认为它们的染色体移动也有微管参与。这点修正了kubai与Ris(1969)的报导。本文根据涡鞭毛虫（甲藻）的核仁在核仁分裂过程中只是拉长和分裂，而并不瓦解和重建的根本原因，探讨了典型有丝分裂过程中核仁的瓦解与重建的起源问题。