The origin of red algae and cryptomonad nucleomorphs: A comparative phylogeny based on small and large subunit rRNA sequences of Palmaria palmata, Gracilaria verrucosa, and the Guillardia theta nucleomorph

The complete large subunit rRNA sequences from the red algae Palmaria palmata and Gracilaria verrucosa, and from the nucleomorph of the cryptomonad Guillardia theta, were determined in order to assess their phylogenetic relationships relative to each other and to other eukaryotes. Neighbor-joining, maximum-parsimony, and maximum-likelihood trees were constructed on the basis of small subunit rRNA, large subunit rRNA, and a combination of both molecules. Our results support the hypothesis that the cryptomonad plastid is derived from a primitive red alga, in that an ancient common ancestor of rhodophytes and cryptomonad nucleomorphs is indicated. This cluster shows some affinity with chlorobionts, which could point to a monophyletic origin of green and red plastids. However, the exact branching order of the crown eukaryotes remains uncertain and further research is required.     

Isolation,physical map and gene map of mitochondrial DNA from the cryptomonad Pyrenomonas salina

Mitochondrial DNA (mtDNA) from the cryptomonad Pyrenomonas salina was isolated by CsCl-buoyant density centrifugation of whole-cell DNA in the presence of Hoechst dye 33258. mtDNA consists of circular molecules about 47 kb in size as estimated from restriction enzyme analysis. A physical map for six restriction enzymes (Bam HI, Bge I, Eco RI, Pst I, Sac I and Sac I) has been constructed. Genes coding for the small subunit of rRNA, cytochrome oxidase subunits I and II, and apocytochrome b were localized on this map using Southern blot hybridization with heterologous gene probes from Oenothera. Genes for 5S rRNA and NADH dehydrogenase subunit 5 are absent from P. salina mtDNA. The mitochondrial genome, being the first analysed to this extent in chromophytic algae, should be valuable for taxonomic and phylogenetic studies.     

PHYLOGENY OF GRACILARIA LEMANEIFORMIS (RHODOPHYTA) BASED ON SEQUENCE ANALYSIS OF ITS SMALL SUBUNIT RIBOSOMAL RNA CODING REGION1

The small subunit ribosomal RNA (rRNA) sequence of Gracilaria lemaneiformis Bory Weber-van Bosse was inferred from analysis of rRNA coding regions that were amplified by the polymerase chain reaction method. Comparison of the G. lemaneiformis small subunit rRNA to homologous genes of diverse eukaryotes demonstrated that the red algal divergence was nearly simultaneous with the separation of plants, fungi, animals and many other protist lineages. This result conflicts with those of 5S rRNA sequence and plastid based phytogenies which suggest that red algae represent an early divergence in the eukaryotic line of descent. Further, algae appear to be of polyphyletic origin and red algae are unrelated to higher fungi.     

Cytoplasmic ribosomal proteins from Chlamydomonas reinhardtii: characterization and immunological comparisons

Summary Experiments were undertaken to characterize the cytoplasmic ribosomal proteins (r-proteins) in Chlamydomonas reinhardtii and to compare immunologically several cytoplasmic r-proteins with those of chloroplast ribosomes of this alga, Escherichia coli, and yeast. The large and small subunits of the C. reinhardtii cytoplasmic ribosomes were shown to contain, respectively, 48 and 45 r-proteins, with apparent molecular weights of 12,000–59,000. No cross-reactivity was seen between antisera made against cytoplasmic r-proteins of Chlamydomonas and chloroplast r-proteins, except in one case where an antiserum made against a large subunit r-protein cross-reacted with an r-protein of the small subunit of the chloroplast ribosome. Antisera made against one out of five small subunit r-proteins and three large subunit r-proteins recognized r-proteins from the yeast large subunit. Each of the yeast r-proteins has been previously identified as an rRNA binding protein. The antiserum to one large subunit r-protein cross-reacted with specific large subunit r-proteins from yeast and E. coli.     

Ribotyping of 16S and 23S rRNA genes and organization of rrn operons in members of the bacterial genera Gemmata,Planctomyces, Thermotoga,Thermus, and Verrucomicrobium

The number of organization of rrn genes of two members of the order Planctomycetales, Planctomyces limnophilus and Gemmata obscuriglobus, as well as three species from other bacterial phyla, namely Thermotoga maritima, Thermus aquaticus and Verrucomicrobium spinosum were examined by Southern blot hybridization analysis of restricted DNA with labeled 16S- and 23S rRNAs. Ribotyping analysis revealed that two species contain unlinked 16S- and 23S rRNA genes. Planctomyces limnophilus possessed two unlinked rrn genes which were separated from each other by at least 4.3 kb, and Thermus aquaticus had to unlinked 16S and 23S rRNA genes, separated from each other by at least 2.5 kb. Gemmata obscuriglobus exhibited five genes for which the organization could as yet not be determined because of the complex hybridization patterns. In the other two species, rrn genes clustered in operons. Thermotoga maritima had a single gene for each rRNA species which were separated by not more than 1.5 kb, while Verrucomicrobium spinosum had four copies of probably linked 16S and 23S rRNA genes with a maximal distance between 16S and 23S rRNA genes of 1.3 kb.     

Nitrobacter winogradskyi cytochrome c oxidase genes are organized in a repeated gene cluster

Cytochrome c oxidase (EC 1.9.3.1) is one of the components of the electron transport chain by which Nitrobacter, a facultative lithoautotrophic bacterium, recovers energy from nitrite oxidation. The genes encoding the two catalytic core subunits of the enzyme were isolated from a Nitrobacter winogradskyi gene library. Sequencing of one of the 14 cloned DNA segments revealed that the subunit genes are side by side in an operon-like cluster. Remarkably the cluster appears to be present in at least two copies per genome. It extends over a 5–6 kb length including, besides the catalytic core subunit genes, other cytochrome oxidase related genes, especially a heme O synthase gene. Noteworthy is the new kind of gene order identified within the cluster. Deduced sequences for the cytochrome oxidase subunits and for the heme O synthase look closest to their counterparts in other -subdivision Proteobacteria, particularly the Rhizobiaceae. This confirms the phylogenetic relationships established only upon 16S rRNA data. Furthermore, interesting similarities exist between N. winogradskyi and mitochondrial cytochrome oxidase subunits while the heme O synthase sequence gives some new insights about the other similar published -subdivision proteobacterial sequences.Abbreviations COI cytochrome oxidase subunit I - COII cytochrome oxidase subunit II - COIII cytochrome oxidase subunit III - HOS Heme O synthase - ORF open reading frame - SDS sodium dodecyl sulfate     

Taxonomy, molecular phylogeny, and ultrastructural morphology of Olpidiopsis porphyrae sp. nov. (Oomycetes, straminipiles), a unicellular obligate endoparasite of Bangia and Porphyra spp. (Bangiales, Rhodophyta)

Olpidiopsis porphyrae sp. nov., a marine oomycete endoparasite that infects the commercially cultivated red alga Porphyra yezoensis, is described and its phylogenetic position based on molecular data and ultrastructural morphology is discussed. O. porphyrae infects the host Porphyra by means of encysted zoospores. Spherical-shaped holocarpic thalli develop within the cytoplasm of its algal host, which produce monoplanetic, subapically biflagellate zoospores. The characteristic features of this isolate are the ellipsoidal, unicellular thallus and simple holocarpic zoosporangial development, which show morphological similarity with the genus Olpidiopsis. Laboratory infection experiments with a wide range of green, brown, and red algae revealed that O. porphyrae infects several stages of the bangialean red algae (the genera Bangia and Porphyra). Molecular phylogenetic analyses inferred from both SSU rRNA and cox2 genes showed O. porphyrae branched before the main saprolegnian and peronosporalean lineages within the monophyletic oomycete clade, indicating its phylogenetic separation from them. A single or double K-body-like organelle, which contains tubular inclusions, is found located to one side of the zoospore nucleus and shows similarities to homologous organelles previously described in O. saprolegniae. The ultrastructural morphology of O. porphyrae with zoospore initials containing K-bodies and tubular mitochondrial cristae is characteristic of oomycetes. Group I intron-like multiple insertions were found in the SSU rRNA gene of O. porphyrae. This is the first report of SSU group I introns in the class Oomycetes.     

Complete modification maps for the cytosolic small and large subunit rRNAs of Euglena gracilis: functional and evolutionary implications of contrasting patterns between the two rRNA components

In the protist Euglena gracilis, the cytosolic small subunit (SSU) rRNA is a single, covalently continuous species typical of most eukaryotes; in contrast, the large subunit (LSU) rRNA is naturally fragmented, comprising 14 separate RNA molecules instead of the bipartite (28S + 5.8S) eukaryotic LSU rRNA typically seen. We present extensively revised secondary structure models of the E. gracilis SSU and LSU rRNAs and have mapped the positions of all of the modified nucleosides in these rRNAs (88 in SSU rRNA and 262 in LSU rRNA, with only 3 LSU rRNA modifications incompletely characterized). The relative proportions of ribose-methylated nucleosides and pseudouridine (∼ 60% and ∼ 35%, respectively) are closely similar in the two rRNAs; however, whereas the Euglena SSU rRNA has about the same absolute number of modifications as its human counterpart, the Euglena LSU rRNA has twice as many modifications as the corresponding human LSU rRNA. The increased levels of rRNA fragmentation and modification in E. gracilis LSU rRNA are correlated with a 3-fold increase in the level of mispairing in helical regions compared to the human LSU rRNA. In contrast, no comparable increase in mispairing is seen in helical regions of the SSU rRNA compared to its homologs in other eukaryotes. In view of the reported effects of both ribose-methylated nucleoside and pseudouridine residues on RNA structure, these correlations lead us to suggest that increased modification in the LSU rRNA may play a role in stabilizing a ‘looser’ structure promoted by elevated helical mispairing and a high degree of fragmentation.     

Comparative Analysis of the Ribosomal Componentsof the Hydrogenosome-Containing Protist, Trichomonas vaginalis

The ribosomes of the amitochondriate but hydrogenosome-containing protist lineage, the trichomonads, have previously been reported to be prokaryotic or primitive eukaryotic, based on evidence that they have a 70S sedimentation coefficient and a small number of proteins, similar to prokaryotic ribosomes. In order to determine whether the components of the trichomonad ribosome indeed differ from those of typical eukaryotic ribosomes, the ribosome of a representative trichomonad, Trichomonas vaginalis, was characterized. The sedimentation coefficient of the T. vaginalis ribosome was smaller than that of Saccharomyces cerevisiae and larger than that of Escherichia coli. Based on two-dimensional PAGE analysis, the number of different ribosomal proteins was estimated to be approximately 80. This number is the same as those obtained for typical eukaryotes (approximately 80) but larger than that of E. coli (approximately 55). N-Terminal amino acid sequencing of 18 protein spots and the complete sequences of 4 ribosomal proteins as deduced from their genes revealed these sequences to display typical eukaryotic features. Phylogenetic analyses of the five ribosomal proteins currently available also clearly confirmed that the T. vaginalis sequences are positioned within a eukaryotic clade. Comparison of deduced secondary structure models of the small and large subunit rRNAs of T. vaginalis with those of other eukaryotes revealed that all helices commonly found in typical eukaryotes are present and conserved in T. vaginalis, while variable regions are shortened or lost. These lines of evidence demonstrate that the T. vaginalis ribosome has no prokaryotic or primitive eukaryotic features but is clearly a typical eukaryotic type.     

Isolation, Crystallization, and Investigation of Ribosomal Protein S8 Complexed with Specific Fragments of rRNA of Bacterial or Archaeal Origin

The core ribosomal protein S8 binds to the central domain of 16S rRNA independently of other ribosomal proteins and is required for assembling the 30S subunit. It has been shown with E. coli ribosomes that a short rRNA fragment restricted by nucleotides 588-602 and 636-651 is sufficient for strong and specific protein S8 binding. In this work, we studied the complexes formed by ribosomal protein S8 from Thermus thermophilus and Methanococcus jannaschii with short rRNA fragments isolated from the same organisms. The dissociation constants of the complexes of protein S8 with rRNA fragments were determined. Based on the results of binding experiments, rRNA fragments of different length were designed and synthesized in preparative amounts in vitro using T7 RNA-polymerase. Stable S8–RNA complexes were crystallized. Crystals were obtained both for homologous bacterial and archaeal complexes and for hybrid complexes of archaeal protein with bacterial rRNA. Crystals of the complex of protein S8 from M. jannaschii with the 37-nucleotide rRNA fragment from the same organism suitable for X-ray analysis were obtained.     

Analysis of the genes encoding the largest subunit of RNA polymerase II in Arabidopsis and soybean

We have cloned and sequenced the gene encoding the largest subunit of RNA polymerase II (RPB1) from Arabidopsis thaliana and partially sequenced genes from soybean (Glycine max). We have also determined the nucleotide sequence for a number of cDNA clones which encode the carboxyl terminal domains (CTDs) of RNA polymerase II from both soybean and Arabidopsis. The Arabidopsis RPB1 gene encodes a polypeptide of approximately 205 kDa, consists of 12 exons, and encompasses more than 8 kb. Predicted amino acid sequence shows eight regions of similarity with the largest subunit of other prokaryotic and eukaryotic RNA polymerases, as well as a highly conserved CTD unique to RNA polymerase II.The CTDs in plants, like those in most other eukaryotes, consist of tandem heptapeptide repeats with the consensus amino acid sequence PTSPSYS. The portion of RPB1 which encodes the CTD in plants differs from that of RPB1 of animals and lower eukaryotes. All the plant genes examined contain 2–3 introns within the CTD encoding regions, and at least two plant genes contain an alternatively spliced intron in the 3 untranslated region. Several clustered amino acid substitutions in the CTD are conserved in the two plant species examined, but are not found in other eukaryotes. RPB1 is encoded by a multigene family in soybean, but a single gene encodes this subunit in Arabidopsis and most other eukaryotes.     

Morphological and molecular investigations of Paramecium schewiakoffi sp. nov. (Ciliophora, Oligohymenophorea) and current status of distribution and taxonomy of Paramecium spp.

Paramecium schewiakoffi sp. nov. is described from a pond in Shanghai, China. It is a freshwater species belonging to the “aurelia” subgroup of the genus. It is of similar size and shape to P. jenningsi, but has a single large micronucleus of the “chromosomal” morphological type, while P. jenningsi has two smaller micronuclei. The general morphology, morphometric characteristics and nuclear reorganization pattern, a random amplified polymorphic DNA (RAPD) fingerprint pattern, and the small subunit rRNA gene sequence are presented for the species. Comparison of P. schewiakoffi with the other species of Paramecium indicates that it is a valid new species of the genus. Geographical locations reported for many Paramecium species do not support the theory that all ciliates have a cosmopolitan distribution. It is proposed that, in an extension of Jankowski's earlier suggestion, the genus Paramecium should be subdivided into four subgenera: Chloroparamecium, Helianter, Cypriostomum and Paramecium, on the basis of morphometric, biological and molecular differences.     

A PROPOSAL FOR A NEW RED ALGAL ORDER,THE THOREALES1

Representatives of the freshwater red algal family Thoreaceae were studied to resolve their taxonomic and phylogenetic status. Three specimens of Nemalionopsis and five collections of Thorea were examined for pit plug ultrastructure and analyzed for the sequences of the genes coding for the large subunit of RUBISCO (rbcL) and the small subunit of rRNA (18S rRNA). The phylogenetic trees generated from the two genes, and a combined tree all showed the Thoreaceae to be contained in a well‐supported monophyletic clade that is separate from the other two families currently classified in the Batrachospermales, the Batrachospermaceae and the Lemaneaceae. In addition, secondary structure elements of the 18S rRNA gene were observed at positions 650 and 1145 (Escherichia coli numbering system) that are not present in other members of the Rhodophyta. The pit plugs of the gametophytic and chantransia stages of the Thoreaceae contain two cap layers, the outer one of which is typically plate‐like, though occasionally inflated ones have been seen. No pit plug cap membrane has been observed. These findings indicate the Thoreaceae has been misclassified in the Batrachospermales and should be placed in its own order, the Thoreales. This order is characterized by having freshwater representatives with multiaxial gametophytes, a uniaxial chantransia stage, and pit plugs with two cap layers, the outer one of which is usually plate‐like.     

Phylogenetic relationships among species of the genus Issatchenkia Kudriavzev

The phylogenetic relatedness of Issatchenkia spp. was estimated from partial rRNA sequences in two regions of the large subunit and one region of the small subunit. I. terricola was the most divergent species of the genus, differing from other members by 18% nucleotide differences in the highly variable 25S-635 region. These data indicate Issatchenkia to be the most divergent ascomycetous yeast genus presently known.     

我国北方岩溶泉域刚毛藻的系统发育及形态学研究

脆弱刚毛藻(Cladophora fracta)是一种大型丝状绿藻,生境分布广泛。然而,对于岩溶泉域分布的刚毛藻研究较少,它们的遗传多样性、生物地理亲缘性和生理特性都有待于深入研究。该研究对我国北方地区五个典型岩溶泉域的50个脆弱刚毛藻样本进行了形态学和分子系统学描述。主要研究目标:(1)对我国北方地区五个典型岩溶泉的刚毛藻生境进行描述;(2)根据形态学特征和分子序列对藻体进行鉴定;(3)探究生境对藻体生理特性的影响。结果表明:基于SSU和LSU序列的结果,发现所分析的50株刚毛藻个体为同一种,同时还发现了13个不同的核糖体基因型。基于SSU和LSU的系统发育树,刚毛藻属均未能形成单系分支,分布在三个不同的分支上。13个样本基因型在SSU和LSU树中的位置相似,与Cladophora vagabunda有很高的序列同源性,但是形态特征却差异很大。从显微结构结果来看,五个岩溶泉域采集到的刚毛藻在细胞直径上无显著差异,藻体的形态特征与脆弱刚毛藻相一致。但是,岩溶泉域采集的藻体细胞直径比文献报道中在湖泊和河流中采集的脆弱刚毛藻直径要大。另外,仅在两个地点(XA和ST)采集的标本中发现有假根状分枝。因此,基于形态学和分子序列的结果,将这五个泉域的刚毛藻鉴定为脆弱刚毛藻(Cladophora fracta)。     

枫香变红过程中叶片组织结构、光合特性及色素含量变化研究

枫香(Liquidambar formosana)因其叶片入秋后逐渐变红而极具观赏价值,是优良的景观生态树种。为了解枫香叶片结构变化与叶色的关系,该文通过连续监测枫香叶片变红过程中组织结构、光合特性及色素含量的变化,分析叶片结构与其光合特性和色素的关系。结果表明：(1)叶片变色过程中,表皮细胞均为椭圆形,紧密排列,未观察到明显的细胞变异,表面未附着绒毛和蜡质,且上表皮细胞与栅栏组织细胞间排列紧密,未出现较大的气室。(2)随着叶片逐渐变红,叶片结构变化显著,其中叶片、上表皮、栅栏组织和海绵组织厚度及气孔开度均逐渐减小,而气孔器长和宽、单个气孔器面积则逐渐增大。(3)随着叶片结构的变化,其叶绿素含量逐渐减少,致使净光合速率逐渐减小,在出现光破坏时,叶片通过在栅栏组织细胞液泡内合成花色苷来自我保护,而大量的花色苷致使叶片表面呈现红色。综上认为,叶绿素含量降低,花色素苷大量积累是导致枫香叶片变红的直接原因,而枫香叶色变红则是其一系列生理结构特征综合作用的结果。     

pZ189质粒DNA体外复制系统的建立

报道了含SV40复制起点的质粒DNA在真核细胞抽提物中进行复制的DNA体外复制系统的建立. 在外源性蛋白质SV40大T抗原(SV40 Tag)的参与下,穿梭质粒pZ189能在猴肾vero细胞胞浆抽提物中,利用其中参与体内DNA复制所需的蛋白质成分,有效地进行体外DNA复制. 从而为研究真核细胞DNA复制系统的结构与功能提供了简单、有效的模型.     

Effects of in vitro tissue culture conditions and acclimatization on the contents of Rubisco, leaf soluble proteins, photosynthetic pigments, and C/N ratio

The levels of two subunits of chloroplast ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), total soluble proteins, carbon and leaf nitrogen content, and photosynthetic pigments in various plants (avocado, oak, olive, and strawberry) grown in vitro and ex vitro were analysed. Compared to ex vitro grown plants, micropropagated avocado, oak, and strawberry showed a markable decrease in large subunit Rubisco. However, the small subunit only decreased in strawberry and oak. Contrary to this, olive did not reveal any difference in the level of either subunit. The C/N ratio increased significantly in in vitro grown plants, except in the case of olive, where an opposite behaviour was found. Leaf chlorophyll concentration on unit mass basis was higher in all the in vitro plants than in those of greenhouse- grown plants. Only avocado plantlets showed a statistically significant decrease in total soluble proteins. Further, overall data suggest that in vitro cultural conditions have a species-specific influence on large and small subunits of Rubisco, independent of the protein, chlorophyll, or nitrogen level.     

Comparison of primary and secondary 26S rRNA structures in twoTetrahymena species: Evidence for a strong evolutionary and structural constraint in expansion segments

Summary We have determined the nucleotide sequence of the 26S large subunit (LSU) rRNA genes for twoTetrahymena species,T. thermophila andT. pyriformis. The inferred rRNA sequences are presented in their most probable secondary structures based on compensatory mutations, energy, and conservation criteria. The majority of the nucleotide changes between the twoTetrahymena LSU rRNAs and the positions of a relatively large deletion and of the processing cleavage sites resulting in the generation of the hidden break are all located within the so-called divergent domains or expansion segments. These are regions within the common core of secondary structure where expansions have taken place during the evolution of the rRNA of higher eukaryotes.The dispensable nature of some of the expansion segments has been taken as evidence of their non-functionality. However, our data show that a considerable selective constraint has operated to presesrve the secondary structure of these segments. Especially in the case of the D2 and D8 segments, the presence of a considerable number of compensatory base changes suggests that the secondary structure of these regions is of functional importance. Alternatively, these expansion segments may have maintained characteristic folding patterns because only such structures are being tolerated within otherwise functionally important regions.     

Implications of complete nuclear large subunit ribosomal RNA molecules from the harmful unarmored dinoflagellate Cochlodinium polykrikoides (Dinophyceae) and relatives

The D1/D2 domains of large subunit (LSU) rDNA have commonly been used for phylogenetic analyses of dinoflagellates; however, their properties have not been evaluated in relation to other D domains due to a deficiency of complete sequences. This study reports the complete LSU rRNA gene sequence in the causative unarmored dinoflagellate Cochlodinium polykrikoides, a member of the order Gymnodiniales, and evaluated the segmented domains and secondary structures when compared with its relatives. Putative LSU rRNA coding regions were recorded to be 3433 bp in length (49.0% GC content). A secondary structure predicted from the LSU and 5.8S rRNAs and parsimony analyses showed that most variation in the LSU rDNA was found in the 12 divergent (D) domains. In particular, the D2 domain was the most informative in terms of recent evolutional and taxonomic aspects, when compared with both the phylogenetic tree topologies and molecular distance (approximately 10 times higher) of the core LSU. Phylogenetic analysis was performed with a matrix of LSU DNA sequences selected from domains D2 to D4 and their flanking core sequences, which showed that C. polykrikoides was placed on the same branch with Akashiwo sanguinea in the “GPP” complex, which is referred to the gymnodinioid, peridinioid and prorocentroid groups. A broad phylogeny showed that armored and unarmored dinoflagellates were never clustered together; instead, they were clearly divided into two groups: the GPP complex and Gonyaulacales. The members of Gymnodiniales were always interspersed with peridinioid, prorocentroid and dinophysoid forms. This supports previous findings showing that the Gymnodiniales are polyphyletic. This study highlights the proper selection of LSU rDNA molecules for molecular phylogeny and signatures.