The phylogenetic position of the pelobiont Mastigamoeba balamuthi based on sequences of rDNA and translation elongation factors EF-1alpha and EF-2

The taxonomic position and phylogenetic relationships of the Pelobionta, an amitochondriate amoeboflagellate group, are not yet completely settled. To provide more information, we obtained sequences for the large subunit rDNA gene, the gene for translation elongation factor 1alpha, and for a large part of the gene encoding translation elongation factor 2 from a representative of this group, Mastigamoeba balamuthi (formerly Phreatamoeba balamuthi). The gene for the large subunit rDNA was unusually large compared to those of other protists, a phenomenon that had previously been observed for the gene encoding the small subunit rDNA. Phylogenetic reconstruction using a maximum likelihood method was performed with these sequences, as well as the gene encoding the small subunit rDNA. When evaluated individually, the M. balamuthi genes for the small and large subunit rDNAs and elongation factor 1alpha had a most recent common ancestor with either the Mycetozoa (slime molds) or with Entamoeba histolytica. A clade formed by M. balamuthi, E. histolytica, and Mycetozoa was not rejected statistically for any of the sequences. A combined maximum likelihood analysis using 3,935 positions from all molecules suggested that these three taxonomic units form a robust clade. We were unable to resolve the closest group to this clade using the combined analysis. These findings support the notion, which had previously been proposed primarily on cytological evidence, that both M. balamuthi and E. histolytica are closely related to the Mycetozoa and that these three together represent a major eukaryotic lineage.     

羊草光合作用相关基因的克隆与分析

在构建了羊草叶片cDNA文库的基础上,利用M13载体通用引物筛选其亚文库,挑选阳性克隆进行测序,将测序结果在NCBI基因库中进行比对,得到一个Rubisco大亚基基因全长序列和Rubisco小亚基基因部分序列,并对其核苷酸及其编码的氨基酸序列进行分析。结果显示,Rubisco大亚基基因长度为1 796 bp,与禾本科大麦、小麦、野雀麦、粗山羊草、旱麦草、异形花草、黑麦等的核苷酸序列同源性达98%以上;羊草的Rubisco小亚基基因部分序列含有一个开放阅读框,其长度为186 bp,编码61个氨基酸,与禾本科的小麦、大麦、燕麦、黑麦以及扁穗雀麦Rubisco小亚基基因氨基酸序列的同源性分别为93%、93%、91%、91%、92%。羊草Rubisco基因的克隆与分析有利于进一步研究其光合作用效率。     

The phylogenetic diversity of metagenomes

Phylogenetic diversity--patterns of phylogenetic relatedness among organisms in ecological communities--provides important insights into the mechanisms underlying community assembly. Studies that measure phylogenetic diversity in microbial communities have primarily been limited to a single marker gene approach, using the small subunit of the rRNA gene (SSU-rRNA) to quantify phylogenetic relationships among microbial taxa. In this study, we present an approach for inferring phylogenetic relationships among microorganisms based on the random metagenomic sequencing of DNA fragments. To overcome challenges caused by the fragmentary nature of metagenomic data, we leveraged fully sequenced bacterial genomes as a scaffold to enable inference of phylogenetic relationships among metagenomic sequences from multiple phylogenetic marker gene families. The resulting metagenomic phylogeny can be used to quantify the phylogenetic diversity of microbial communities based on metagenomic data sets. We applied this method to understand patterns of microbial phylogenetic diversity and community assembly along an oceanic depth gradient, and compared our findings to previous studies of this gradient using SSU-rRNA gene and metagenomic analyses. Bacterial phylogenetic diversity was highest at intermediate depths beneath the ocean surface, whereas taxonomic diversity (diversity measured by binning sequences into taxonomically similar groups) showed no relationship with depth. Phylogenetic diversity estimates based on the SSU-rRNA gene and the multi-gene metagenomic phylogeny were broadly concordant, suggesting that our approach will be applicable to other metagenomic data sets for which corresponding SSU-rRNA gene sequences are unavailable. Our approach opens up the possibility of using metagenomic data to study microbial diversity in a phylogenetic context.     

Complete sequence and gene organization of the Nosema spodopterae rRNA gene

By sequencing the entire ribosomal RNA (rRNA) gene of Nosema spodopterae, we show here that its gene organization follows a pattern similar to the Nosema type species, Nosema bombycis, i.e. 5'-large subunit rRNA (2,497 bp)-internal transcribed spacer (185 bp)-small subunit rRNA (1,232 bp)-intergenic spacer (277 bp)-5S rRNA (114 bp)-3'. Gene sequences and the secondary structures of large subunit rRNA, small subunit rRNA, and 5S rRNA are compared with the known corresponding sequences and structures of closely related microsporidia. The results suggest that the Nosema genus may be heterogeneous and that the rRNA gene organization may be a useful characteristic for determining which species are closely related to the type species.     

Species divergence and the measurement of microbial diversity

Diversity measurement is important for understanding community structure and dynamics, but has been particularly challenging for microorganisms. Microbial community characterization using small subunit rRNA (SSU rRNA) gene sequences has revealed an extensive, previously unsuspected diversity that we are only now beginning to understand, especially now that advanced sequencing technologies are producing datasets containing hundreds of thousands of sequences from hundreds of samples. Efforts to quantify microbial diversity often use taxon-based methods that ignore the fact that not all species are equally related, which can therefore obscure important patterns in the data. For example, alpha-diversity (diversity within communities) is often estimated as the number of species in a community (species richness), and beta-diversity (partitioning of diversity among communities) is often based on the number of shared species. Methods for measuring alpha- and beta-diversity that account for different levels of divergence between individuals have recently been more widely applied. These methods are more powerful than taxon-based methods because microorganisms in a community differ dramatically in sequence similarity, which also often correlates with phenotypic similarity in key features such as metabolic capabilities. Consequently, divergence-based methods are providing new insights into microbial community structure and function.     

Sequence analysis of ribosomal and mitochondrial genes of the giant liver fluke Fascioloides magna (Trematoda: Fasciolidae): intraspecific variation and differentiation from Fasciola hepatica

Complete sequences of ribosomal and mitochondrial genes of the giant liver fluke Fascioloides magna are presented. In particular, small subunit (18S) and internal transcribed spacers (ITS1 and ITS2) of the ribosomal gene (rDNA), as well as cytochrome c oxidase subunit I (cox1) and nicotinamide dehydrogenase subunit I (nad1) of the mitochondrial DNA (mtDNA), were analyzed. The 18S and ITS sequences were compared with previously published sequences of the liver fluke Fasciola hepatica. Fixed interspecific genetic differences were determined that allow molecular differentiation of F. magna and F. hepatica using either the PCR-RFLP method or PCR amplification of species-specific DNA regions. Additionally, intraspecific sequence polymorphism of the complete cox1 and nad1 mitochondrial genes in geographically distinct F. magna populations was determined. Based on the sequence divergences, short (< 500 bp) variable regions suitable for broader biogeographical studies of giant liver fluke were designed.     

New subgroup of Bacteroidetes and diverse microorganisms in Tibetan plateau glacial ice provide a biological record of environmental conditions

We recovered microorganisms from five ice core samples from three glaciers (Puruogangri, Malan, and Dunde) located in the Tibetan Plateau in China and analyzed their small subunit rRNA gene sequences. Most of the bacterial sequences were unknown previously; the most closely related known sequences were from bacteria of the Proteobacteria, Bacteroidetes , and Actinobacteria phyla. Chlorophyta, Streptophyta, Ciliophora , and fungal groups were represented among the 18S rRNA gene sequences that we obtained. The most abundantly represented glacial bacteria were Bacteroidetes , and Chlamydomonas was the predominant eukaryote. Comparative analysis showed that the Bacteroidetes sequences obtained from this study were highly similar to one another but most were only distantly related to previously characterized Bacteroidetes (<92% identity). We propose that our Bacteroidetes sequences represent two novel subgroups: one at the family level and one at the genus level. The unique ice environment and the high abundance of Bacteroidetes , combined with the coexistence of a high abundance of psychrophilic Chlamydomonas , strongly suggests that there is a viable ecosystem on the surface of Tibetan glaciers. Comparisons of microbial community structures in the five ice samples showed distinct differences, likely due to environmental differences in the locations in which the samples were obtained.     

Exploring Microbial Diversity and Taxonomy Using SSU rRNA Hypervariable Tag Sequencing

Massively parallel pyrosequencing of hypervariable regions from small subunit ribosomal RNA (SSU rRNA) genes can sample a microbial community two or three orders of magnitude more deeply per dollar and per hour than capillary sequencing of full-length SSU rRNA. As with full-length rRNA surveys, each sequence read is a tag surrogate for a single microbe. However, rather than assigning taxonomy by creating gene trees de novo that include all experimental sequences and certain reference taxa, we compare the hypervariable region tags to an extensive database of rRNA sequences and assign taxonomy based on the best match in a Global Alignment for Sequence Taxonomy (GAST) process. The resulting taxonomic census provides information on both composition and diversity of the microbial community. To determine the effectiveness of using only hypervariable region tags for assessing microbial community membership, we compared the taxonomy assigned to the V3 and V6 hypervariable regions with the taxonomy assigned to full-length SSU rRNA sequences isolated from both the human gut and a deep-sea hydrothermal vent. The hypervariable region tags and full-length rRNA sequences provided equivalent taxonomy and measures of relative abundance of microbial communities, even for tags up to 15% divergent from their nearest reference match. The greater sampling depth per dollar afforded by massively parallel pyrosequencing reveals many more members of the “rare biosphere” than does capillary sequencing of the full-length gene. In addition, tag sequencing eliminates cloning bias and the sequences are short enough to be completely sequenced in a single read, maximizing the number of organisms sampled in a run while minimizing chimera formation. This technique allows the cost-effective exploration of changes in microbial community structure, including the rare biosphere, over space and time and can be applied immediately to initiatives, such as the Human Microbiome Project.     

Development and application of a PCR-denaturing gradient gel electrophoresis tool to study the diversity of Nitrobacter-like nxrA sequences in soil

A new PCR-denaturing gel gradient electrophoresis (DGGE) tool based on the functional gene nxrA encoding the catalytic subunit of the nitrite oxidoreductase in nitrite-oxidizing bacteria (NOB) has been developed. The first aim was to determine if the primers could target representatives of NOB genera: Nitrococcus and Nitrospira. The primers successfully amplified nxrA gene sequences from Nitrococcus mobilis, but not from Nitrospira marina. The second aim was to develop a PCR-DGGE tool to characterize NOB community structure on the basis of Nitrobacter-like partial nrxA gene sequences (Nb-nxrA). We tested (1) the ability of this tool to discriminate between Nitrobacter strains, and (2) its ability to reveal changes in the community structure of NOB harbouring Nb-nrxA sequences induced by light grazing or intensive grazing in grassland soils. The DGGE profiles clearly differed between the four Nitrobacter strains tested. Differences in the structure of NOB community were revealed between grazing regimes. Phylogenetic analysis of the sequences corresponding to different DGGE bands showed that Nb-nxrA sequences did not group in management-specific clusters. Most of the nxrA sequences obtained from soils differed from nxrA sequences of NOB strains. Along with existing tools for characterizing the community structure of nitrifiers, this new approach is a significant step forward to performing comprehensive studies on nitrification.     

Molecular analysis of arbuscular mycorrhizal fungi colonising a semi-natural grassland along a fertilisation gradient

The community of arbuscular mycorrhizal fungi (AMF) colonizing the roots of Festuca pratensis and Achillea millefolium was characterized in a Swedish pasture at different times, along a gradient of fertilization. The small subunit ribosomal RNA gene was subjected to PCR and denaturing gradient gel electrophoresis (DGGE), sequencing and phylogenetic analysis. The sequences found in this study clustered in 10 discrete sequence groups, seven belonging to Glomus, two to Scutellospora and one to Diversispora. A negative correlation was observed between soil mineral nitrogen and the number of AMF sequence groups in the roots. The frequency of occurrence of AMF in roots decreased dramatically between June and September. No plant-host specificity could be detected.     

Organization of the mitochondrial genome of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> (anamorphic Hypocreales)

A 33.4-kb fragment of the mitochondrial genome of Fusarium oxysporum has been sequenced. The fragment contains the complete gene sequences for 13 of the 14 proteins typically encoded by the mitochondrial genome of filamentous ascomycetes. Similarity searching revealed all encoded proteins to be most similar to those from other members of the Hypocreales. The fragment contains the complete small subunit rRNA gene, partial large rRNA subunit gene, and 12 tRNAs. Two introns were present, one in the nad5 gene and one in the large rRNA subunit gene, the latter containing a ribosomal protein gene.     

Efficiency of ligation-mediated PCR and TAIL-PCR methods for isolation of RbcS promoter sequences from green microalgae Ankistrodesmus convolutus

Isolation of promoter sequences from known gene sequences is a tedious task in genome-related research. An efficient method of obtaining the promoter sequences is necessary in order to successfully use targeted promoters for genetic manipulations. Here, efficiency and usefulness of two PCR-based methods, namely: ligation-mediated PCR and thermal asymmetric interlaced (TAIL) PCR, for isolation of promoter sequences of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) gene from green microalgae Ankistrodesmus convolutus (A. convolutus) were evaluated. The results showed that the amplification efficiency of TAIL-PCR was higher than that of the ligation-mediated PCR method, i.e. the amplified promoter fragments of 1.2 and 0.8 kb in length or promoter sequences of 813 and 606 bp (after eliminating the unreadable sequences). The use of TAIL-PCR described here presents a low cost and efficient strategy for the isolation of promoter sequences of known genes, especially in GC-rich regions, and species with little or no available genome information such as A. convolutus.     

Paramecium mitochondrial genes. I. Small subunit rRNA gene sequence and microevolution

The sequences of the small subunit mitochondrial rRNA genes from two divergent species of Paramecium (primaurelia and tetraurelia) were determined. The gene lies near the center of the linear mitochondrial genome, on the same strand as are all other currently identified genes. The sequences generally resemble their counterparts found in cytoplasmic, procaryotic, and other mitochondrial sources. The rDNA gene boundaries were located by nuclease S1 protection. Small subunit rDNA spans about 1680 nucleotides, including an extraneous 83-base pair sequence very near the 3' end which is unique to Paramecium mitochondria. This "insert" occurs at the apex of the highly variable in length penultimate helix, according to proposed models for small subunit rRNA secondary structure. A discontinuity occurs in isolated rRNA near the start of the insert, resulting in a stable 13 S RNA species and a small segment containing the remaining 3' portion of the gene. The overall rRNA gene sequence was 94% conserved between the two species, and the nucleotide differences consisted of 53% transitions, 37% transversions, and 9% insertions plus deletions. These substitutions were somewhat clustered, and the two most divergent regions coincided with the gene boundaries. The sequence was aligned with Escherichia coli 16 S rRNA for direct comparison of sequence and structure.     

野生大豆rbcS基因的克隆及结构分析

核酮糖１,５二磷酸羧化酶（Ｒｕｂｉｓｃｏ,Ｅ．Ｃ．４．１．１．３９）是光合碳代谢中的关键酶,也是植物中研究最为广泛深入的一种酶。高等植物的Ｒｕｂｉｓｃｏ大、小亚基分别由叶绿体和核基因组编码。迄今已有几十种光合生物的Ｒｕｂｉｓｃｏ大、小亚基的基因（ｒｂｃＬ、ｒｂｃＳ）结构得到阐明［１］。在高等植物中ｒｂｃＳ基因由多基因家族编码,结构较为复杂,但它同时又是一种相对保守的基因,且同一物种内各ｒｂｃＳ基因成员是协同进化的,因此ｒｂｃＳ基因适合于植物分子进化及系统分类的研究［２］。我国是栽培大豆（Ｇｌｙｃ…     

Single‐cell sequencing of dinoflagellate (Dinophyceae) nuclear ribosomal genes

Genetic sequences from dinoflagellates offer valuable information regarding taxonomies, phylogenies and population genetics that generally require the growth of these organisms in culture. We have developed a quick and simple method to obtain small and large subunit ribosomal gene sequences from dinoflagellates using single cells. This method, based on freeze–thaw cell lysis and a simple two‐step polymerase chain reaction, provides template for sequencing in 6–8 h. We have sequenced five dinoflagellate species, including unculturable Dinophysis and Ceratium species, using fresh and frozen samples.     

Molecular Identification of Nanoplanktonic Protists Based on Small Subunit Ribosomal RNA Gene Sequences for Ecological Studies1

Nanoplanktonic protists are comprised of a diverse assemblage of species which are responsible for a variety of trophic processes in marine and freshwater ecosystems. Current methods for identifying small protists by electron microscopy do not readily permit both identification and enumeration of nanoplanktonic protists in field samples. Thus, one major goal in the application of molecular approaches in protistan ecology has been the detection and quantification of individual species in natural water samples. Sequences of small subunit ribosomal RNA (SSU rRNA) genes have proven to be useful towards achieving this goal. Comparison of sequences from clone libraries of protistan SSU rRNA genes amplified from natural assemblages of protists by the polymerase chain reaction (PCR) can be used to examine protistan diversity. Furthermore, oligonucleotide probes complementary to short sequence regions unique to species of small protists can be designed by comparative analysis of rRNA gene sequences. These probes may be used to either detect the RNA of particular species of protists in total nucleic acid extracts immobilized on membranes, or the presence of target species in water samples via in situ hybridization of whole cells. Oligonucleotide probes may also serve as primers for the selective amplification of target sequences from total population DNA by PCR. Thus, molecular sequence information is becoming increasingly useful for identifying and enumerating protists, and for studying their spatial and temporal distribution in nature. Knowledge of protistan species composition, abundance and variability in an environment can ultimately be used to relate community structure to various aspects of community function and biogeochemical activity.     

Phylogenetic analysis of the small subunit ribosomal RNA of Marteilia refringens validates the existence of phylum Paramyxea (Desportes and Perkins, 1990)

Marteilia refringens is recognized as one of the most significant pathogens of bivalve molluscs. The nucleotide sequence of the small subunit ribosomal RNA gene of Marteilia refringens is used to elucidate the phylogenetic position of the phylum Paramyxea. Genomic DNA was extracted from sporangia of Marteilia, purified from infected blue mussels, Mytilus edulis, and flat oysters, Ostrea edulis. The sequences obtained from Marteilia species purified from both oysters and mussels were identical. The sequence identity was confirmed by in situ hybridization using a DNA probe targeted to a variable region of the ribosomal DNA. The small subunit ribosomal RNA gene sequence of M. refringens is very different from all known sequences of eukaryotic organisms, including those of myxosporeans and haplosporeans. Therefore, the phylum Paramyxea should continue to be recognized as an independent eukaryotic phylum.     

Discrimination Amongst Leishmania by Polymerase Chain Reaction and Hybridization with Small Subunit Ribosomal DNA Derived Oligonucleotides

ABSTRACT. A method for discriminating among Leishmania is described, based upon small subunit ribosomal DNA sequence differences. The method was to amplify the entire 2.2 kb small subunit rDNA by polymerase chain reaction using conserved primers specific for the 5' and 3' termini of the small subunit ribosomal RNA, and then hybridize the product dotted onto nylon membranes with labeled oligonucleotides. The design of the hybridization probes was based upon complete small subunit rDNA sequences from L. amazonensis, L. major and L. guyanensis and partial sequences of L. mexicana, L. braziliensis, L. tropica and L. chagasi. A high degree of sequence similarity (> 99%) among species was found. However, sufficient sequence divergence occurred to permit the design of internal oligonucleotide probes specific for species complexes. This procedure successfully discriminated amongst a wide range of Leishmania isolates. The method detected as few as 10 cultured organisms and detected parasites in tissue samples from experimentally infected animals. Non-radioactive labeling showed the same specificity and sensitivity as radioactive probes.