Home About us Contact
|
Home About us Contact | ||
|
|
||
Small Subunit (small + subunit)
Terms modified by Small Subunit Selected AbstractsWrinkles in the rare biosphere: pyrosequencing errors can lead to artificial inflation of diversity estimatesENVIRONMENTAL MICROBIOLOGY, Issue 1 2010Victor Kunin Summary Massively parallel pyrosequencing of the small subunit (16S) ribosomal RNA gene has revealed that the extent of rare microbial populations in several environments, the ,rare biosphere', is orders of magnitude higher than previously thought. One important caveat with this method is that sequencing error could artificially inflate diversity estimates. Although the per-base error of 16S rDNA amplicon pyrosequencing has been shown to be as good as or lower than Sanger sequencing, no direct assessments of pyrosequencing errors on diversity estimates have been reported. Using only Escherichia coli MG1655 as a reference template, we find that 16S rDNA diversity is grossly overestimated unless relatively stringent read quality filtering and low clustering thresholds are applied. In particular, the common practice of removing reads with unresolved bases and anomalous read lengths is insufficient to ensure accurate estimates of microbial diversity. Furthermore, common and reproducible homopolymer length errors can result in relatively abundant spurious phylotypes further confounding data interpretation. We suggest that stringent quality-based trimming of 16S pyrotags and clustering thresholds no greater than 97% identity should be used to avoid overestimates of the rare biosphere. [source] Assembly of cytochrome f into the cytochrome bf complex in isolated pea chloroplastsFEBS JOURNAL, Issue 3 2001Ruth M. Mould Structural features of cytochrome f necessary for assembly into the cytochrome bf complex were examined in isolated pea chloroplasts following import of 35S-labelled chimeric precursor proteins, consisting of the presequence of the small subunit of Rubisco fused to the turnip cytochrome f precursor. Assembly was detected by nondenaturing gel electrophoresis of dodecyl maltoside-solubilized thylakoid membranes. A cytochrome f polypeptide unable to bind haem because of mutagenesis of Cys21 and Cys24 to alanine residues was assembled into the complex and had similar stability to the wild-type polypeptide. This indicates that covalent haem binding to cytochrome f is not necessary for assembly of the protein into the cytochrome bf complex. A truncated protein lacking the C-terminal 33 amino acid residues, including the transmembrane span and the stroma-exposed region, was translocated across the thylakoid membrane, had a similar stability to wild-type cytochrome f but was not assembled into the complex. This indicates that the C-terminal region of cytochrome f is important for assembly into the complex. A mutant cytochrome f unable to bind haem and lacking the C-terminal region was also translocated across the thylakoid membrane but was extremely labile, indicating that, in the absence of the C-terminal membrane anchor, haem-less cytochrome f is recognized by a thylakoid proteolytic system. [source] Dominance of a clonal green sulfur bacterial population in a stratified lakeFEMS MICROBIOLOGY ECOLOGY, Issue 1 2009Lea H. Gregersen Abstract For many years, the chemocline of the meromictic Lake Cadagno, Switzerland, was dominated by purple sulfur bacteria. However, following a major community shift in recent years, green sulfur bacteria (GSB) have come to dominate. We investigated this community by performing microbial diversity surveys using FISH cell counting and population multilocus sequence typing [clone library sequence analysis of the small subunit (SSU) rRNA locus and two loci involved in photosynthesis in GSB: fmoA and csmCA]. All bacterial populations clearly stratified according to water column chemistry. The GSB population peaked in the chemocline (c. 8 × 106 GSB cells mL,1) and constituted about 50% of all cells in the anoxic zones of the water column. At least 99.5% of these GSB cells had SSU rRNA, fmoA, and csmCA sequences essentially identical to that of the previously isolated and genome-sequenced GSB Chlorobium clathratiforme strain BU-1 (DSM 5477). This ribotype was not detected in Lake Cadagno before the bloom of GSB. These observations suggest that the C. clathratiforme population that has stabilized in Lake Cadagno is clonal. We speculate that such a clonal bloom could be caused by environmental disturbance, mutational adaptation, or invasion. [source] Potentiality of the cox1 gene in the taxonomic resolution of soil fungiFEMS MICROBIOLOGY LETTERS, Issue 1 2010Claire Molitor Abstract We explored the potential of the cox1 gene in the species resolution of soil fungi and compared it with the nuclear internal transcribed spacer (ITS) and small subunit (SSU)-rDNA. Conserved primers allowing the amplification of the fungal cox1 gene were designed, and a total of 47 isolates of Zygomycota and Ascomycota were investigated. The analysis revealed a lack of introns in >90% of the isolates. Comparison of the species of each of the six studied genera showed high interspecific sequence polymorphisms. Indeed, the average of nucleotide variations (4.2,11%) according to the genus, due mainly to the nucleotide substitutions, led to the taxonomic resolution of all the species studied regarding both ITS and SSU-rDNA, in which <88% were discriminated. The phylogenetic analysis performed after alignment of the cox1 gene across distant fungal species was in accordance with the well-known taxonomic position of the species studied and no overlap was observed between intra- and interspecific variations. These results clearly demonstrated that the cox1 sequences could provide good molecular markers for the determination of the species composition of environmental samples and constitute an important advance to study soil fungal biodiversity. [source] Phylogeographic variation among isolates of the Sirococcus conigenus P groupFOREST PATHOLOGY, Issue 1 2007H. Konrad Summary In this study the phylogeographic variation among isolates of the Sirococcus conigenus P group and the phylogenetic relationships of S. conigenus with Sirococcus clavigignenti-juglandacearum and other species previously placed in the genus Sirococcus were investigated. A collection of 33 isolates originating from Picea, Pinus and Larix in Europe, North America and Bhutan were characterized by sequence analyses of the internal transcribed spacer (ITS) region (including ITS1, 5.8S ribosomal DNA, ITS2) of the nuclear rDNA and a portion of the , -tubulin gene. In phylogenetic analyses most isolates from pine, spruce and larch formed a distinct clade, representing the P group of S. conigenus, which was separated from the T group of this pathogen. Four isolates from Picea in Europe and Canada formed a third clade within S. conigenus and these isolates are referred to as the S group. The P group consisted of five distinct ITS haplotypes, which partly differed in their optimum growth temperature and their growth rates at 25°C on malt extract agar. Nested clade analysis resolved the five haplotypes into three distinct clades and revealed significant genetic/geographic associations for some of the haplotypes. Parsimony analysis of the small subunit (18S) ribosomal DNA sequences confirmed the phylogenetic affinities between S. conigenus and S. clavigignenti-juglandacearum. In contrast, Godronia cassandrae and Hormococcus conorum, which formerly had been placed in the genus Sirococcus, were found to be only distantly related to S. conigenus and S. clavigignenti-juglandacearum. [source] Molecular diagnosis of Phytophthora lateralis in trees, water, and foliage baits using multiplex polymerase chain reactionFOREST PATHOLOGY, Issue 5 2001L. M. Winton A polymerase chain reaction (PCR)-based protocol for detection of Phytophthora lateralis in plant tissues and water is described. Base-pair (bp) deletions in both of the ribosomal DNA internal transcribed spacer (ITS) regions in P. lateralis were used to design complementary PCR primer sequences that amplify a 738 bp fragment only if P. lateralis DNA is present in the sample. Universal control primers based on conserved sequences of the nuclear ribosomal small subunit are included in a multiplexed reaction, providing an internal check on the procedure. The universal primers amplify an approximately 550 bp fragment that is common to plants, protists, and true fungi. The procedure reliably detects P. lateralis in cedar stem tissues and in roots. Positive reactions were obtained with as few as 200 P. lateralis zoospores in water. Diagnostic moléculaire par PCR multiplex pour détecter Phytophthora lateralis dans les arbres, l'eau et le feuillage utilisé comme piège Un protocole basé sur la PCR est décrit pour détecter Phytophthora lateralis dans les tissus végétaux et l'eau. Des délétions de paires de bases dans chacune des régions ITS de l'ADN ribosomal de P. lateralis ont été utilisées pour définir des amorces de PCR qui n'amplifient un fragment de 738 paires de bases que si l'ADN de P. lateralis est présent dans l'échantillon. Des amorces universelles basées sur des régions conservées de la petite sous-unité de l'ADN ribosomal nucléaire ont été incluses dans une réaction de PCR multiplex, fournissant ainsi un témoin interne de la réaction. Ces amorces universelles amplifient un fragment de 550 pb qui est commun aux plantes, aux protistes et aux champignons vrais. Ce protocole permet la détection de P. lateralis dans les tiges et dans les racines du Chamaecyparis. Des réactions positives ont été obtenues avec seulement 200 zoospores de P. lateralis dans l'eau. Molekulare Diagnose von Phytophthora lateralis in Bäumen, Wasser und als Köder benutzten Blättern mittels Multiplex-PCR Eine auf der PCR beruhende Methode zum Nachweis von Phytophthora lateralis in Pflanzengeweben und Wasser wird beschrieben. Deletionen in den beiden ITS Regionen der ribosomalen DNA von P. lateralis wurden zur Synthese von PCR-Primern ausgenutzt, die ein 738 Basenpaare langes Fragment nur dann amplifizieren, wenn P. lateralis in der Probe vorhanden ist. Universelle Primer, die konservierten Sequenzen der kleinen Unterheit der ribosomalen Kern-DNA entsprechen, wurden als interne Kontrollen in die Multiplex-PCR miteinbezogen. Diese Primer amplifizieren ein ungefähr 550 Basenpaare langes Fragment, das sowohl bei Pflanzen als auch bei Protisten und höheren Pilzen vorkommt. Mit der Methode liess sich P. lateralis im Stamm und in den Wurzeln von Lawsons Scheinzypresse verlässlich nachweisen. Für den Nachweis von P. lateralis im Wasser waren mindestens 200 Zoosporen nötig. [source] Stimulation of RNA polymerase II transcript cleavage activity contributes to maintain transcriptional fidelity in yeastGENES TO CELLS, Issue 5 2007Hiroshi Koyama The transcription elongation factor S-II, also designated TFIIS, stimulates the nascent transcript cleavage activity intrinsic to RNA polymerase II. Rpb9, a small subunit of RNA polymerase II, enhances the cleavage stimulation activity of S-II. Here, we investigated the role of nascent transcript cleavage stimulation activity on the maintenance of transcriptional fidelity in yeast. In yeast, S-II is encoded by the DST1 gene. Disruption of the DST1 gene decreased transcriptional fidelity in cells. Mutations in the DST1 gene that reduce the S-II cleavage stimulation activity led to decreased transcriptional fidelity in cells. A disruption mutant of the RPB9 gene also had decreased transcriptional fidelity. Expression of mutant Rpb9 proteins that are unable to enhance the S-II cleavage stimulation activity failed to restore the phenotype. These results suggest that both S-II and Rpb9 maintain transcriptional fidelity by stimulating the cleavage activity intrinsic to RNA polymerase II. Also, a DST1 and RPB9 double mutant had more severe transcriptional fidelity defect compared with the DST1 gene deletion mutant, suggesting that Rpb9 maintains transcriptional fidelity via two mechanisms, enhancement of S-II dependent cleavage stimulation and S-II independent function(s). [source] Phylogeny of Medusozoa and the evolution of cnidarian life cyclesJOURNAL OF EVOLUTIONARY BIOLOGY, Issue 3 2002A. G. Collins To investigate the evolution of cnidarian life cycles, data from the small subunit of the ribosome are used to derive a phylogenetic hypothesis for Medusozoa. These data indicate that Cnidaria is monophyletic and composed of Anthozoa and Medusozoa. While Cubozoa and Hydrozoa are well supported clades, Scyphozoa appears to be paraphyletic. Stauromedusae is possibly the sister group of either Cubozoa or all other medusozoans. The phylogenetic results suggest that: the polyp probably preceded the medusa in the evolution of Cnidaria; within Hydrozoa, medusa development involving the entocodon is ancestral; within Trachylina, the polyp was lost and subsequently regained in the parasitic narcomedusans; within Siphonophorae, the float originated prior to swimming bells; stauromedusans are not likely to be descended from ancestors that produced medusae by strobilation; and cubozoan polyps are simplified from those of their ancestors, which possessed polyps with gastric septa and four mesogleal muscle bands and peristomial pits. [source] Hepatitis B virus X protein upregulates expression of calpain small subunit 1 via nuclear facter-,B/p65 in hepatoma cellsJOURNAL OF MEDICAL VIROLOGY, Issue 6 2010Feng Zhang Abstract Hepatitis B virus (HBV) infection is closely correlated with the development of hepatocellular carcinoma (HCC), in which hepatitis B virus X protein (HBx) plays crucial roles. HBx is believed to be a multifunctional oncoprotein. It has been reported that the calpain small subunit 1 (Capn4) is upregulated in the HCC tissues and involved in the metastasis of HCC. Therefore, we suppose that HBx may promote hepatoma cell migration through Capn4. In the present study, we investigated the effect of HBx on regulating Capn4 expression in human HCC cells. Our data showed that HBx could increase promoter activity of Capn4 and upregulate the expression of Capn4 at the levels of mRNA and protein in human hepatoma HepG2 (or H7402) cells using luciferase reporter gene assay, real-time quantitative RT-PCR assay and Western blot analysis. While, the RNA interference targeting HBx mRNA was able to abolish the upregulation. Interestingly, we found that the inhibition of nuclear factor-,B (NF-,B) mediated by siRNA targeting NF-,B/p65 mRNA or PDTC (an inhibitor of NF-,B) could attenuate the upregulation of Capn4. While, HBx failed to increase the promoter activity of Capn4 in hepatoma cells when the putative NF-,B binding site of the Capn4 promoter was mutant, suggesting that NF-,B is involved in the activation of Capn4 mediated by HBx. In function, wound healing assay showed that HBx could significantly enhance the migration ability of HepG2 cells through upregulating Capn4. Thus, we conclude that HBx upregulate Capn4 through NF-,B/p65 to promote migration of hepatoma cells. J. Med. Virol. 82:920,928, 2010. © 2010 Wiley-Liss, Inc. [source] PHYLOGENY OF THE EUGLENALES BASED UPON COMBINED SSU AND LSU RDNA SEQUENCE COMPARISONS AND DESCRIPTION OF DISCOPLASTIS GEN.JOURNAL OF PHYCOLOGY, Issue 3 2006NOV. (EUGLENOPHYTA) A Bayesian analysis, utilizing a combined data set developed from the small subunit (SSU) and large subunit (LSU) rDNA gene sequences, was used to resolve relationships and clarify generic boundaries among 84 strains of plastid-containing euglenophytes representing 11 genera. The analysis produced a tree with three major clades: a Phacus and Lepocinlis clade, a Discoplastis clade, and a Euglena, Colacium, Trachelomonas, Strombomonas, Monomorphina, and Cryptoglena clade. The majority of the species in the genus Euglena formed a well-supported clade, but two species formed a separate clade near the base of the tree. A new genus, Discoplastis, was erected to accommodate these taxa, thus making the genus Euglena monophyletic. The analysis also supported the monophyly of Colacium, Trachelomonas, Strombomonas, Monomorphina, and Cryptoglena, which formed two subclades sister to the Euglena clade. Colacium, Trachelomonas, and Strombomonas, all of which produce copious amounts of mucilage to form loricas or mucilaginous stalks, formed a well-supported lineage. Our analysis supported retaining Strombomonas and Trachelomonas as separate genera. Monomorphina and Cryptoglena formed two well-supported clades that were sister to the Colacium, Trachelomonas, and Strombomonas clade. Phacus and Lepocinclis, both of which have numerous small discoid chloroplasts without pyrenoids and lack peristaltic euglenoid movement (metaboly), formed a well-supported monophyletic lineage that was sister to the larger Euglena through Cryptoglena containing clade. This study demonstrated that increased taxon sampling, multiple genes, and combined data sets provided increased support for internal nodes on the euglenoid phylogenetic tree and resolved relationships among the major genera in the photosynthetic euglenoid lineage. [source] EVIDENCE FOR LATERAL TRANSFER OF AN IE INTRON BETWEEN FUNGAL AND RED ALGAL SMALL SUBUNIT RRNA GENES,JOURNAL OF PHYCOLOGY, Issue 2 2005Kirsten M. Müller A previous study of the North American biogeography of the red algal genus Hildenbrandia noted the presence of group I introns in the nuclear small subunit (SSU) rRNA gene of the marine species H. rubra (Sommerf.) Menegh. Group IC1 introns have been previously reported at positions 516 and 1506 in the nuclear SSU RNA genes in the Bangiales and Hildenbrandiales. However, the presence of an unclassified intron at position 989 in a collection of H. rubra from British Columbia was noted. This intron is a member of the IE subclass and is the first report of this intron type in the red algae. Phylogenetic analyses of the intron sequences revealed a close relationship between this IE intron inserted at position 989 and similar fungal IE introns in positions 989 and 1199. The 989 IE introns formed a moderately to well-supported clade, whereas the 1199 IE introns are weakly supported. Unique structural helices in the P13 domain of the 989 and 1199 IE introns also point to a close relationship between these two clades and provide further evidence for the value of secondary structural characteristics in identifying homologous introns in evolutionarily divergent organisms. The absence of the 989 IE intron in all other red algal nuclear SSU rRNA genes suggests that it is unlikely that this intron was vertically inherited from the common ancestor of the red algal and fungal lineages but rather is the result of lateral transfer between fungal and red algal nuclear SSU rRNA genes. [source] PHYLOGENETIC PLACEMENT OF BOTRYOCOCCUS BRAUNII (TREBOUXIOPHYCEAE) AND BOTRYOCOCCUS SUDETICUS ISOLATE UTEX 2629 (CHLOROPHYCEAE),JOURNAL OF PHYCOLOGY, Issue 2 2004Hoda H. Senousy The phylogenetic placement of four isolates of Botryococcus braunii Kützing and of Botryococcus sudeticus Lemmermann isolate UTEX 2629 was investigated using sequences of the nuclear small subunit (18S) rRNA gene. The B. braunii isolates represent the A (two isolates), B, and L chemical races. One isolate of B. braunii (CCAP 807/1; A race) has a group I intron at Escherichia coli position 1046 and isolate UTEX 2629 has group I introns at E. coli positions 516 and 1512. The rRNA sequences were aligned with 53 previously reported rRNA sequences from members of the Chlorophyta, including one reported for B. braunii (Berkeley strain). Phylogenetic trees were constructed using distance, weighted maximum parsimony, and maximum likelihood, and their reliability was estimated using bootstrap analysis for distance and parsimony and Bayesian inference for likelihood. All methods showed, with high bootstrap or credibility support, that the four isolates of B. braunii form a monophyletic group whose closest relatives are in the genus Choricystis in the Trebouxiophyceae, whereas the previously reported B. braunii sequence is from a member of the Chlamydomonadales in the Chlorophyceae and isolate UTEX 2629 is a member of the Sphaeropleales in the Chlorophyceae. Polyphyly of these sequences was confirmed by Kishino-Hasegawa tests on artificial trees in which sequences were moved to a single lineage. [source] PHYLOGENY OF PHAGOTROPHIC EUGLENIDS (EUGLENOZOA): A MOLECULAR APPROACH BASED ON CULTURE MATERIAL AND ENVIRONMENTAL SAMPLES,JOURNAL OF PHYCOLOGY, Issue 4 2003Ingo Busse Molecular studies based on small subunit (SSU) rDNA sequences addressing euglenid phylogeny hitherto suffered from the lack of available data about phagotrophic species. To extend the taxon sampling, SSU rRNA genes from species of seven genera of phagotrophic euglenids were investigated. Sequence analyses revealed an increasing genetic diversity among euglenid SSU rDNA sequences compared with other well-known eukaryotic groups, reflecting an equally broad diversity of morphological characters among euglenid phagotrophs. Phylogenetic inference using standard parsimony and likelihood approaches as well as Bayesian inference and spectral analyses revealed no clear support for euglenid monophyly. Among phagotrophs, monophyly of Petalomonas cantuscygni and Notosolenus ostium, both comprising simple ingestion apparatuses, is strongly supported. A moderately supported clade comprises phototrophic euglenids and primary osmotrophic euglenids together with phagotrophs, exhibiting a primarily flexible pellicle composed of numerous helically arranged strips and a complex ingestion apparatus with two supporting rods and four curved vanes. Comparison of molecular and morphological data is used to demonstrate the difficulties to formulate a hypothesis about how the ingestion apparatus evolved in this group. [source] EFFECT OF TAXON SAMPLING, CHARACTER WEIGHTING, AND COMBINED DATA ON THE INTERPRETATION OF RELATIONSHIPS AMONG THE HETEROKONT ALGAE,JOURNAL OF PHYCOLOGY, Issue 2 2003Leslie R. Goertzen Nuclear ribosomal small subunit and chloroplast rbcL sequence data for heterokont algae and potential outgroup taxa were analyzed separately and together using maximum parsimony. A series of taxon sampling and character weighting experiments was performed. Traditional classes (e.g. diatoms, Phaeophyceae, etc.) were monophyletic in most analyses of either data set and in analyses of combined data. Relationships among classes and of heterokont algae to outgroup taxa were sensitive to taxon sampling. Bootstrap (BS) values were not always predictive of stability of nodes in taxon sampling experiments or between analyses of different data sets. Reweighting sites by the rescaled consistency index artificially inflates BS values in the analysis of rbcL data. Inclusion of the third codon position from rbcL enhanced signal despite the superficial appearance of mutational saturation. Incongruence between data sets was largely due to placement of a few problematic taxa, and so data were combined. BS values for the combined analysis were much higher than for analyses of each data set alone, although combining data did not improve support for heterokont monophyly. [source] MONOPHYLY OF THE GENUS CLOSTERIUM AND THE ORDER DESMIDIALES (CHAROPHYCEAE, CHLOROPHYTA) INFERRED FROM NUCLEAR SMALL SUBUNIT rDNA DATAJOURNAL OF PHYCOLOGY, Issue 6 2001Takashi Denboh We newly sequenced the nuclear-encoded small subunit (SSU) rDNA coding region for 21 taxa of the genus Closterium. The new sequences were integrated into an alignment with 13 known sequences of conjugating green algae representing six traditional families (i.e. Zygnemataceae, Mesotaeniaceae, Gonatozygaceae, Peniaceae, Closteriaceae, and Desmidiaceae) and five known charophycean sequences as outgroups. Both maximum likelihood and maximum parsimony analyses supported with high bootstrap values one large clade containing all placoderm desmids (Desmidiales). All the Closterium taxa formed one clade with 100% bootstrap support, indicating their monophyly, but not paraphyly, as suggested earlier. As to the taxa within the genus Closterium, we found two clades of morphologically closely related taxa in both maximum likelihood and maximum parsimony trees. They corresponded to the C. calosporum species complex and the C. moniliferum-ehrenbergii species complex. It is of particular interest that the homothallic entity of C. moniliferum v. moniliferum was distinguished from and ancestral to all other entities of the C. moniliferum-ehrenbergii species complex. Superimposing all 50 charophycean sequences on the higher order SSU rRNA structure model of Closterium, we investigated degrees of nucleotide conservation at a given position in the nucleotide sequence. A characteristic "signature" structure to the genus Closterium was found as an additional helix at the tip of V1 region. In addition, eight base deletions at the tip of helix 10 were found to be characteristic of the C. calosporum species complex, C. gracile, C. incurvum, C. pleurodermatum, and C. pusillum v. maius. These taxa formed one clade with an 82% bootstrap value in maximum parsimony analysis. [source] Two Genetically Distinct Populations of Colletotrichum gloeosporioides Penz.JOURNAL OF PHYTOPATHOLOGY, Issue 3 2005Causing Anthracnose Disease of Yam (Dioscorea spp.) Abstract Variation within Colletotrichum gloeosporioides, the causal agent of yam anthracnose disease, is still poorly defined and this hinders breeding for resistance. Two morphotypes of C. gloeosporioides, designated slow-growing grey (SGG) and fast-growing salmon (FGS), are associated with anthracnose disease of yam in Nigeria. The morphotypes are distinguishable based on colony and conidial morphology, growth rate, virulence, as well as vegetative compatibility, but molecular differentiation of SGG and FGS strains is needed to facilitate epidemiological studies. Denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified small subunit (18S) rDNA fragments, and microsatellite-primed PCR (MP-PCR) genomic fingerprinting were employed to provide a basis for molecular differentiation of the morphotypes. DGGE analysis revealed patterns that clearly differentiated isolates of the aggressive defoliating SGG from the moderately virulent non-defoliating FGS strains. Genetic analysis based on 52 MP-PCR markers revealed highly significant differentiation between the SGG and FGS populations on yam (GST = 0.22; Nei's genetic identity = 0.85; , = 0.28, P < 0.001), indicating that the SGG and FGS morphotypes represent genetically differentiated populations. The results of the molecular typing using DGGE and MP-PCR analyses were consistent with the disease phenotype caused by the two morphotypes. Consequently, these molecular techniques might be used, at least partly, to replace time-consuming virulence studies on yam. [source] Molecular diversity of arbuscular mycorrhizal fungi and patterns of host association over time and space in a tropical forestMOLECULAR ECOLOGY, Issue 12 2002R. Husband Abstract We have used molecular techniques to investigate the diversity and distribution of the arbuscular mycorrhizal (AM) fungi colonizing tree seedling roots in the tropical forest on Barro Colorado Island (BCI), Republic of Panama. In the first year, we sampled newly emergent seedlings of the understory treelet Faramea occidentalis and the canopy emergent Tetragastris panamensis, from mixed seedling carpets at each of two sites. The following year we sampled surviving seedlings from these cohorts. The roots of 48 plants were analysed using AM fungal-specific primers to amplify and clone partial small subunit (SSU) ribosomal RNA gene sequences. Over 1300 clones were screened for random fragment length polymorphism (RFLP) variation and 7% of these were sequenced. Compared with AM fungal communities sampled from temperate habitats using the same method, the overall diversity was high, with a total of 30 AM fungal types identified. Seventeen of these types have not been recorded previously, with the remainder being similar to types reported from temperate habitats. The tropical mycorrhizal population showed significant spatial heterogeneity and nonrandom associations with the different hosts. Moreover there was a strong shift in the mycorrhizal communities over time. AM fungal types that were dominant in the newly germinated seedlings were almost entirely replaced by previously rare types in the surviving seedlings the following year. The high diversity and huge variation detected across time points, sites and hosts, implies that the AM fungal types are ecologically distinct and thus may have the potential to influence recruitment and host composition in tropical forests. [source] Calpain 11 is unique to mouse spermatogenic cells,MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 6 2006Irit Ben-Aharon Abstract The calpains are a family of calcium-dependent thiol proteases involved in intracellular processing of proteins. They occur as heterodimers containing one of various large subunits and a common small subunit. Some of the large subunits are expressed ubiquitously and others are expressed in a restricted set of tissues. We have cloned the cDNA for mouse calpain 11 and demonstrated that it is expressed specifically in the mouse testis. The mRNA begins to accumulate in the testis between days 14 and 16 after birth, corresponding to the period of pachytene spermatocyte development. The protein is detected by day 18 after birth, during mid to late pachytene spermatocyte development, and is present in the acrosomal region of spermatozoa from the cauda epididymis. The expression of calpain 11 during spermatogenesis and its localization in spermatozoa suggest that it is involved in regulating calcium-dependent signal transduction events during meiosis and sperm functional processes. Mol. Reprod. Dev. Published 2006 Wiley-Liss, Inc. [source] Development of insect-resistant transgenic rice with Cry1C*-free endospermPEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 9 2009Rongjian Ye Abstract BACKGROUND: Yellow stem borer (Tryporyza incertulas Walker), striped stem borer (Chilo suppressalis Walker) and leaf folder (Cnaphalocrocis medinalis Guenec) are three lepidopteran pests that cause severe damage to rice in many areas of the world. In this study, novel insect-resistant transgenic rice was developed in which Bt protein expression was nearly absent in the endosperm. The resistant gene, cry1C*, driven by the rice rbcS promoter (small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase), was introduced into Zhonghua 11 (Oryza sativa L. ssp. japonica) by Agrobacterium -mediated transformation. RESULTS: A total of 83 independent transformants were obtained, 19 of which were characterised as single-copy foreign gene insertion. After preliminary screening of the T1 families of these 19 transformants in the field, six highly insect-resistant homozygous lines were selected. These six homozygous transgenic lines were field tested for resistance to leaf folders and stem borers, and for their agronomic performance. The Cry1C* protein levels in leaves and endosperm were measured by ELISA. Subsequently, the elite transgenic line RJ5 was selected; this line not only possessed high resistance to leaf folders and stem borers, normal agronomic performance, but also Cry1C* expression was only 2.6 ng g,1 in the endosperm. CONCLUSION: These results indicated that RJ5 has the potential for widespread utility in rice production. Copyright © 2009 Society of Chemical Industry [source] Serial replacement of a diatom endosymbiont in the marine dinoflagellate Peridinium quinquecorne (Peridiniales, Dinophyceae)PHYCOLOGICAL RESEARCH, Issue 3 2006Takeo Horiguchi SUMMARY To infer the phylogeny of both the host and the endosymbiont of Peridinium quinquecorne Abé, the small subunit (SSU) ribosomal DNA (rDNA) from the host and two genes of endosymbiont origin (plastid-encoded rbcL and nuclear-encoded SSU rDNA) were determined. The phylogenetic analysis of the host revealed that the marine dinoflagellate P. quinquecorne formed a clade with other diatom-harbouring dinoflagellates, including Kryptoperidinium foliaceum (Stein) Lindeman, Durinskia baltica (Levander) Carty et Cox and Galeidinium rugatum Tamura et Horiguchi, indicating a single endosymbiotic event for this lineage. Phylogenetic analyses of the endosymbiont in these organisms revealed that the endosymbiont of P. quinquecorne formed a clade with a centric diatom (SSU data indicated it to be closely related to Chaetoceros), whereas the endosymbionts of other three dinoflagellates formed a clade with a pennate diatom. The discrepancy between the host and the endosymbiont phylogenies suggests a secondary replacement of the endosymbiont from a pennate to a centric diatom in P. quinquecorne. [source] Transgenic maize lines with cell-type specific expression of fluorescent proteins in plastidsPLANT BIOTECHNOLOGY JOURNAL, Issue 2 2010Amir Sattarzadeh Summary Plastid number and morphology vary dramatically between cell types and at different developmental stages. Furthermore, in C4 plants such as maize, chloroplast ultrastructure and biochemical functions are specialized in mesophyll and bundle sheath cells, which differentiate acropetally from the proplastid form in the leaf base. To develop visible markers for maize plastids, we have created a series of stable transgenics expressing fluorescent proteins fused to either the maize ubiquitin promoter, the mesophyll-specific phosphoenolpyruvate carboxylase (PepC) promoter, or the bundle sheath-specific Rubisco small subunit 1 (RbcS) promoter. Multiple independent events were examined and revealed that maize codon-optimized versions of YFP and GFP were particularly well expressed, and that expression was stably inherited. Plants carrying PepC promoter constructs exhibit YFP expression in mesophyll plastids and the RbcS promoter mediated expression in bundle sheath plastids. The PepC and RbcS promoter fusions also proved useful for identifying plastids in organs such as epidermis, silks, roots and trichomes. These tools will inform future plastid-related studies of wild-type and mutant maize plants and provide material from which different plastid types may be isolated. [source] Bioengineered ,golden' indica rice cultivars with ,-carotene metabolism in the endosperm with hygromycin and mannose selection systemsPLANT BIOTECHNOLOGY JOURNAL, Issue 2 2003Karabi Datta Summary Vitamin-A deficiency (VAD) is a major malnutrition problem in South Asia, where indica rice is the staple food. Indica-type rice varieties feed more than 2 billion people. Hence, we introduced a combination of transgenes using the biolistic system of transformation enabling biosynthesis of provitamin A in the endosperm of several indica rice cultivars adapted to diverse ecosystems of different countries. The rice seed-specific glutelin promoter (Gt-1 P) was used to drive the expression of phytoene synthase (psy), while lycopene ,-cyclase (lcy) and phytoene desaturase (crtI), fused to the transit peptide sequence of the pea-Rubisco small subunit, were driven by the constitutive cauliflower mosaic virus promoter (CaMV35S P). Transgenic plants were recovered through selection with either CaMV35S P driven hph (hygromycin phosphotransferase) gene or cestrum yellow leaf curling virus promoter (CMP) driven pmi (phophomannose isomerase) gene. Molecular and biochemical analyses demonstrated stable integration and expression of the transgenes. The yellow colour of the polished rice grain evidenced the carotenoid accumulation in the endosperm. The colour intensity correlated with the estimated carotenoid content by spectrophotometric and HPLC analysis. Carotenoid level in cooked polished seeds was comparable (with minor loss of xanthophylls) to that in non-cooked seeds of the same transgenic line. The variable segregation pattern in T1 selfing generation indicated single to multiple loci insertion of the transgenes in the genome. This is the first report of using nonantibiotic pmi driven by a novel promoter in generating transgenic indica rice for possible future use in human nutrition. [source] Long-range allosteric transitions in carbamoyl phosphate synthetasePROTEIN SCIENCE, Issue 9 2004James B. Thoden Abstract Carbamoyl phosphate synthetase plays a key role in both pyrimidine and arginine biosynthesis by catalyzing the production of carbamoyl phosphate from one molecule of bicarbonate, two molecules of MgATP, and one molecule of glutamine. The enzyme from Escherichia coli consists of two polypeptide chains referred to as the small and large subunits, which contain a total of three separate active sites that are connected by an intramolecular tunnel. The small subunit harbors one of these active sites and is responsible for the hydrolysis of glutamine to glutamate and ammonia. The large subunit binds the two required molecules of MgATP and is involved in assembling the final product. Compounds such as L-ornithine, UMP, and IMP allosterically regulate the enzyme. Here, we report the three-dimensional structure of a site-directed mutant protein of carbamoyl phosphate synthetase from E. coli, where Cys 248 in the small subunit was changed to an aspartate. This residue was targeted for a structural investigation because previous studies demonstrated that the partial glutaminase activity of the C248D mutant protein was increased 40-fold relative to the wild-type enzyme, whereas the formation of carbamoyl phosphate using glutamine as a nitrogen source was completely abolished. Remarkably, although Cys 248 in the small subunit is located at ,100 Å from the allosteric binding pocket in the large subunit, the electron density map clearly revealed the presence of UMP, although this ligand was never included in the purification or crystallization schemes. The manner in which UMP binds to carbamoyl phosphate synthetase is described. [source] Structure and dynamics of translation initiation factor aIF-1A from the archaeon Methanococcus jannaschii determined by NMR spectroscopyPROTEIN SCIENCE, Issue 12 2001Wei Li Abstract Translation initiation factor 1A (aIF-1A) from the archaeon Methanococcus jannaschii was expressed in Escherichia coli, purified, and characterized in terms of its structure and dynamics using multidimensional NMR methods. The protein was found to be a member of the OB-fold family of RNA-associated proteins, containing a barrel of five beta-strands, a feature that is shared with the homologous eukaryotic translation initiation factor 1A (eIF-1A), as well as the prokaryotic translation initiation factor IF1. External to the , barrel, aIF-1A contains an ,-helix at its C-terminal and a flexible loop at its N-terminal, features that are qualitatively similar to those found in eIF-1A, but not present in prokaryotic IF1. The structural model of aIF-1A, when used in combination with primary sequence information for aIF-1A in divergent species, permitted the most-conserved residues on the protein surface to be identified, including the most likely candidates for direct interaction with the 16S ribosomal RNA and other components of the translational apparatus. Several of the conserved surface residues appear to be unique to the archaea. Nitrogen-15 relaxation and amide exchange rate data were used to characterize the internal motions within aIF-1A, providing evidence that the protein surfaces that are most likely to participate in intermolecular interactions are relatively flexible. A model is proposed, suggesting some specific interactions that may occur between aIF-1A and the small subunit of the archaeal ribosome. [source] Identification of four proteins from the small subunit of the mammalian mitochondrial ribosome using a proteomics approachPROTEIN SCIENCE, Issue 3 2001Emine Cavdar Koc Abstract Proteins in the small subunit of the mammalian mitochondrial ribosome were separated by two-dimensional polyacrylamide gel electrophoresis. Four individual proteins were subjected to in-gel Endoprotease Lys-C digestion. The sequences of selected proteolytic peptides were obtained by electrospray tandem mass spectrometry. Peptide sequences obtained from in-gel digestion of individual spots were used to screen human, mouse, and rat expressed sequence tag databases, and complete consensus cDNAs for these species were deduced in silico. The corresponding protein sequences were characterized by comparison to known ribosomal proteins in protein databases. Four different classes of mammalian mitochondrial small subunit ribosomal proteins were identified. Only two of these proteins have significant sequence similarities to ribosomal proteins from prokaryotes. These proteins are homologs to Escherichia coli S9 and S5 proteins. The presence of these newly identified mitochondrial ribosomal proteins are also investigated in the Drosophila melanogaster, Caenorhabditis elegans, and in the genomes of several fungi. [source] Euplotespora binucleata n. gen., n. sp. (Protozoa: Microsporidia), a Parasite Infecting the Hypotrichous Ciliate Euplotes woodruffi, with Observations on Microsporidian Infections in CiliophoraTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 3 2008SERGEI I. FOKIN ABSTRACT. A new microsporidian species, Euplotespora binucleata n. gen., n. sp., from the brackish-water ciliate Euplotes woodruffi is described and defined on the basis of life history characteristics, light and electron microscopic features, and small subunit (SSU) ribosomal DNA (rDNA) sequencing. The life cycle of E. binucleata n. sp. probably has rather short merogonic and relatively long sporogonic phases. Some uninuclear meronts and sporonts, along with diplokaryotic sporoblasts and spores, were found in experimentally infected host cells. Such a peculiar life cycle has been induced experimentally in Euplotes eurystomus and constitutively microsporidian-free stocks of E. woodruffi. Spores of E. binucleata n. sp. are monomorphic, ovoid,cylindrical in shape, 3.44±0.17 × 1.65±0.22 ,m in size, and characterized by a diplokaryotic condition and a large posterior vacuole. The polar tube is isofilar, 4.5,5.5 ,m in length when ejected, and lacking a distinctive coiled region (half-coiled). The polaroplast is divided into two regions: the anterior part has a few lamellae close to the anchoring disc; and the posterior part is a rounded body (sack), about one-quarter of the spore length. Spores do not appear to cluster together as a group. Each spore is surrounded by a sporophorous membrane closely adjacent to the exospore layer. A phylogenetic analysis of SSU rDNA sequences by different methods placed E. binucleata n. sp. in a clade with representatives of the microsporidian genera Cystosporogenes and Vittaforma. Observations of microsporidia in several other ciliates are discussed in view of the microsporidian infection frequency in the phylum Ciliophora. [source] Phylogenetic Analysis of Nosema antheraeae (Microsporidia) Isolated from Chinese Oak Silkworm, Antheraea pernyiTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 4 2006LIN-LING WANG ABSTRACT. The microsporidian Nosema antheraeae is a pathogen that infects the Chinese oak silkworm, Antheraea pernyi. We sequenced the complete small subunit (SSU) rRNA gene and the internal transcribed spacer (ITS) of N. antheraeae, and compared the SSU rRNA sequences in other microsporidia. The results indicated that Nosema species, including N. antheraeae, formed two distinct clades, consistent with previous observations. Furthermore, N. antheraeae is clustered with N. bombycis with high bootstrap support. The organization of the rRNA gene of N. antheraeae is LSU,ITS1,SSU,ITS2-5S, also following a pattern similar to the Nosema type species, N. bombycis. Thus, N. antheraeae is a Nosema species and has a close relationship to N. bombycis. [source] Identification of a New Microsporidian Parasite Related to Vittaforma corneae in HIV-Positive and HIV-Negative Patients from PortugalTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 2003IRSHAD M. SULAIMAN ABSTRACT. Fecal samples from 22 HIV-positive and 3 HIV-negative patients from Portugal with symptomatic diarrhea were diagnosed positive for microsporidia by microscopy, with most parasites detected significantly bigger than Enterocytozoon bieneusi and Encephalitozoon spp. Sequence characterization of the small subunit (SSU) rRNA gene identified a microsporidian parasite with 96% homology to two published Vittaforma corneae sequences. Phylogenetic analysis confirmed the genetic relatedness of this new microsporidian parasite to Vittaforma corneae as well as Cystosporogenes operophterae. Results of the study demonstrate the presence of a new human-pathogenic microsporidian species, which is responsible for significant number of infections in HIV-positive and HIV-negative patients in Portugal. [source] The maize viviparous15 locus encodes the molybdopterin synthase small subunitTHE PLANT JOURNAL, Issue 2 2006Masaharu Suzuki Summary A new Zea mays viviparous seed mutant, viviparous15 (vp15), was isolated from the UniformMu transposon-tagging population. In addition to precocious germination, vp15 has an early seedling lethal phenotype. Biochemical analysis showed reduced activities of several enzymes that require molybdenum cofactor (MoCo) in vp15 mutant seedlings. Because MoCo is required for abscisic acid (ABA) biosynthesis, the viviparous phenotype is probably caused by ABA deficiency. We cloned the vp15 mutant using a novel high-throughput strategy for analysis of high-copy Mu lines: We used MuTAIL PCR to extract genomic sequences flanking the Mu transposons in the vp15 line. The Mu insertions specific to the vp15 line were identified by in silico subtraction using a database of MuTAIL sequences from 90 UniformMu lines. Annotation of the vp15 -specific sequences revealed a Mu insertion in a gene homologous to human MOCS2A, the small subunit of molybdopterin (MPT) synthase. Molecular analysis of two allelic mutations confirmed that Vp15 encodes a plant MPT synthase small subunit (ZmCNX7). Our results, and a related paper reporting the cloning of maize viviparous10, demonstrate robust cloning strategies based on MuTAIL-PCR. The Vp15/CNX7, together with other CNX genes, is expressed in both embryo and endosperm during seed maturation. Expression of Vp15 appears to be regulated independently of MoCo biosynthesis. Comparisons of Vp15 loci in genomes of three cereals and Arabidopsis thaliana identified a conserved sequence element in the 5, untranslated region as well as a micro-synteny among the cereals. [source] Expression, purification, crystallization and preliminary X-ray analysis of ORF60, the small subunit (R2) of ribonucleotide reductase from Kaposi's sarcoma-associated herpesvirus (KSHV)ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2010Daniel Gurmu Ribonucleotide reductase (RNR) is responsible for converting ribonucleotides to deoxyribonucleotides, which are the building blocks of DNA. The enzyme is present in all life forms as well as in some large DNA viruses such as herpesviruses. The ,-herpesviruses and ,-herpesviruses encode two class Ia RNR subunits, R1 and R2, while the ,-herpesvirus subfamily only encode an inactive R1 subunit. Here, the crystallization of the R2 subunit of RNR encoded by the ORF60 gene from the oncovirus Kaposi's sarcoma-associated ,-herpesvirus (KSHV) is reported. These are the first crystals of a viral R2 subunit; the use of in situ proteolysis with chymotrypsin and the addition of hexamine cobalt(III) chloride that were necessary to obtain crystals are described. Optimization of the crystallization conditions yielded crystals that diffracted to 2.0,Å resolution. The crystals belonged to space group P21, with unit-cell parameters a = 63.9, b = 71.2, c = 71.8,Å, , = 90, , = 106.7, , = 90°. The data set collected was 95.3% complete, with an Rmerge of 9.6%. There are two molecules in the asymmetric unit, corresponding to a solvent content of 43.4%. [source] | ||||||||||||||||||||||||||||||||||