DNA-based approaches to identify forest fungi in Pacific Islands: A pilot study

Treesearch

Anna E. Case; Sara M. Ashiglar; Phil G. Cannon; Ernesto P. Militante; Edwin R. Tadiosa; Mutya Quintos-Manalo; Nelson M. Pampolina; John W. Hanna; Fred E. Brooks; Amy L. Ross-Davis; Mee-Sook Kim; Ned B. Klopfenstein

2013-01-01

DNA-based diagnostics have been successfully used to characterize diverse forest fungi (e.g., Hoff et al. 2004, Kim et al. 2006, Glaeser & Lindner 2011). DNA sequencing of the internal transcribed spacer (ITS) and large subunit (LSU) regions of nuclear ribosomal DNA (rDNA) has proved especially useful (Sonnenberg et al. 2007, Seifert 2009, Schoch et al. 2012) for...

Mitochondrial cytochrome c oxidase subunit 1 gene and nuclear rDNA regions of Enterobius vermicularis parasitic in captive chimpanzees with special reference to its relationship with pinworms in humans.

PubMed

Nakano, Tadao; Okamoto, Munehiro; Ikeda, Yatsukaho; Hasegawa, Hideo

2006-12-01

Sequences of mitochondrial cytochrome c oxidase subunit 1 (CO1) gene, nuclear internal transcribed spacer 2 (ITS2) region of ribosomal DNA (rDNA), and 5S rDNA of Enterobius vermicularis from captive chimpanzees in five zoos/institutions in Japan were analyzed and compared with those of pinworm eggs from humans in Japan. Three major types of variants appearing in both CO1 and ITS2 sequences, but showing no apparent connection, were observed among materials collected from the chimpanzees. Each one of them was also observed in pinworms in humans. Sequences of 5S rDNA were identical in the materials from chimpanzees and humans. Phylogenetic analysis of CO1 gene revealed three clusters with high bootstrap value, suggesting considerable divergence, presumably correlated with human evolution, has occurred in the human pinworms. The synonymy of E. gregorii with E. vermicularis is supported by the molecular evidence.

First report of the epiphytic benthic dinoflagellates Coolia canariensis and Coolia malayensis in the waters off Jeju Island, Korea: morphology and rDNA sequences.

PubMed

Jeong, Hae Jin; Yih, Wonho; Kang, Nam Seon; Lee, Sung Yeon; Yoon, Eun Young; Yoo, Yeong Du; Kim, Hyung Seop; Kim, Jong Hyeok

2012-01-01

Coolia spp. are epiphytic and benthic dinoflagellates. Herein, we report for the first time, the occurrence of Coolia canariensis and Coolia malayensis in Korean waters. The morphology of the Korean strains of C. canariensis and C. malayensis isolated from the waters off Jeju Island, Korea was similar to that of the original Canary lslands strains and Malaysian strains, respectively. We found several pores and a line of small knobs on the pore plate, and perforations within the large pores of both C. canariensis and C. malayensis. The plates of the Korean strains of C. canariensis and C. malayensis were arranged in a Kofoidian series of Po, 3', 7'', 6c, 6s, 5''', and 2'''', and Po, 3', 7'', 7c, 6-7s, 5''', and 2'''', respectively. When properly aligned, the large subunit (LSU) rDNA sequence of the Korean strain of C. canariensis was identical to that of the Biscayan strains, but it was 2-3% different from the Canary lslands strain VGO0775 and the Australian strain. In addition, the sequences of small subunit (SSU) and/or LSU rDNA from the two Korean strains of C. malayensis were < 1% different from the Malaysian strains of C. malayensis and the Florida strain CCMP1345 and New Zealand strain CAWD39 ("Coolia monotis"). In phylogenetic trees based on LSU rDNA sequences, the Korean strains of C. malayensis belonged to a clade including the Malaysian strains and these two strains. Therefore, based on genealogical analyses, we suggest that the Korean strain of C. canariensis is closely related to two Atlantic strains and the Australian strain, whereas the Korean strains of C. malayensis are related to the Malaysian strains of C. malayensis and the Florida and New Zealand strains. © 2012 The Author(s) Journal of Eukaryotic Microbiology © 2012 International Society of Protistologists.

Confirmation of Two Undescribed Fungal Species from Dokdo of Korea Based on Current Classification System Using Multi Loci

PubMed Central

Lee, Hye Won; Nguyen, Thi Thuong Thuong; Mun, Hye Yeon; Lee, Haengsub; Kim, Changmu

2015-01-01

Using dilution plating method, 47 fungal isolates were obtained from a soil sample collected from Dokdo in the East Sea of Korea in 2013. In this study, two fungal isolates, EML-MFS30-1 and EML-DDSF4, were confirmed as undescribed species, Metarhizium guizhouense and Mortierella oligospora in Korea based on current classification system using multi loci including rDNA internal transcribed spacer, large subunit, small subunit, and β-tubulin (BTUB) genes. Herein, detailed morphological descriptions on characters of the undescribed fungal species as well as their molecular phylogenetic status are provided with comparisons to related species. PMID:26839498

Molecular phylogeny of the Cladophoraceae (Cladophorales, Ulvophyceae), with the resurrection of Acrocladus Nägeli and Willeella Børgesen, and the description of Lurbica gen. nov. and Pseudorhizoclonium gen. nov.

PubMed

Boedeker, Christian; Leliaert, Frederik; Zuccarello, Giuseppe C

2016-12-01

The taxonomy of the Cladophoraceae, a large family of filamentous green algae, has been problematic for a long time due to morphological simplicity, parallel evolution, phenotypic plasticity, and unknown distribution ranges. Partial large subunit (LSU) rDNA sequences were generated for 362 isolates, and the analyses of a concatenated dataset consisting of unique LSU and small subunit (SSU) rDNA sequences of 95 specimens greatly clarified the phylogeny of the Cladophoraceae. The phylogenetic reconstructions showed that the three currently accepted genera Chaetomorpha, Cladophora, and Rhizoclonium are polyphyletic. The backbone of the phylogeny is robust and the relationships of the main lineages were inferred with high support, only the phylogenetic position of both Chaetomorpha melagonium and Cladophora rupestris could not be inferred unambiguously. There have been at least three independent switches between branched and unbranched morphologies within the Cladophoraceae. Freshwater environments have been colonized twice independently, namely by the freshwater Cladophora species as well as by several lineages of the Rhizoclonium riparium clade. In an effort to establish monophyletic genera, the genera Acrocladus and Willeella are resurrected and two new genera are described: Pseudorhizoclonium and Lurbica. © 2016 Phycological Society of America.

Morphology and molecular characterization of the epiphytic dinoflagellate Prorocentrum cf. rhathymum in temperate waters off Jeju Island, Korea

NASA Astrophysics Data System (ADS)

Lim, An Suk; Jeong, Hae Jin; Jang, Tae Young; Kang, Nam Seon; Lee, Sung Yeon; Yoo, Yeong Du; Kim, Hyung Seop

2013-03-01

Prorocentrum spp. are planktonic and/or benthic species. Benthic Prorocentrum species are of primary concern to scientists and the public because some of them are toxic. We established clonal cultures of 3 strains of Prorocentrum species that were collected from the thalli of a macroalga in the coastal waters off Jeju Island, located at the southern end of Korea. The Korean strains of P. cf. rhathymum, which are morphologically almost identical to the Virgin Island strain of P. rhathymum, were different from P. mexicanum because the former dinoflagellate has one simple collar-like spine in the periflagellar area, while the latter dinoflagellate has a 2- or 3-horned spine. In addition, the sequences of the small subunit (SSU) rDNA of the Korean strains were identical to those of the Malaysian and Floridian strains of P. rhathymum, while the sequences of the large subunit (LSU) rDNA of the Korean strains were 0.1-0.9% different from those of the Iranian and Malaysian strains of P. rhathymum. In phylogenetic trees based on the SSU rDNA sequences, the Korean strains of P. rhathymum formed a clade with the Malaysian and Floridian strains of P. rhathymum and the Vietnamese and Polynesian strains of P. mexicanum. However, in phylogenetic trees based on the LSU rDNA sequences, the Korean strains of P. rhathymum formed a clade with the Iranian strain of P. rhathymum and the Spanish and Mexican strains of P. mexicanum. Therefore, the molecular characterization of the Korean strains does not allow us to clearly classify them as P. rhathymum, nor P. mexicanum, although their morphology has so far been reported to be closer to that of P. rhathymum than P. mexicanum and thus we designated them as P. cf. rhathymum.

PCR tools for the verification of the specific identity of ascaridoid nematodes from dogs and cats.

PubMed

Li, M W; Lin, R Q; Chen, H H; Sani, R A; Song, H Q; Zhu, X Q

2007-01-01

Based on the sequences of the internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA) of Toxocara canis, Toxocara cati, Toxocara malaysiensis and Toxascaris leonina, specific forward primers were designed in the ITS-1 or ITS-2 for each of the four ascaridoid species of dogs and cats. These primers were used individually together with a conserved primer in the large subunit of rDNA to amplify partial ITS-1 and/or ITS-2 of rDNA from 107 DNA samples from ascaridoids from dogs and cats in China, Australia, Malaysia, England and the Netherlands. This approach allowed their specific identification, with no amplicons being amplified from heterogeneous DNA samples, and sequencing confirmed the identity of the sequences amplified. The minimum amounts of DNA detectable using the PCR assays were 0.13-0.54ng. These PCR assays should provide useful tools for the diagnosis and molecular epidemiological investigations of toxocariasis in humans and animals.

Acetylation of TAF(I)68, a subunit of TIF-IB/SL1, activates RNA polymerase I transcription.

PubMed

Muth, V; Nadaud, S; Grummt, I; Voit, R

2001-03-15

Mammalian rRNA genes are preceded by a terminator element that is recognized by the transcription termination factor TTF-I. In exploring the functional significance of the promoter-proximal terminator, we found that TTF-I associates with the p300/CBP-associated factor PCAF, suggesting that TTF-I may target histone acetyltransferase to the rDNA promoter. We demonstrate that PCAF acetylates TAF(I)68, the second largest subunit of the TATA box-binding protein (TBP)-containing factor TIF-IB/SL1, and acetylation enhances binding of TAF(I)68 to the rDNA promoter. Moreover, PCAF stimulates RNA polymerase I (Pol I) transcription in a reconstituted in vitro system. Consistent with acetylation of TIF-IB/SL1 being required for rDNA transcription, the NAD(+)-dependent histone deacetylase mSir2a deacetylates TAF(I)68 and represses Pol I transcription. The results demonstrate that acetylation of the basal Pol I transcription machinery has functional consequences and suggest that reversible acetylation of TIF-IB/SL1 may be an effective means to regulate rDNA transcription in response to external signals.

Two Endophytic Diaporthe Species Isolated from the Leaves of Astragalus membranaceus in Korea

PubMed Central

Kim, Jin-Hee; Kim, Dong-Yeo; Park, Hyeok

2017-01-01

We characterized two endophyte fungi from the leaves of Astragalus membranaceus in Korea. The isolated strains were identified on the basis of the morphological characters and sequences analysis of the internal transcribed spacer and large subunit regions of the rDNA and β-tubulin gene. To the best of our knowledge, this is the first report of Diaporthe oncostoma and Diaporthe infecunda in Korea, and we have provided descriptions and figures. PMID:29371813

Molecular phylogenetic lineage of Plagiopogon and Askenasia (Protozoa, Ciliophora) revealed by their gene sequences

NASA Astrophysics Data System (ADS)

Liu, An; Yi, Zhenzhen; Lin, Xiaofeng; Hu, Xiaozhong; Al-Farraj, Saleh A.; Al-Rasheid, Khaled A. S.

2015-08-01

Prostomates and haptorians are two basal groups of ciliates with limited morphological characteristics available for taxonomy. Morphologically, the structures used to identify prostomates and haptorians are similar or even identical, which generate heavy taxonomic and phylogenetic confusion. In present work, phylogenetic positions lineage of two rare genera, Plagiopogon and Askenasia, were investigated. Three genes including small subunit ribosomal RNA gene (hereafter SSU rDNA), internal transcribed spacer region (ITS region), and large subunit ribosomal RNA gene (LSU rDNA) were analyzed, 10 new sequences five species each. Our findings included 1) class Prostomatea and order Haptorida are multiphyletic; 2) it may not be appropriate to place order Cyclotrichiida in subclass Haptoria, and the systematic lineage of order Cyclotrichiida needs to be verified further; 3) genus Plagiopogon branches consistently within a clade covering most prostomes and is basal of clade Colepidae, implying its close lineage to Prostomatea; and 4) Askenasia is phylogenetically distant from the subclass Haptoria but close to classes Prostomatea, Plagiopylea and Oligohymenophorea. We supposed that the toxicyst of Askenasia may be close to taxa of prostomes instead of haptorians, and the dorsal brush is a more typical morphological characteristics of haptorians than toxicysts.

Morphology and Small-Subunit Ribosomal DNA Sequence of Henneguya Adiposa (Myxosporea) From Ictalurus punctatus (Siluriformes)

USDA-ARS?s Scientific Manuscript database

The original description of Henneguya adiposa, a myxozoan parasitizing channel catfish Ictalurus punctatus, is supplemented with new data on spore morphology, including photomicrographs and line drawings, as well as 18S small-subunit (SSU) ribosomal DNA (rDNA) sequence. Elongate, translucent, linear...

Taxonomy and phylogeny of Lopharia s.s., Dendrodontia, Dentocorticium and Fuscocerrena (Basidiomycota, Polyporales)

Treesearch

Shi-Liang Liu; Karen K. Nakasone; Sheng-Hua Wu; Shuang-Hui He; Yu-Cheng Dai

2018-01-01

Eleven taxa of Lopharia s.s., Dendrodontia, Dentocorticium and Fuscocerrena in Polyporales are included in the phylogenetic analyses of nuc rDNA ITS1-5.8S-ITS2 (ITS), D1-D2 domains of nuc 28S rDNA (28S) and RNA polymerase II second-largest subunit (rpb2) sequences. New species Lopharia resupinata...

Asexual-sexual morph connection in the type species of Berkleasmium.

PubMed

Tanney, Joey; Miller, Andrew N

2017-06-01

Berkleasmium is a polyphyletic genus comprising 37 dematiaceous hyphomycetous species. In this study, independent collections of the type species, B. concinnum , were made from Eastern North America. Nuclear internal transcribed spacer rDNA (ITS) and partial nuc 28S large subunit rDNA (LSU) sequences obtained from collections and subsequent cultures showed that Berkleasmium concinnum is the asexual morph of Neoacanthostigma septoconstrictum ( Tubeufiaceae , Tubeufiales ). Phylogenies inferred from Bayesian inference and maximum likelihood analyses of ITS-LSU sequence data confirmed this asexual-sexual morph connection and a re-examination of fungarium reference specimens also revealed the co-occurrence of N. septoconstrictum ascomata and B. concinnum sporodochia. Neoacanthostigma septoconstrictum is therefore synonymized under B. concinnum on the basis of priority. A specimen identified as N. septoconstrictum from Thailand is described as N. thailandicum sp. nov., based on morphological and genetic distinctiveness.

The nucleotide sequence of the entire ribosomal DNA operon and the structure of the large subunit rRNA of Giardia muris.

PubMed

van Keulen, H; Gutell, R R; Campbell, S R; Erlandsen, S L; Jarroll, E L

1992-10-01

The total nucleotide sequence of the rDNA of Giardia muris, an intestinal protozoan parasite of rodents, has been determined. The repeat unit is 7668 basepairs (bp) in size and consists of a spacer of 3314 bp, a small-subunit rRNA (SSU-rRNA) gene of 1429, and a large-subunit rRNA (LSU-rRNA) gene of 2698 bp. The spacer contains long direct repeats and is heterogeneous in size. The LSU-rRNA of G. muris was compared to that of the human intestinal parasite Giardia duodenalis, to the bird parasite Giardia ardeae, and to that of Escherichia coli. The LSU-rRNA has a size comparable to the 23S rRNA of E. coli but shows structural features typical for eukaryotes. Some variable regions are typically small and account for the overall smaller size of this rRNA. The structure of the G. muris LSU-rRNA is similar to that of the other Giardia rRNA, but each rRNA has characteristic features residing in a number of variable regions.

Morphological and molecular characterisation of Aporocotyle margolisi Smith, 1967 (Digenea: Aporocotylidae) from the North Pacific hake Merluccius productus (Ayres) (Gadiformes: Merlucciidae) off Oregon, USA.

PubMed

Hernández-Orts, Jesus S; Hernández-Mena, David I; Alama-Bermejo, Gema; Kuchta, Roman; Jacobson, Kym C

2017-09-01

Aporocotylid blood flukes conspecific with Aporocotyle margolisi Smith, 1967 were collected from the bulbus arteriosus of the North Pacific hake Merluccius productus (Ayres). This study revisits the morphology of A. margolisi, including drawings, measurements and scanning electron microscopy images, and provides for the first time molecular data for the large subunit of the ribosomal RNA (28S rDNA) and the mitochondrial cytochrome c oxidase subunit 1 (cox1) genes for this species. A 28S rDNA phylogenetic study of A. margolisi, and all available Aporocotyle spp., was also performed. The distribution range of A. margolisi is extended to the Pacific coast of the USA. We provide a morphological comparison of Aporocotyle spp. from the Pacific coast in North America as well as other Aporocotyle spp. infecting hake. Comparisons with the original description revealed that the new specimens of A. margolisi were considerably larger with respect to all morphological features, except for shorter spines. Molecular results showed a close relationship between A. margolisi and A. argentinensis Smith, 1969 from the Argentine hake Merluccius hubbsi Marini. The phylogenetic relationships of Aporocotyle spp. point to a possible co-speciation of hakes species and these blood fluke parasites.

Intraspecific differentiation of Paramecium novaurelia strains (Ciliophora, Protozoa) inferred from phylogenetic analysis of ribosomal and mitochondrial DNA variation.

PubMed

Tarcz, Sebastian

2013-01-01

Paramecium novaurelia Beale and Schneller, 1954, was first found in Scotland and is known to occur mainly in Europe, where it is the most common species of the P. aurelia complex. In recent years, two non-European localities have been described: Turkey and the United States of America. This article presents the analysis of intraspecific variability among 25 strains of P. novaurelia with the application of ribosomal and mitochondrial loci (ITS1-5.8S-ITS2, 5' large subunit rDNA (5'LSU rDNA) and cytochrome c oxidase subunit 1 (COI) mtDNA). The mean distance observed for all of the studied P. novaurelia sequence pairs was p=0.008/0.016/0.092 (ITS1-5.8S-ITS2/5'LSU rDNA/COI). Phylogenetic trees (NJ/MP/BI) based on a comparison of all of the analysed sequences show that the studied strains of P. novaurelia form a distinct clade, separate from the P. caudatum outgroup, and are divided into two clusters (A and B) and two branches (C and D). The occurrence of substantial genetic differentiation within P. novaurelia, confirmed by the analysed DNA fragments, indicates a rapid evolution of particular species within the Paramecium genus. Copyright © 2012 Elsevier GmbH. All rights reserved.

Phylogenetic Analysis of the Synnema-Producing Genus Synnemapestaloides

PubMed Central

Watanabe, Kyoko; Sekiguchi, Mao; Sato, Toyozo; Hsiang, Tom; Kaneko, Shigeru; Tanaka, Kazuaki; Kanda, Masaru; Fujita, Naoko; Nozawa, Shunsuke

2016-01-01

Synnemapestaloides rhododendri, the type species of the genus Synnemapestaloides, is a pathogen of Rhododendron brachycarpum. This fungus produces six-celled conidia with appendages at both end cells, and are generated by annellidic conidiogenous cells on the synnema. These conidial structures are similar to those of the genus Pestalotia. The monotypic genus Synnemapestaloides is currently classified in the family Amphisphaeriaceae solely based on conidial morphology. Here we demonstrate that Synnemapestaloides represents a distinct genus in the family Sporocadaceae (Amphisphaeriales) based on differences in the nucleotide sequences of the partial large subunit rDNA gene, the rDNA internal transcribed spacer, and the partial β-tubulin. The genus most closely related to Synnemapestaloides is Seimatosporium and the species most similar to Synnemapestaloides rhododendri is Seim. foliicola which produces short synnema-like conidiomata (sporodochia). These results demonstrate that Seim. foliicola should be transferred to Synnemapestaloides, and also demonstrate that Sporocadaceae can have synnematal in addition to pycnidial and acervular conidiomata. PMID:29376945

Discrimination of closely related species in tintinnid ciliates: new insights on crypticity and polymorphism in the genus Helicostomella.

PubMed

Santoferrara, Luciana F; Tian, Michael; Alder, Viviana A; McManus, George B

2015-02-01

This study focuses on the utility of molecular markers for the discrimination of closely related species in tintinnid ciliates. We analyzed the ecologically important genus Helicostomella by sequencing part of the large-subunit rDNA (LSU rDNA) and the 5.8S rDNA combined with the internally transcribed spacer regions 1 and 2 (5.8S rDNA-ITS) from forty-five individuals collected in NW and SW Atlantic waters and after culturing. Although all described Helicostomella species represent a continuum of morphologies, forms with shorter or longer loricae would correspond to different species according to previous molecular data. Here we observed that long forms show both crypticity (i.e. two almost identical long forms with different DNA sequences) and polymorphism (i.e. some long forms develop significantly shorter loricae after culturing). Reviewing all available tintinnid sequences, we found that 1) three Helicostomella clusters are consistent with different species from a molecular perspective, although these clusters are neither clearly differentiated by their loricae nor unambiguously linked to described species, 2) Helicostomella is closely related (probably to the family or genus level) to four "Tintinnopsis-like" morphospecies, and 3) if considered separately, neither LSU rDNA nor 5.8S rDNA-ITS completely discriminate closely related species, thus supporting the use of multi-gene barcodes for tintinnids. Copyright © 2014 Elsevier GmbH. All rights reserved.

First report of the epiphytic dinoflagellate Gambierdiscus caribaeus in the temperate waters off Jeju Island, Korea: morphology and molecular characterization.

PubMed

Jeong, Hae Jin; Lim, An Suk; Jang, Se Hyeon; Yih, Won Ho; Kang, Nam Seon; Lee, Sung Yeon; Yoo, Yeong Du; Kim, Hyung Seop

2012-01-01

Gambierdiscus spp. are epiphytic, benthic dinoflagellates. Some species have been shown to be toxic and cause ciguatera fish poisoning. We report, for the first time, the occurrence of Gambierdiscus caribaeus isolated from the waters off Jeju Island in Korea. Its morphology was similar to that of the original Belize strains of G. caribaeus. Gambierdiscus caribaeus has been reported in the tropical and subtropical waters of the Pacific, Gulf of Mexico, Caribbean Sea, and Floridian coast. Our report extends its range to the North Pacific Ocean. The plates of the Korean strain were arranged in a Kofoidian series of Po, 3', 7'', 6c, 6s, 5''', 1p, and 2'''', morphologically closer to other strains of G. caribaeus than to G. carpenteri. When properly aligned, its small subunit (SSU) rDNA was 0.5% different from those of Gambierdiscus sp. C-1, a strain that was isolated from the waters off eastern Japan, but was 2.4-4.0% different from those of the NOAA strains of G. caribaeus and 3.1-3.4% different from those of the NOAA strains of G. carpenteri. Additionally, the D1-D3 large subunit (LSU) rDNA sequence of the Korean strain of G. caribaeus was 4.7-5.3% different from those of the NOAA strains of G. caribaeus and 7.1-7.5% different from those of all reported G. carpenteri strains, including the NOAA strains. In phylogenetic trees based on SSU and LSU rDNA sequences, our Korean strain was basal to the clade consisting of the NOAA strains of G. caribaeus, which in turn was sister clade to all reported G. carpenteri strains. © 2012 The Author(s) Journal of Eukaryotic Microbiology © 2012 International Society of Protistologists.

Biogeography and diversity of Collodaria (Radiolaria) in the global ocean.

PubMed

Biard, Tristan; Bigeard, Estelle; Audic, Stéphane; Poulain, Julie; Gutierrez-Rodriguez, Andres; Pesant, Stéphane; Stemmann, Lars; Not, Fabrice

2017-06-01

Collodaria are heterotrophic marine protists that exist either as large colonies composed of hundreds of cells or as large solitary cells. All described species so far harbour intracellular microalgae as photosymbionts. Although recent environmental diversity surveys based on molecular methods demonstrated their consistently high contribution to planktonic communities and their worldwide occurrence, our understanding of their diversity and biogeography is still very limited. Here we estimated the 18S ribosomal DNA (rDNA) gene copies per collodarian cell for solitary (5770±1960 small subunit (SSU) rDNA copies) and colonial specimens (37 474±17 799 SSU rDNA copies, for each individual cell within a colony) using single-specimen quantitative PCR. We then investigated the environmental diversity of Collodaria within the photic zone through the metabarcoding survey from the Tara Oceans expedition and found that the two collodarian families Collosphaeridae and Sphaerozoidae contributed the most to the collodarian diversity and encompassed mostly cosmopolitan taxa. Although the biogeographical patterns were homogeneous within each biogeochemical biome considered, we observed that coastal biomes were consistently less diverse than oceanic biomes and were dominated by the Sphaerozoidae while the Collosphaeridae were dominant in the open oceans. The significant relationships with six environmental variables suggest that collodarian diversity is influenced by the trophic status of oceanic provinces and increased towards more oligotrophic regions.

Phylogenetic Status of an Unrecorded Species of Curvularia, C. spicifera, Based on Current Classification System of Curvularia and Bipolaris Group Using Multi Loci.

PubMed

Jeon, Sun Jeong; Nguyen, Thi Thuong Thuong; Lee, Hyang Burm

2015-09-01

A seed-borne fungus, Curvularia sp. EML-KWD01, was isolated from an indigenous wheat seed by standard blotter method. This fungus was characterized based on the morphological characteristics and molecular phylogenetic analysis. Phylogenetic status of the fungus was determined using sequences of three loci: rDNA internal transcribed spacer, large ribosomal subunit, and glyceraldehyde 3-phosphate dehydrogenase gene. Multi loci sequencing analysis revealed that this fungus was Curvularia spicifera within Curvularia group 2 of family Pleosporaceae.

Ribosomal DNA sequence divergence and group I introns within the Leucostoma species L. cinctum, L. persoonii, and L. parapersoonii sp. nov., ascomycetes that cause Cytospora canker of fruit trees.

PubMed

Adams, Gerard C; Surve-Iyer, Rupa S; Iezzoni, Amy F

2002-01-01

Leucostoma species that are the causal agents of Cytospora canker of stone and pome fruit trees were studied in detail. DNA sequence of the internal transcribed spacer regions and the 5.8S of the nuclear ribosomal DNA operon (ITS rDNA) supplied sufficient characters to assess the phylogenetic relationships among species of Leucostoma, Valsa, Valsella, and related anamorphs in Cytospora. Parsimony analysis of the aligned sequence divided Cytospora isolates from fruit trees into clades that generally agreed with the morphological species concepts, and with some of the phenetic groupings (PG 1-6) identified previously by isozyme analysis and cultural characteristics. Phylogenetic analysis inferred that isolates of L. persoonii formed two well-resolved clades distinct from isolates of L. cinctum. Phylogenetic analysis of the ITS rDNA, isozyme analysis, and cultural characteristics supported the inference that L. persoonii groups PG 2 and PG 3 were populations of a new species apparently more genetically different from L. persoonii PG 1 than from isolates representative of L. massariana, L. niveum, L. translucens, and Valsella melastoma. The new species, L. parapersoonii, was described. A diverse collection of isolates of L. cinctum, L. persoonii, and L. parapersoonii were examined for genetic variation using restriction fragment length polymorphism (RFLP) analysis of the ITS rDNA and the five prime end of the large subunit of the rDNA (LSU rDNA). HinfI and HpaII endonucleases were each useful in dividing the Leucostoma isolates into RFLP profiles corresponding to the isozyme phenetic groups, PG 1-6. RFLP analysis was more effective than isozyme analysis in uncovering variation among isolates of L. persoonii PG 1, but less effective within L. cinctum populations. Isolates representative of seven of the L. persoonii formae speciales proposed by G. Défago in 1935 were found to be genetically diverse isolates of PG 1. Two large insertions, 415 and 309 nucleotides long, in the small subunit (SSU) of the nuclear rDNA of L. cinctum were identified as Group 1 introns; intron 1 at position 943 and intron 2 at position 1199. The two introns were found to be consistently present in isolates of L. cinctum PG 4 and PG 5 and absent from L. cinctum PG 6 isolates, despite the similarity of the ITS sequence and teleomorph morphology. Intron 1 was of subgroup 1C1 whereas intron 2 was of an unknown subgroup. RFLP patterns and presence/absence of introns were useful characters for expediting the identification of cultures of Leucostoma isolated from stone and pome fruit cankers. RFLP patterns from 13 endonucleases provided an effective method for selecting an array of diverse PG 1 isolates useful in screening plant germplasm for disease-resistance.

Prototheca miyajii sp. nov., isolated from a patient with systemic protothecosis.

PubMed

Masuda, Michiaki; Hirose, Noriyuki; Ishikawa, Tomohiro; Ikawa, Yoshiya; Nishimura, Kazuko

2016-03-01

Species of the genus Prototheca are achlorophyllous algae and ubiquitous in nature, and so far, six species have been listed in this genus: Prototheca wickerhamii, Prototheca zopfii, Prototheca blaschkeae, Prototheca cutis, Prototheca stagnora and Prototheca ulmea. A strain of the genus Prototheca, IFM 53848T, was isolated in Japan from a patient with systemic protothecosis and had been designated P. wickerhamii. Our previous study, by using PCR analysis, revealed that its SSU rRNA gene (rDNA) was distinctively larger than that of P. wickerhamii and other species of the genus Prototheca. In this study, molecular analysis showed that the exceptionally large SSU rDNA of IFM 53848T contains four group I introns. The morphology of IFM 53848T was indistinguishable from those of P. wickerhamii or P. cutis, and phylogenetic analyses, based on the sequences of the SSU rDNA exons and the D1/D2 region of the large subunit rDNA, indicated that IFM 53848T was closely related to P. cutis. On the other hand, unlike P. cutis, IFM 53848T failed to assimilate fructose or lysine and grew well at higher temperatures of up to 42 °C. In addition, the nucleotide sequence of the ribosomal internal transcribed spacer and the matrix assisted laser desorption ionization time-of-flight mass spectrometry profile of IFM 53848T were clearly distinct from those of P. cutis. The results strongly suggest that IFM 53848T represents a novel species, and so the seventh member of the genus Prototheca, which we have named Prototheca miyajii sp. nov. The unique characteristics of the strain may provide useful insights into the systematic taxonomy of the genus Prototheca.

Fine organization of genomic regions tagged to the 5S rDNA locus of the bread wheat 5B chromosome.

PubMed

Sergeeva, Ekaterina M; Shcherban, Andrey B; Adonina, Irina G; Nesterov, Michail A; Beletsky, Alexey V; Rakitin, Andrey L; Mardanov, Andrey V; Ravin, Nikolai V; Salina, Elena A

2017-11-14

The multigene family encoding the 5S rRNA, one of the most important structurally-functional part of the large ribosomal subunit, is an obligate component of all eukaryotic genomes. 5S rDNA has long been a favored target for cytological and phylogenetic studies due to the inherent peculiarities of its structural organization, such as the tandem arrays of repetitive units and their high interspecific divergence. The complex polyploid nature of the genome of bread wheat, Triticum aestivum, and the technically difficult task of sequencing clusters of tandem repeats mean that the detailed organization of extended genomic regions containing 5S rRNA genes remains unclear. This is despite the recent progress made in wheat genomic sequencing. Using pyrosequencing of BAC clones, in this work we studied the organization of two distinct 5S rDNA-tagged regions of the 5BS chromosome of bread wheat. Three BAC-clones containing 5S rDNA were identified in the 5BS chromosome-specific BAC-library of Triticum aestivum. Using the results of pyrosequencing and assembling, we obtained six 5S rDNA- containing contigs with a total length of 140,417 bp, and two sets (pools) of individual 5S rDNA sequences belonging to separate, but closely located genomic regions on the 5BS chromosome. Both regions are characterized by the presence of approximately 70-80 copies of 5S rDNA, however, they are completely different in their structural organization. The first region contained highly diverged short-type 5S rDNA units that were disrupted by multiple insertions of transposable elements. The second region contained the more conserved long-type 5S rDNA, organized as a single tandem array. FISH using probes specific to both 5S rDNA unit types showed differences in the distribution and intensity of signals on the chromosomes of polyploid wheat species and their diploid progenitors. A detailed structural organization of two closely located 5S rDNA-tagged genomic regions on the 5BS chromosome of bread wheat has been established. These two regions differ in the organization of both 5S rDNA and the neighboring sequences comprised of transposable elements, implying different modes of evolution for these regions.

Isolation of the Entomopathogenic Fungal Strain Cod-MK1201 from a Cicada Nymph and Assessment of Its Antibacterial Activities.

PubMed

Sangdee, Kusavadee; Nakbanpote, Woranan; Sangdee, Aphidech

2015-01-01

The entomopathogenic fungus Cod-MK1201 was isolated from a dead cicada nymph. Three regions of ribosomal nuclear DNA, the internal transcribed spacers of nuclear ribosomal DNA repeats (ITS), the partial small subunit of rDNA (nrSSU) , and the partial large subunit of rDNA (nrLSU), and two protein-coding regions, the elongation factor 1α (EF-1α), and the largest subunit of the RNA polymerase II (rpb1) gene, were sequenced and used for fungal identification. The phylogenetic analysis of the ITS and the combined data set of the five genes indicated that the fungal isolate Cod-MK1201 is a new strain of Cordyceps sp. that is closely related to Cordyceps nipponica and C. kanzashiana. Crude extracts of mycelium-cultured Cod-MK1201 were obtained using distilled water and 50% (v/v) ethanol, and the antibacterial activity of each was determined. Both extracts had activity against Gram-positive and Gram-negative bacteria, but the ethanol extract was the more potent of the two. The antibacterial activity of the protein fractions of these extracts was also determined. The protein fraction from the ethanol extract was more antibacterial than the protein fraction from the aqueous extract. Three antibacterial constituents including adenosine, the total phenolic content (TPC), and the total flavonoid content (TFC) was also determined. The results showed that the adenosine content, the TPC, and the TFC of the ethanol extract were more active than those of the aqueous extract. Moreover, synergism was detected between these antibacterial constituents. In conclusion, the entomopathogenic fungal isolate Cod-MK1201 represents a natural source of antibacterial agents.

Phylogeny and genetic diversity of Bridgeoporus nobilissimus inferred using mitochondrial and nuclear rDNA sequences

USGS Publications Warehouse

Redberg, G.L.; Hibbett, D.S.; Ammirati, J.F.; Rodriguez, R.J.

2003-01-01

The genetic diversity and phylogeny of Bridgeoporus nobilissimus have been analyzed. DNA was extracted from spores collected from individual fruiting bodies representing six geographically distinct populations in Oregon and Washington. Spore samples collected contained low levels of bacteria, yeast and a filamentous fungal species. Using taxon-specific PCR primers, it was possible to discriminate among rDNA from bacteria, yeast, a filamentous associate and B. nobilissimus. Nuclear rDNA internal transcribed spacer (ITS) region sequences of B. nobilissimus were compared among individuals representing six populations and were found to have less than 2% variation. These sequences also were used to design dual and nested PCR primers for B. nobilissimus-specific amplification. Mitochondrial small-subunit rDNA sequences were used in a phylogenetic analysis that placed B. nobilissimus in the hymenochaetoid clade, where it was associated with Oxyporus and Schizopora.

Molecular phylogenetic study at the generic boundary between the lichen-forming fungi Caloplaca and Xanthoria (Ascomycota, Teloschistaceae).

PubMed

Søchting, Ulrik; Lutzoni, François

2003-11-01

A molecular phylogenetic analysis of rDNA was performed for seven Caloplaca, seven Xanthoria, one Fulgensia and five outgroup species. Phylogenetic hypotheses are constructed based on nuclear small and large subunit rDNA, separately and in combination. Three strongly supported major monophyletic groups were revealed within the Teloschistaceae. One group represents the Xanthoria fallax-group. The second group includes three subgroups: (1) X. parietina and X. elegans; (2) basal placodioid Caloplaca species followed by speciations leading to X. polycarpa and X. candelaria; and (3) a mixture of placodioid and endolithic Caloplaca species. The third main monophyletic group represents a heterogeneous assemblage of Caloplaca and Fulgensia species with a drastically different metabolite content. We report here that the two genera Caloplaca and Xanthoria, as well as the subgenus Gasparrinia, are all polyphyletic. The taxonomic significance of thallus morphology in Teloschistaceae and the current delimitation of the genus Xanthoria is discussed in light of these results.

Identification of EhTIF-IA: The putative E. histolytica orthologue of the human ribosomal RNA transcription initiation factor-IA.

PubMed

Srivastava, Ankita; Bhattacharya, Alok; Bhattacharya, Sudha; Jhingan, Gagan Deep

2016-03-01

Initiation of rDNA transcription requires the assembly of a specific multi-protein complex at the rDNA promoter containing the RNA Pol I with auxiliary factors. One of these factors is known as Rrn3P in yeast and Transcription Initiation Factor IA (TIF-IA) in mammals. Rrn3p/TIF-IA serves as a bridge between RNA Pol I and the pre-initiation complex at the promoter. It is phosphorylated at multiple sites and is involved in regulation of rDNA transcription in a growth-dependent manner. In the early branching parasitic protist Entamoeba histolytica, the rRNA genes are present exclusively on circular extra chromosomal plasmids. The protein factors involved in regulation of rDNA transcription in E. histolytica are not known. We have identified the E. histolytica equivalent of TIF-1A (EhTIF-IA) by homology search within the database and was further cloned and expressed. Immuno-localization studies showed that EhTIF-IA co-localized partially with fibrillarin in the peripherally localized nucleolus. EhTIF-IA was shown to interact with the RNA Pol I-specific subunit RPA12 both in vivo and in vitro. Mass spectroscopy data identified RNA Pol I-specific subunits and other nucleolar proteins to be the interacting partners of EhTIF-IA. Our study demonstrates for the first time a conserved putative RNA Pol I transcription factor TIF-IA in E. histolytica.

Genetic diversity of Entamoeba: Novel ribosomal lineages from cockroaches

PubMed Central

Kawano, Tetsuro; Imada, Mihoko; Chamavit, Pennapa; Kobayashi, Seiki; Hashimoto, Tetsuo

2017-01-01

Our current taxonomic perspective on Entamoeba is largely based on small-subunit ribosomal RNA genes (SSU rDNA) from Entamoeba species identified in vertebrate hosts with minor exceptions such as E. moshkovskii from sewage water and E. marina from marine sediment. Other Entamoeba species have also been morphologically identified and described from non-vertebrate species such as insects; however, their genetic diversity remains unknown. In order to further disclose the diversity of the genus, we investigated Entamoeba spp. in the intestines of three cockroach species: Periplaneta americana, Blaptica dubia, and Gromphadorhina oblongonota. We obtained 134 Entamoeba SSU rDNA sequences from 186 cockroaches by direct nested PCR using the DNA extracts of intestines from cockroaches, followed by scrutinized BLASTn screening and phylogenetic analyses. All the sequences identified in this study were distinct from those reported from known Entamoeba species, and considered as novel Entamoeba ribosomal lineages. Furthermore, they were positioned at the base of the clade of known Entamoeba species and displayed remarkable degree of genetic diversity comprising nine major groups in the three cockroach species. This is the first report of the diversity of SSU rDNA sequences from Entamoeba in non-vertebrate host species, and should help to understand the genetic diversity of the genus Entamoeba. PMID:28934335

Evolution of a Planktonic Foraminifer during Environmental Changes in the Tropical Oceans.

PubMed

Ujiié, Yurika; Ishitani, Yoshiyuki

2016-01-01

Ecological adaptation to environmental changes is a strong driver of evolution, enabling speciation of pelagic plankton in the open ocean without the presence of effective physical barriers to gene flow. The tropical ocean environment, which plays an important role in shaping marine biodiversity, has drastically and frequently changed since the Pliocene. Nevertheless, the evolutionary history of tropical pelagic plankton has been poorly understood, as phylogeographic investigations are still in the developing state and paleontological approaches are insufficient to obtain a sequential record from the deep-sea sediments. The planktonic foraminifer Pulleniatina obliquiloculata is widely distributed in the tropical area throughout the world's oceans, and its phylogeography is well established. It is thus one of the best candidates to examine how past environmental changes may have shifted the spatial distribution and affected the diversification of tropical pelagic plankton. Such an examination requires the divergence history of the planktonic foraminifers, yet the gene marker (partial small subunit (SSU) rDNA) previously used for phylogeographic studies was not powerful enough to achieve a high accuracy in estimating the divergence times. The present study focuses on improving the precision of divergence time estimates for the splits between sibling species (genetic types) of planktonic foraminifers by increasing the number of genes as well as the number of nucleotide bases used for molecular clock estimates. We have amplified the entire coding regions of two ribosomal RNA genes (SSU rDNA and large subunit (LSU) rDNA) of three genetic types of P. obliquiloculata and two closely related species for the first time and applied them to the Bayesian relaxed clock method. The comparison of the credible intervals of the four datasets consisting either of sequences of the partial SSU rDNA, the complete SSU rDNA, LSU rDNA, or a combination of both genes (SSU+LSU) clearly demonstrated that the two-gene dataset improved the accuracy of divergence time estimates. The P. obliquiloculata lineage diverged twice, first at the end of the Pliocene (3.1 Ma) and again in the middle Pleistocene (1.4 Ma). Both timings coincided with the environmental changes, which indirectly involved geographic separation of populations. The habitat of P. obliquiloculata was expanded toward the higher latitudinal zones during the stable warm periods and subsequently placed on the steep environmental gradients following the global cooling. Different environmental conditions in the stable warm tropics and unstable higher latitudes may have triggered ecological divergence among the populations, leading to adaptive differentiation and eventually speciation. A comprehensive analysis of divergence time estimates combined with phylogeography enabled us to reveal the evolutionary history of the pelagic plankton and to find the potential paleoenvironmental events, which could have changed their biogeography and ecology.

Recent radiation in a marine and freshwater dinoflagellate species flock

PubMed Central

Annenkova, Nataliia V; Hansen, Gert; Moestrup, Øjvind; Rengefors, Karin

2015-01-01

Processes of rapid radiation among unicellular eukaryotes are much less studied than among multicellular organisms. We have investigated a lineage of cold-water microeukaryotes (protists) that appear to have diverged recently. This lineage stands in stark contrast to known examples of phylogenetically closely related protists, in which genetic difference is typically larger than morphological differences. We found that the group not only consists of the marine-brackish dinoflagellate species Scrippsiella hangoei and the freshwater species Peridinium aciculiferum as discovered previously but also of a whole species flock. The additional species include Peridinium euryceps and Peridinium baicalense, which are restricted to a few lakes, in particular to the ancient Lake Baikal, Russia, and freshwater S. hangoei from Lake Baikal. These species are characterized by relatively large conspicuous morphological differences, which have given rise to the different species descriptions. However, our scanning electron microscopic studies indicate that they belong to a single genus according to traditional morphological characterization of dinoflagellates (thecal plate patterns). Moreover, we found that they have identical SSU (small subunit) rDNA fragments and distinct but very small differences in the DNA markers LSU (large subunit) rDNA, ITS2 (internal transcribed spacer 2) and COB (cytochrome b) gene, which are used to delineate dinoflagellates species. As some of the species co-occur, and all four have small but species–specific sequence differences, we suggest that these taxa are not a case of phenotypic plasticity but originated via recent adaptive radiation. We propose that this is the first clear example among free-living microeukaryotes of recent rapid diversification into several species followed by dispersion to environments with different ecological conditions. PMID:25603395

Characterization of Metarhizium viride Mycosis in Veiled Chameleons (Chamaeleo calyptratus), Panther Chameleons (Furcifer pardalis), and Inland Bearded Dragons (Pogona vitticeps).

PubMed

Schmidt, Volker; Klasen, Linus; Schneider, Juliane; Hübel, Jens; Pees, Michael

2017-03-01

Metarhizium viride has been associated with fatal systemic mycoses in chameleons, but subsequent data on mycoses caused by this fungus in reptiles are lacking. The aim of this investigation was therefore to obtain information on the presence of M. viride in reptiles kept as pets in captivity and its association with clinical signs and pathological findings as well as improvement of diagnostic procedures. Beside 18S ribosomal DNA (rDNA) (small subunit [SSU]) and internal transcribed spacer region 1 (ITS-1), a fragment of the large subunit (LSU) of 28S rDNA, including domain 1 (D1) and D2, was sequenced for the identification of the fungus and phylogenetic analysis. Cultural isolation and histopathological examinations as well as the pattern of antifungal drug resistance, determined by using agar diffusion testing, were additionally used for comparison of the isolates. In total, 20 isolates from eight inland bearded dragons ( Pogona vitticeps ), six veiled chameleons ( Chamaeleo calyptratus ), and six panther chameleons ( Furcifer pardalis ) were examined. Most of the lizards suffered from fungal glossitis, stomatitis, and pharyngitis or died due to visceral mycosis. Treatment with different antifungal drugs according to resistance patterns in all three different lizard species was unsuccessful. Sequence analysis resulted in four different genotypes of M. viride based on differences in the LSU fragment, whereas the SSU and ITS-1 were identical in all isolates. Sequence analysis of the SSU fragment revealed the first presentation of a valid large fragment of the SSU of M. viride According to statistical analysis, genotypes did not correlate with differences in pathogenicity, antifungal susceptibility, or species specificity. Copyright © 2017 American Society for Microbiology.

Characterization of Metarhizium viride Mycosis in Veiled Chameleons (Chamaeleo calyptratus), Panther Chameleons (Furcifer pardalis), and Inland Bearded Dragons (Pogona vitticeps)

PubMed Central

Klasen, Linus; Schneider, Juliane; Hübel, Jens; Pees, Michael

2016-01-01

ABSTRACT Metarhizium viride has been associated with fatal systemic mycoses in chameleons, but subsequent data on mycoses caused by this fungus in reptiles are lacking. The aim of this investigation was therefore to obtain information on the presence of M. viride in reptiles kept as pets in captivity and its association with clinical signs and pathological findings as well as improvement of diagnostic procedures. Beside 18S ribosomal DNA (rDNA) (small subunit [SSU]) and internal transcribed spacer region 1 (ITS-1), a fragment of the large subunit (LSU) of 28S rDNA, including domain 1 (D1) and D2, was sequenced for the identification of the fungus and phylogenetic analysis. Cultural isolation and histopathological examinations as well as the pattern of antifungal drug resistance, determined by using agar diffusion testing, were additionally used for comparison of the isolates. In total, 20 isolates from eight inland bearded dragons (Pogona vitticeps), six veiled chameleons (Chamaeleo calyptratus), and six panther chameleons (Furcifer pardalis) were examined. Most of the lizards suffered from fungal glossitis, stomatitis, and pharyngitis or died due to visceral mycosis. Treatment with different antifungal drugs according to resistance patterns in all three different lizard species was unsuccessful. Sequence analysis resulted in four different genotypes of M. viride based on differences in the LSU fragment, whereas the SSU and ITS-1 were identical in all isolates. Sequence analysis of the SSU fragment revealed the first presentation of a valid large fragment of the SSU of M. viride. According to statistical analysis, genotypes did not correlate with differences in pathogenicity, antifungal susceptibility, or species specificity. PMID:28003420

Molecular identification and description of the female of Nothoaspis reddelli (Ixodida: Argasidae) from a cave in southeastern Mexico.

PubMed

Guzmán-Cornejo, Carmen; Paredes-León, Ricardo; Labruna, Marcelo B; Nava, Santiago; Venzal, José M

2012-10-01

Nothoaspis reddelli Keirans and Clifford, 1975 , was described from 3 males collected in Grutas de Xtacumbilxunaán, Campeche, Mexico, although females have remained undescribed for 37 yr. Recently adult females of this species were collected from Cueva de Villa Luz ( =  Cueva de las Sardinas, Cueva del Azufre), in Tapijulapa, Tabasco, Mexico. Here we present a morphological description of the female stage, together with 16S rDNA sequences that confirm the conspecificity of our female, male, and nymphal specimens. The female integument of the anterior portion of the dorsal surface is smooth (nothoaspis), appearing to consist of 3 large "subunits," 1 anterior and 2 posterior, each with a small sublateral "subunit" on either side. The remaining dorsal covered integument is a cell-like configuration. The hood is large and bluntly rounded, and visible dorsally. The spiracular plate is oval. It possesses 1 pair of posthypostomal setae. The palpal trochanter has 1 pair of setae and a 5/5 hypostome decreasing to 4/4 at the apex. There is a single central pore at the base of the hypostome.

Description of a new planktonic mixotrophic dinoflagellate Paragymnodinium shiwhaense n. gen., n. sp. from the coastal waters off Western Korea: morphology, pigments, and ribosomal DNA gene sequence.

PubMed

Kang, Nam Seon; Jeong, Hae Jin; Moestrup, Øjvind; Shin, Woongghi; Nam, Seung Won; Park, Jae Yeon; De Salas, Miguel F; Kim, Ki Woo; Noh, Jae Hoon

2010-01-01

The mixotrophic dinoflagellate Paragymnodinium shiwhaense n. gen., n. sp. is described from living cells and from cells prepared by light, scanning electron, and transmission electron microscopy. In addition, sequences of the small subunit (SSU) and large subunit (LSU) rDNA and photosynthetic pigments are reported. The episome is conical, while the hyposome is hemispherical. Cells are covered with polygonal amphiesmal vesicles arranged in 16 rows and containing a very thin plate-like component. There is neither an apical groove nor apical line of narrow plates. Instead, there is a sulcal extension-like furrow. The cingulum is as wide as 0.2-0.3 x cell length and displaced by 0.2-0.3 x cell length. Cell length and width of live cells fed Amphidinium carterae were 8.4-19.3 and 6.1-16.0 microm, respectively. Paragymnodinium shiwhaense does not have a nuclear envelope chamber nor a nuclear fibrous connective (NFC). Cells contain chloroplasts, nematocysts, trichocysts, and peduncle, though eyespots, pyrenoids, and pusules are absent. The main accessory pigment is peridinin. The sequence of the SSU rDNA of this dinoflagellate (GenBank AM408889) is 4% different from that of Gymnodinium aureolum, Lepidodinium viride, and Gymnodinium catenatum, the three closest species, while the LSU rDNA was 17-18% different from that of G. catenatum, Lepidodinium chlorophorum, and Gymnodinium nolleri. The phylogenetic trees show that this dinoflagellate belongs within the Gymnodinium sensu stricto clade. However, in contrast to Gymnodinium spp., cells lack nuclear envelope chambers, NFC, and an apical groove. Unlike Polykrikos spp., which have a taeniocyst-nematocyst complex, P. shiwhaense has nematocysts without taeniocysts. In addition, P. shiwhaense does not have ocelloids in contrast to Warnowia spp. and Nematodinium spp. Therefore, based on morphological and molecular analyses, we suggest that this taxon is a new species, also within a new genus.

Post-Miocene expansion, colonization, and host switching drove speciation among extant nematodes of the archaic genus Trichinella

PubMed Central

Zarlenga, D. S.; Rosenthal, B. M.; La Rosa, G.; Pozio, E.; Hoberg, E. P.

2006-01-01

Parasitic nematodes of the genus Trichinella cause significant food-borne illness and occupy a unique evolutionary position at the base of the phylum Nematoda, unlike the free-living nematode Caenorhabditis elegans. Although the forthcoming genome sequence of Trichinella spiralis can provide invaluable comparative information about nematode biology, a basic framework for understanding the history of the genus Trichinella is needed to maximize its utility. We therefore developed the first robust and comprehensive analysis of the phylogeny and biogeographic history of Trichinella using the variation in three genes (nuclear small-subunit rDNA, and second internal transcribed spacer, mitochondrial large-subunit rDNA, and cytochrome oxidase I DNA) from all 11 recognized taxa. We conclude that (i) although Trichinellidae may have diverged from their closest extant relatives during the Paleozoic, all contemporary species of Trichinella diversified within the last 20 million years through geographic colonization and pervasive host switching among foraging guilds of obligate carnivores; (ii) mammalian carnivores disseminated encapsulated forms from Eurasia to Africa during the late Miocene and Pliocene, and to the Nearctic across the Bering Land Bridge during the Pliocene and Pleistocene, when crown species ultimately diversified; (iii) the greatest risk to human health is posed by those species retaining an ancestral capacity to parasitize a wide range of hosts; and (iv) early hominids may have first acquired Trichinella on the African savannah several million years before swine domestication as their diets shifted from herbivory to facultative carnivory. PMID:16651518

Homology-dependent repair is involved in 45S rDNA loss in plant CAF-1 mutants

PubMed Central

Muchová, Veronika; Amiard, Simon; Mozgová, Iva; Dvořáčková, Martina; Gallego, Maria E; White, Charles; Fajkus, Jiří

2015-01-01

Arabidopsis thaliana mutants in FAS1 and FAS2 subunits of chromatin assembly factor 1 (CAF1) show progressive loss of 45S rDNA copies and telomeres. We hypothesized that homology-dependent DNA damage repair (HDR) may contribute to the loss of these repeats in fas mutants. To test this, we generated double mutants by crossing fas mutants with knock-out mutants in RAD51B, one of the Rad51 paralogs of A. thaliana. Our results show that the absence of RAD51B decreases the rate of rDNA loss, confirming the implication of RAD51B-dependent recombination in rDNA loss in the CAF1 mutants. Interestingly, this effect is not observed for telomeric repeat loss, which thus differs from that acting in rDNA loss. Involvement of DNA damage repair in rDNA dynamics in fas mutants is further supported by accumulation of double-stranded breaks (measured as γ-H2AX foci) in 45S rDNA. Occurrence of the foci is not specific for S-phase, and is ATM-independent. While the foci in fas mutants occur both in the transcribed (intranucleolar) and non-transcribed (nucleoplasmic) fraction of rDNA, double fas rad51b mutants show a specific increase in the number of the intranucleolar foci. These results suggest that the repair of double-stranded breaks present in the transcribed rDNA region is RAD51B dependent and that this contributes to rDNA repeat loss in fas mutants, presumably via the single-stranded annealing recombination pathway. Our results also highlight the importance of proper chromatin assembly in the maintenance of genome stability. PMID:25359579

Bacillus nealsonii sp. nov., isolated from a spacecraft-assembly facility, whose spores are gamma-radiation resistant

NASA Technical Reports Server (NTRS)

Venkateswaran, Kasthuri; Kempf, Michael; Chen, Fei; Satomi, Masataka; Nicholson, Wayne; Kern, Roger

2003-01-01

One of the spore-formers isolated from a spacecraft-assembly facility, belonging to the genus Bacillus, is described on the basis of phenotypic characterization, 16S rDNA sequence analysis and DNA-DNA hybridization studies. It is a Gram-positive, facultatively anaerobic, rod-shaped eubacterium that produces endospores. The spores of this novel bacterial species exhibited resistance to UV, gamma-radiation, H2O2 and desiccation. The 18S rDNA sequence analysis revealed a clear affiliation between this strain and members of the low G+C Firmicutes. High 16S rDNA sequence similarity values were found with members of the genus Bacillus and this was supported by fatty acid profiles. The 16S rDNA sequence similarity between strain FO-92T and Bacillus benzoevorans DSM 5391T was very high. However, molecular characterizations employing small-subunit 16S rDNA sequences were at the limits of resolution for the differentiation of species in this genus, but DNA-DNA hybridization data support the proposal of FO-92T as Bacillus nealsonii sp. nov. (type strain is FO-92T =ATCC BAAM-519T =DSM 15077T).

The Evolutionary Relationships between Endosymbiotic Green Algae of Paramecium bursaria Syngens Originating from Different Geographical Locations.

PubMed

Zagata, Patrycja; Greczek-Stachura, Magdalena; Tarcz, Sebastian; Rautian, Maria

2016-01-01

Paramecium bursaria (Ehrenberg 1831), a freshwater ciliate, typically harbors hundreds of green algal symbionts inside the cell. The aim of present study was the molecular identification of newly analyzed P. bursaria symbionts. The second aspect of the present survey was testing a hypothesis whether endosymbionts prefer the specified syngen of the host, and the specified geographical distribution. Ten strains of endosymbionts isolated from strains of P. bursaria originating from different geographical locations were studied. We analyzed for the first time, both the fragment of plastid genome containing 3'rpl36-5' infA genes and a fragment of a nuclear gene encoding large subunit ribosomal RNA (LSU rDNA). The analysis of the LSU rDNA sequences showed the existence of 3 haplotypes and the haplotype diversity of 0.733, and 8 haplotypes for the 3'rpl36-5' infA gene fragment and haplotype diversity of 0.956. The endosymbionts isolated from P. bursaria strains were identified as Chlorella vulgaris, Ch. variabilis and Micractinium conductrix. There was no correlation between the syngen of P. bursaria and the species of endosymbiont.

Identifying the true oysters (Bivalvia: Ostreidae) with mitochondrial phylogeny and distance-based DNA barcoding.

PubMed

Liu, Jun; Li, Qi; Kong, Lingfeng; Yu, Hong; Zheng, Xiaodong

2011-09-01

Oysters (family Ostreidae), with high levels of phenotypic plasticity and wide geographic distribution, are a challenging group for taxonomists and phylogenetics. As a useful tool for molecular species identification, DNA barcoding might offer significant potential for oyster identification and taxonomy. This study used two mitochondrial fragments, cytochrome c oxidase I (COI) and the large ribosomal subunit (16S rDNA), to assess whether oyster species could be identified by phylogeny and distance-based DNA barcoding techniques. Relationships among species were estimated by the phylogenetic analyses of both genes, and then pairwise inter- and intraspecific genetic divergences were assessed. Species forming well-differentiated clades in the molecular phylogenies were identical for both genes even when the closely related species were included. Intraspecific variability of 16S rDNA overlapped with interspecific divergence. However, average intra- and interspecific genetic divergences for COI were 0-1.4% (maximum 2.2%) and 2.6-32.2% (minimum 2.2%), respectively, indicating the existence of a barcoding gap. These results confirm the efficacy of species identification in oysters via DNA barcodes and phylogenetic analysis. © 2011 Blackwell Publishing Ltd.

Discopersicus n. gen., a New Member of the Family Tylenchidae Örley, 1880 with Detailed SEM Study on Two Known Species of the Genus Discotylenchus Siddiqi, 1980 (Nematoda; Tylenchidae) from Iran.

PubMed

Yaghoubi, Ali; Pourjam, Ebrahim; Álvarez-Ortega, Sergio; Liébanas, Gracia; Atighi, Mohammad Reza; Pedram, Majid

2016-09-01

Discopersicus iranicus n. gen., n. comb., previously described from Iran as a new species under the genus Discotylenchus , is illustrated using light microscope and scanning electron microscope (SEM) observations and further studied using molecular characters. SEM studies revealed the newly proposed genus has oblique amphidial apertures on the lateral sides of the lip region. SEM images are also provided for two species of Discotylenchus , namely D. discretus and D. brevicaudatus , as the first SEM study of the genus . These results confirmed longitudinal amphidial aperture type on lateral sides of the lip region in genus Discotylenchus , as noted by Siddiqi while erecting the genus with D. discretus as the type species . Molecular phylogenetic analyses using partial small subunit (SSU) and large subunit (LSU) rDNA sequences revealed the affinity of the genus Discopersicus n. gen. with members of the subfamily Boleodorinae, as supported by morphological characters (mainly, the oblique amphidial opening).

Microbial Composition of Near-Boiling Silica-Depositing Thermal Springs throughout Yellowstone National Park

PubMed Central

Blank, Carrine E.; Cady, Sherry L.; Pace, Norman R.

2002-01-01

The extent of hyperthermophilic microbial diversity associated with siliceous sinter (geyserite) was characterized in seven near-boiling silica-depositing springs throughout Yellowstone National Park using environmental PCR amplification of small-subunit rRNA genes (SSU rDNA), large-subunit rDNA, and the internal transcribed spacer (ITS). We found that Thermocrinis ruber, a member of the order Aquificales, is ubiquitous, an indication that primary production in these springs is driven by hydrogen oxidation. Several other lineages with no known close relatives were identified that branch among the hyperthermophilic bacteria. Although they all branch deep in the bacterial tree, the precise phylogenetic placement of many of these lineages is unresolved at this time. While some springs contained a fair amount of phylogenetic diversity, others did not. Within the same spring, communities in the subaqueous environment were not appreciably different than those in the splash zone at the edge of the pool, although a greater number of phylotypes was found along the pool's edge. Also, microbial community composition appeared to have little correlation with the type of sinter morphology. The number of cell morphotypes identified by fluorescence in situ hybridization and scanning electron microscopy was greater than the number of phylotypes in SSU clone libraries. Despite little variation in Thermocrinis ruber SSU sequences, abundant variation was found in the hypervariable ITS region. The distribution of ITS sequence types appeared to be correlated with distinct morphotypes of Thermocrinis ruber in different pools. Therefore, species- or subspecies-level divergences are present but not detectable in highly conserved SSU sequences. PMID:12324363

Molecular detection of fungal pathogens in clinical specimens by 18S rDNA high-throughput screening in comparison to ITS PCR and culture.

PubMed

Wagner, K; Springer, B; Pires, V P; Keller, P M

2018-05-03

The rising incidence of invasive fungal infections and the expanding spectrum of fungal pathogens makes early and accurate identification of the causative pathogen a daunting task. Diagnostics using molecular markers enable rapid identification of fungi, offer new insights into infectious disease dynamics, and open new possibilities for infectious disease control and prevention. We performed a retrospective study using clinical specimens (N = 233) from patients with suspected fungal infection previously subjected to culture and/or internal transcribed spacer (ITS) PCR. We used these specimens to evaluate a high-throughput screening method for fungal detection using automated DNA extraction (QIASymphony), fungal ribosomal small subunit (18S) rDNA RT-PCR and amplicon sequencing. Fungal sequences were compared with sequences from the curated, commercially available SmartGene IDNS database for pathogen identification. Concordance between 18S rDNA RT-PCR and culture results was 91%, and congruence between 18S rDNA RT-PCR and ITS PCR results was 94%. In addition, 18S rDNA RT-PCR and Sanger sequencing detected fungal pathogens in culture negative (N = 13) and ITS PCR negative specimens (N = 12) from patients with a clinically confirmed fungal infection. Our results support the use of the 18S rDNA RT-PCR diagnostic workflow for rapid and accurate identification of fungal pathogens in clinical specimens.

Invasion of the French paleolithic painted cave of Lascaux by members of the Fusarium solani species complex.

PubMed

Dupont, Joëlle; Jacquet, Claire; Dennetière, Bruno; Lacoste, Sandrine; Bousta, Faisl; Orial, Geneviève; Cruaud, Corinne; Couloux, Arnaud; Roquebert, Marie-France

2007-01-01

A major fungal invasion was discovered in the prehistoric painted cave of Lascaux in France in Sep 2001. At least three species of the Fusarium solani complex were isolated and identified with a portion of the translation elongation factor 1alpha gene (EF-1alpha), a portion of the nuclear large subunit rDNA (LSU) and nuclear ribosomal intergenic spacer region (ITS). This study represents the first time that Fusarium species have been reported from a cave containing prehistoric paintings. Significant interspecific molecular variability was observed, suggesting that there might have been repeated introduction of the species, possibly carried by water from soils above the cave.

Divergent nuclear 18S rDNA paralogs in a turkey coccidium, Eimeria meleagrimitis, complicate molecular systematics and identification.

PubMed

El-Sherry, Shiem; Ogedengbe, Mosun E; Hafeez, Mian A; Barta, John R

2013-07-01

Multiple 18S rDNA sequences were obtained from two single-oocyst-derived lines of each of Eimeria meleagrimitis and Eimeria adenoeides. After analysing the 15 new 18S rDNA sequences from two lines of E. meleagrimitis and 17 new sequences from two lines of E. adenoeides, there were clear indications that divergent, paralogous 18S rDNA copies existed within the nuclear genome of E. meleagrimitis. In contrast, mitochondrial cytochrome c oxidase subunit I (COI) partial sequences from all lines of a particular Eimeria sp. were identical and, in phylogenetic analyses, COI sequences clustered unambiguously in monophyletic and highly-supported clades specific to individual Eimeria sp. Phylogenetic analysis of the new 18S rDNA sequences from E. meleagrimitis showed that they formed two distinct clades: Type A with four new sequences; and Type B with nine new sequences; both Types A and B sequences were obtained from each of the single-oocyst-derived lines of E. meleagrimitis. Together these rDNA types formed a well-supported E. meleagrimitis clade. Types A and B 18S rDNA sequences from E. meleagrimitis had a mean sequence identity of only 97.4% whereas mean sequence identity within types was 99.1-99.3%. The observed intraspecific sequence divergence among E. meleagrimitis 18S rDNA sequence types was even higher (approximately 2.6%) than the interspecific sequence divergence present between some well-recognized species such as Eimeria tenella and Eimeria necatrix (1.1%). Our observations suggest that, unlike COI sequences, 18S rDNA sequences are not reliable molecular markers to be used alone for species identification with coccidia, although 18S rDNA sequences have clear utility for phylogenetic reconstruction of apicomplexan parasites at the genus and higher taxonomic ranks. Copyright © 2013. Published by Elsevier Ltd.

Gyrodiniellum shiwhaense n. gen., n. sp., a new planktonic heterotrophic dinoflagellate from the coastal waters of western Korea: morphology and ribosomal DNA gene sequence.

PubMed

Kang, Nam Seon; Jeong, Hae Jin; Moestrup, Ojvind; Park, Tae Gyu

2011-01-01

The heterotrophic dinoflagellate Gyrodiniellum shiwhaense n. gen., n. sp. is described from live cells and from cells prepared for light, scanning electron, and transmission electron microscopy. Also, sequences of the small subunit (SSU) and large subunit (LSU) of rDNA have been analyzed. The episome is conical, while the hyposome is ellipsoid. Cells are covered with polygonal amphiesmal vesicles arranged in 16 horizontal rows. Unlike other Gyrodinium-like dinoflagellates, the apical end of the cell shows a loop-shaped row of five elongate amphiesmal vesicles. The cingulum is displaced by 0.3-0.5 × cell length. Cells that were feeding on the dinoflagellate Amphidinium carterae Hulburt were 9.1-21.6 μm long and 6.6-15.7 μm wide. Cells of G. shiwhaense contain nematocysts, trichocysts, a peduncle, and pusule systems, but they lack chloroplasts. The SSU rDNA sequence is >3% different from that of the six most closely related species: Warnowia sp. (FJ947040), Lepidodinium viride Watanabe, Suda, Inouye, Sawaguchi & Chihara, Gymnodinium aureolum (Hulburt) Hansen, Gymnodinium catenatum Graham, Nematodinium sp. (FJ947039), and Gymnodinium sp. MUCC284 (AF022196), while the LSU rDNA is 11-12% different from that of Warnowia sp., G. aureolum, and Nematodinium sp. (FJ947041). The phylogenetic trees show that the species belongs in the Gymnodinium sensu stricto clade. However, in contrast to Gymnodinium spp., cells lack nuclear envelope chambers and a nuclear fibrous connective. Unlike Polykrikos spp., cells of which possess a taeniocyst-nematocyst complex, G. shiwhaense has nematocysts but lacks taeniocysts. It differs from Paragymnodinium shiwhaense Kang, Jeong, Moestrup & Shin by possessing nematocysts with stylets and filaments. Gyrodiniellum shiwhaense n. gen., n. sp. furthermore lacks ocelloids, in contrast to Warnowia spp., Nematodinium spp., and Proterythropsis spp. Based on morphological and molecular data, we suggest that the taxon represents a new species within a new genus. © 2011 The Author(s). Journal of Eukaryotic Microbiology © 2011 International Society of Protistologists.

Characterisation of Oscheius onirici (Nematoda: Rhabditidae), a hermaphroditic nematode from the marshlands of Wisconsin

USDA-ARS?s Scientific Manuscript database

An Oscheius (Rhabditidae) was recovered through the Galleria bait method from a wild cranberry marsh in Jackson County, Wisconsin, USA. Morphological studies with light microscopy and scanning electron microscopy, as well as molecular analyses of the near-full-length small subunit rDNA gene (SSU), D...

Intra-isolate genome variation in arbuscular mycorrhizal fungi persists in the transcriptome.

PubMed

Boon, E; Zimmerman, E; Lang, B F; Hijri, M

2010-07-01

Arbuscular mycorrhizal fungi (AMF) are heterokaryotes with an unusual genetic makeup. Substantial genetic variation occurs among nuclei within a single mycelium or isolate. AMF reproduce through spores that contain varying fractions of this heterogeneous population of nuclei. It is not clear whether this genetic variation on the genome level actually contributes to the AMF phenotype. To investigate the extent to which polymorphisms in nuclear genes are transcribed, we analysed the intra-isolate genomic and cDNA sequence variation of two genes, the large subunit ribosomal RNA (LSU rDNA) of Glomus sp. DAOM-197198 (previously known as G. intraradices) and the POL1-like sequence (PLS) of Glomus etunicatum. For both genes, we find high sequence variation at the genome and transcriptome level. Reconstruction of LSU rDNA secondary structure shows that all variants are functional. Patterns of PLS sequence polymorphism indicate that there is one functional gene copy, PLS2, which is preferentially transcribed, and one gene copy, PLS1, which is a pseudogene. This is the first study that investigates AMF intra-isolate variation at the transcriptome level. In conclusion, it is possible that, in AMF, multiple nuclear genomes contribute to a single phenotype.

Molecular Diagnostics of Arthroconidial Yeasts, Frequent Pulmonary Opportunists.

PubMed

Kaplan, Engin; Al-Hatmi, Abdullah M S; Ilkit, Macit; Gerrits van den Ende, A H G; Hagen, Ferry; Meis, Jacques F; de Hoog, G Sybren

2018-01-01

Magnusiomyces capitatus and Saprochaete clavata are members of the clade of arthroconidial yeasts that represent emerging opportunistic pulmonary pathogens in immunocompromised patients. Given that standard ribosomal DNA (rDNA) identification often provides confusing results, in this study, we analyzed 34 isolates with the goal of finding new genetic markers for classification using multilocus sequencing and amplified fragment length polymorphism (AFLP). The interspecific similarity obtained using rDNA markers (the internal transcribed spacer [ITS] and large subunit regions) was in the range of 96 to 99%, whereas that obtained using protein-coding loci ( Rbp2 , Act , and Tef1α ) was lower at 89.4 to 95.2%. Ultimately, Rbp2 was selected as the best marker for species distinction. On the basis of cloned ITS data, some strains proved to be misidentified in comparison with the identities obtained with phenotypic characters, protein sequences, and AFLP profiles, indicating that different copies of the ribosomal operon were present in a single species. Antifungal susceptibility testing revealed that voriconazole had the lowest MIC against M. capitatus , while amphotericin B had the lowest MIC against S. clavata Both species exhibited in vitro resistance to fluconazole and micafungin. Copyright © 2017 American Society for Microbiology.

Three Group-I introns in 18S rDNA of Endosymbiotic Algae of Paramecium bursaria from Japan

NASA Astrophysics Data System (ADS)

Hoshina, Ryo; Kamako, Shin-ichiro; Imamura, Nobutaka

2004-08-01

In the nuclear encoded small subunit ribosomal DNA (18S rDNA) of symbiotic alga of Paramecium bursaria (F36 collected in Japan) possesses three intron-like insertions (Hoshina et al., unpubl. data, 2003). The present study confirmed these exact lengths and insertion sites by reverse transcription-PCR. Two of them were inserted at Escherichia coli 16S rRNA genic position 943 and 1512 that are frequent intron insertion positions, but another insertion position (nearly 1370) was the first finding. Their secondary structures suggested they belong to Group-I intron; one belongs to subgroup IE, others belong to subgroup IC1. Similarity search indicated these introns are ancestral ones.

Morphology and Description of Bursaphelenchus platzeri n. sp. (Nematoda: Parasitaphelenchidae), an Associate of Nitidulid Beetles

PubMed Central

Giblin-Davis, Robin M.; Kanzaki, Natsumi; Ye, Weimin; Mundo-Ocampo, Manuel; Baldwin, James G.; Thomas, W. Kelley

2006-01-01

Bursaphelenchus platzeri n. sp., an associate of nitidulid beetles in southern California, is described and illustrated. Adult males and females of B. platzeri n. sp. were examined by scanning electron microscopy for ultrastructural comparisons with other members of the genus. Bursaphelenchus cocophilus (red ring nematode) appears to be the closest related taxon to B. platzeri n. sp. based upon shared morphological features of the fused spicules, female tail shape, phoresy with non-scolytid beetles, and molecular analysis of the near full-length small subunit (SSU) rDNA. Unfortunately, sequence data from the D2D3 expansion segments of the large subunit (LSU) rDNA and partial mitochondrial DNA COI did not help resolve the relationship of nearest relative. In addition to significant molecular sequence differences in SSU, LSU, and COI, B. platzeri n. sp., which is an obligate fungal feeder, can be differentiated from B. cocophilus because it is an obligate parasite of palms. Bursaphelenchus platzeri n. sp. can be differentiated from all other species of Bursaphelenchus by the length and shape of the female tail and spicule morphology. The spicules are fused along the ventral midline and possess unfused cucullae; the fused unit appears to function as a conduit for sperm. Population growth of B. platzeri n. sp. was measured in a time-course experiment at 25°C in the laboratory on cultures of the fungus Monilinia fructicola grown on 5% glycerol-supplemented potato dextrose agar (GPDA). Nematode population densities rapidly increased from 25 to approximately 200,000/culture within 14 d and then plateaued for up to 28 d. PMID:19259440

A phylogenetic analysis of armored scale insects (Hemiptera: Diaspididae), based upon nuclear, mitochondrial, and endosymbiont gene sequences.

PubMed

Andersen, Jeremy C; Wu, Jin; Gruwell, Matthew E; Gwiazdowski, Rodger; Santana, Sharlene E; Feliciano, Natalie M; Morse, Geoffrey E; Normark, Benjamin B

2010-12-01

Armored scale insects (Hemiptera: Diaspididae) are among the most invasive insects in the world. They have unusual genetic systems, including diverse types of paternal genome elimination (PGE) and parthenogenesis. Intimate relationships with their host plants and bacterial endosymbionts make them potentially important subjects for the study of co-evolution. Here, we expand upon recent phylogenetic work (Morse and Normark, 2006) by analyzing armored scale and endosymbiont DNA sequences from 125 species of armored scale insect, represented by 253 samples and eight outgroup species. We used fragments of four different gene regions: the nuclear protein-coding gene Elongation Factor 1α (EF1α), the large ribosomal subunit (28S) rDNA, a mitochondrial region spanning parts of cytochrome oxidase I (COI) and cytochrome oxidase II (COII), and the small ribosomal subunit (16S) rDNA from the primary bacterial endosymbiont Uzinura diaspidicola. Maximum likelihood, and Bayesian analyses were performed producing highly congruent topological results. A comparison of two datasets, one with and one without missing data, found that missing data had little effect on topology. Our results broadly corroborate several major features of the existing classification, although we do not find any of the subfamilies, tribes or subtribes to be monophyletic as currently constituted. Using ancestral state reconstruction we estimate that the ancestral armored scale had the late PGE sex system, and it may as well have been pupillarial, though results differed between reconstruction methods. These results highlight the need for a complete revision of this family, and provide the groundwork for future taxonomic work in armored scale insects. Copyright © 2010 Elsevier Inc. All rights reserved.

Flooding greatly affects the diversity of arbuscular mycorrhizal fungi communities in the roots of wetland plants.

PubMed

Wang, Yutao; Huang, Yelin; Qiu, Qiu; Xin, Guorong; Yang, Zhongyi; Shi, Suhua

2011-01-01

The communities of arbuscular mycorrhizal fungi (AMF) colonizing the roots of three mangrove species were characterized along a tidal gradient in a mangrove swamp. A fragment, designated SSU-ITS-LSU, including part of the small subunit (SSU), the entire internal transcribed spacer (ITS) and part of the large subunit (LSU) of rDNA from samples of AMF-colonized roots was amplified, cloned and sequenced using AMF-specific primers. Similar levels of AMF diversity to those observed in terrestrial ecosystems were detected in the roots, indicating that the communities of AMF in wetland ecosystems are not necessarily low in diversity. In total, 761 Glomeromycota sequences were obtained, which grouped, according to phylogenetic analysis using the SSU-ITS-LSU fragment, into 23 phylotypes, 22 of which belonged to Glomeraceae and one to Acaulosporaceae. The results indicate that flooding plays an important role in AMF diversity, and its effects appear to depend on the degree (duration) of flooding. Both host species and tide level affected community structure of AMF, indicating the presence of habitat and host species preferences.

Flooding Greatly Affects the Diversity of Arbuscular Mycorrhizal Fungi Communities in the Roots of Wetland Plants

PubMed Central

Wang, Yutao; Huang, Yelin; Qiu, Qiu; Xin, Guorong; Yang, Zhongyi; Shi, Suhua

2011-01-01

The communities of arbuscular mycorrhizal fungi (AMF) colonizing the roots of three mangrove species were characterized along a tidal gradient in a mangrove swamp. A fragment, designated SSU-ITS-LSU, including part of the small subunit (SSU), the entire internal transcribed spacer (ITS) and part of the large subunit (LSU) of rDNA from samples of AMF-colonized roots was amplified, cloned and sequenced using AMF-specific primers. Similar levels of AMF diversity to those observed in terrestrial ecosystems were detected in the roots, indicating that the communities of AMF in wetland ecosystems are not necessarily low in diversity. In total, 761 Glomeromycota sequences were obtained, which grouped, according to phylogenetic analysis using the SSU-ITS-LSU fragment, into 23 phylotypes, 22 of which belonged to Glomeraceae and one to Acaulosporaceae. The results indicate that flooding plays an important role in AMF diversity, and its effects appear to depend on the degree (duration) of flooding. Both host species and tide level affected community structure of AMF, indicating the presence of habitat and host species preferences. PMID:21931734

Natural Endophytic Occurrence of Acetobacter diazotrophicus in Pineapple Plants.

PubMed

Tapia-Hernández; Bustillos-Cristales; Jiménez-Salgado; Caballero-Mellado; Fuentes-Ramírez

2000-01-01

The presence of endophytic Acetobacter diazotrophicus was tested for pineapple plants (Ananas comosus [L.] Merr.) grown in the field. Diazotrophic bacteria were isolated from the inner tissues of surface sterilized roots, stems, and leaves of pineapple plants. Phenotypic tests permitted the selection of presumptive nitrogen-fixing A. diazotrophicus isolates. Restriction fragment length polymorphisms (RFLPs) of small subunit (SSU) rDNA using total DNA digested with endonuclease SphI and with endonuclease NcoI, hybridizations of RNA with an A. diazotrophicus large subunit (LSU) rRNA specific probe, as well as patterns in denaturing protein electrophoresis (SDS-PAGE) and multilocus enzyme tests allowed the identification of A. diazotrophicus isolates. High frequencies of isolation were obtained from propagative buds that had not been nitrogen-fertilized, and lower frequencies from 3-month-old plants that had been nitrogen-fertilized. No isolates were recovered from 5- to 7-month-old nitrogen-fertilized plants. All the A. diazotrophicus isolates recovered from pineapple plants belonged to the multilocus genotype which shows the most extensive distribution among all host species previously analyzed.

Blastocystis phylogeny among various isolates from humans to insects.

PubMed

Yoshikawa, Hisao; Koyama, Yukiko; Tsuchiya, Erika; Takami, Kazutoshi

2016-12-01

Blastocystis is a common unicellular eukaryotic parasite found not only in humans, but also in various kinds of animal species worldwide. Since Blastocystis isolates are morphologically indistinguishable, many molecular biological approaches have been applied to classify these isolates. The complete or partial sequences of the small subunit rRNA gene (SSU rDNA) are mainly used for comparisons and phylogenetic analyses among Blastocystis isolates. However, various lengths of the partial SSU rDNA sequence have been used for phylogenetic inference among genetically different isolates. Based on the complete SSU rDNA sequences, consensus terminology of nine subtypes (STs) of Blastocystis sp. that were supported by phylogenetically monophyletic nine clades was proposed in 2007. Thereafter, eight additional kinds of STs comprising non-human mammalian Blastocystis isolates have been reported based on the phylogeny of SSU rDNA sequences, while STs 11 and 12 were only proposed on the base of partial sequences. Although many sequence data from mammalian and avian Blastocystis are registered in GenBank, only limited data on SSU rDNA are available for poikilotherm-derived Blastocystis isolates. Therefore, the phylogenetic positions of the reptilian/amphibian Blastocystis clades are unstable. The phylogenetic inference of various STs comprising mammalian and/or avian Blastocystis isolates was verified herein based on comparisons between partial and complete SSU rDNA sequences, and the phylogenetic positions of reptilian and amphibian Blastocystis isolates were also investigated using 14 new Blastocystis isolates from reptiles with all known isolates from other reptilians, amphibians, and insects registered in GenBank. Copyright © 2016. Published by Elsevier Ireland Ltd.

Phylogenetic Analyses of Meloidogyne Small Subunit rDNA.

PubMed

De Ley, Irma Tandingan; De Ley, Paul; Vierstraete, Andy; Karssen, Gerrit; Moens, Maurice; Vanfleteren, Jacques

2002-12-01

Phylogenies were inferred from nearly complete small subunit (SSU) 18S rDNA sequences of 12 species of Meloidogyne and 4 outgroup taxa (Globodera pallida, Nacobbus abberans, Subanguina radicicola, and Zygotylenchus guevarai). Alignments were generated manually from a secondary structure model, and computationally using ClustalX and Treealign. Trees were constructed using distance, parsimony, and likelihood algorithms in PAUP* 4.0b4a. Obtained tree topologies were stable across algorithms and alignments, supporting 3 clades: clade I = [M. incognita (M. javanica, M. arenaria)]; clade II = M. duytsi and M. maritima in an unresolved trichotomy with (M. hapla, M. microtyla); and clade III = (M. exigua (M. graminicola, M. chitwoodi)). Monophyly of [(clade I, clade II) clade III] was given maximal bootstrap support (mbs). M. artiellia was always a sister taxon to this joint clade, while M. ichinohei was consistently placed with mbs as a basal taxon within the genus. Affinities with the outgroup taxa remain unclear, although G. pallida and S. radicicola were never placed as closest relatives of Meloidogyne. Our results show that SSU sequence data are useful in addressing deeper phylogeny within Meloidogyne, and that both M. ichinohei and M. artiellia are credible outgroups for phylogenetic analysis of speciations among the major species.

Phylogenetic Analyses of Meloidogyne Small Subunit rDNA

PubMed Central

De Ley, Irma Tandingan; De Ley, Paul; Vierstraete, Andy; Karssen, Gerrit; Moens, Maurice; Vanfleteren, Jacques

2002-01-01

Phylogenies were inferred from nearly complete small subunit (SSU) 18S rDNA sequences of 12 species of Meloidogyne and 4 outgroup taxa (Globodera pallida, Nacobbus abberans, Subanguina radicicola, and Zygotylenchus guevarai). Alignments were generated manually from a secondary structure model, and computationally using ClustalX and Treealign. Trees were constructed using distance, parsimony, and likelihood algorithms in PAUP* 4.0b4a. Obtained tree topologies were stable across algorithms and alignments, supporting 3 clades: clade I = [M. incognita (M. javanica, M. arenaria)]; clade II = M. duytsi and M. maritima in an unresolved trichotomy with (M. hapla, M. microtyla); and clade III = (M. exigua (M. graminicola, M. chitwoodi)). Monophyly of [(clade I, clade II) clade III] was given maximal bootstrap support (mbs). M. artiellia was always a sister taxon to this joint clade, while M. ichinohei was consistently placed with mbs as a basal taxon within the genus. Affinities with the outgroup taxa remain unclear, although G. pallida and S. radicicola were never placed as closest relatives of Meloidogyne. Our results show that SSU sequence data are useful in addressing deeper phylogeny within Meloidogyne, and that both M. ichinohei and M. artiellia are credible outgroups for phylogenetic analysis of speciations among the major species. PMID:19265950

Two myxozoans from the urinary tract of topsmelt, Atherinops affinis

USGS Publications Warehouse

Sanders, Justin L.; Jaramillo, Alejandra G.; Ashford, Jacob E.; Feist, Stephen W.; Lafferty, Kevin D.; Kent, Michael L.

2015-01-01

Two myxozoan species were observed in the kidney of topsmelt, Atherinops affinis, during a survey of parasites of estuarine fishes in the Carpinteria Salt Marsh Reserve, California. Fish collected on three dates in 2012 and 2013 were sectioned and examined histologically. Large extrasporogonic stages occurred in the renal interstitium of several fish from the first two collections (5/8, 11/20, respectively), and, in some fish, these replaced over 80% of the kidney. In addition, presporogonic and polysporogonic stages occurred in the lumen of the renal tubules, collecting and mesonephric ducts. The latter contained subspherical spores with up to 4 polar capsules, consistent with the genus Chloromyxum. For the third collection (15 May 2013, n=30), we portioned kidneys for examination by histology, wet mount, and DNA extraction for small subunit ribosomal gene sequencing. Histology showed the large extrasporogonic forms in the kidney interstitium of 3 fish, and 2 other fish with subspherical myxospores in the lumen of the renal tubules with smooth valves and two spherical polar capsules consistent with the genus Sphaerospora. Chloromyxum-type myxospores were observed in the renal tubules of one fish by wet mount. Sequencing of the kidney tissue from this fish yielded a partial SSU rDNA sequence of 1769 bp. Phylogenetic reconstruction suggested this organism to be a novel species of Chloromyxum, most similar to Chloromyxum careni (84% similarity). In addition, subspherical myxospores with smooth valves and two spherical polar capsules consistent with the genus Sphaerospora were observed in wet mounts of 2 fish. Sequencing of the kidney tissue from 1 fish yielded a partial SSU rDNA sequence of 1937 bp. Phylogenetic reconstruction suggests this organism to be a novel species of Sphaerospora most closely related to Sphaerospora epinepheli (93%). We conclude that these organisms represent novel species of the genera Chloromyxum and Sphaerospora based on host, location, and SSU rDNA sequence. We further conclude that the formation of large, histozoic extrasprogonic stages in the renal interstitium represent developmental stages of the Chloromyxum species for the following reasons: 1. Large extrasporogonic stages stages were only observed in fish with Chloromyxum-type spores developing within the renal tubules, 2. DNA sequence consistent with the Chloromyxum sp. was only detected in fish with the large extrasporogonic stages and 3.Sphaerospora species have extrasporogonic forms, but they are considerably smaller and are comprised of much fewer cells.

Acanthocotyle gurgesiella n. sp. (Monogenea: Acanthocotylidae) from the deep-sea skate Gurgesiella furvescens (Rajidae) in the south-eastern Pacific.

PubMed

Ñacari, L A; Sepulveda, F A; Escribano, R; Oliva, M E

2018-03-01

Little is known about the diversity of parasites of the deep-sea fish of the world's oceans. Here, a new species of monogenean parasite of the deep-sea skate Gurgesiella furvescens is described. Specimens of parasites were obtained from the skin of two specimens of the dusky finless skate, G. furvescens (Rajidae), in the vicinity of Valparaiso (33°S, 72°W), central Chile, from midwater trawl fishing at depths of 350-450 m. Both morphological and molecular analyses were conducted to provide a full description of the new species, named Acanthocotyle gurgesiella. For the molecular analyses, nuclear large subunit (LSU) rDNA and the mitochondrial gene cytochrome c oxidase 1 (COI) were used. From the morphological analysis and a comparison with the known species of the genus, A. gurgesiella can be identified by a combination of morphological characteristics, including the number of testes, number of radial rows of sclerites in the pseudohaptor, aperture of the genital pore and shape of the vitelline follicles. The results from the DNA analysis indicated that A. gurgesiella has a genetic divergence of 3.2-3.7% (LSU rDNA gene) from A. urolophi, the only congener species for which molecular data are available.

Nuclear and mitochondrial rDNA variability in Crinipellis perniciosa from different geographic origins and hosts.

PubMed

de Arruda, Maricília C C; Ferreira, Marisa A S V; Miller, Robert N G; Resende, Mário Lúcio V; Felipe, Maria Sueli S

2003-01-01

Genetic variability in Crinipellis perniciosa, the causal organism of witches' broom disease in Theobroma cacao, was determined in strains originating from T. cacao and other susceptible host species Heteropterys acutifolia and Solanum lycocarpum in Brazil, in order to clarify host specificity and geographical variability. RFLP analysis of the ribosomal DNA ITS regions (rDNA ITS), and the mitochondrial DNA small subunit ribosomal DNA gene (mtDNA SSU rDNA) did not reveal any genetic variability in 120 tested strains, possibly serving only as species level markers. Genetic variability was observed in the ribosomal DNA IGS spacer region, in terms of IGS size, RFLPs and sequence data. Phylogenetic analyses (using CLUSTAL W, PHYLIP and TREEVIEW) indicated considerable differences between C. perniciosa strains from T. cacao and those from H. acutifolia (85-86%) and S. lycocarpum (95-96%). Sequence differences also indicated that C. perniciosa from T. cacao in Bahia is less variable (98%) when compared to the pathogen on T. cacao in Amazonas (97-98%), perhaps reflecting a recent introduction to T. cacao in Bahia.

Detection of trypanosomatid Phytomonas parasitic in plants by polymerase chain reaction amplification of small subunit ribosomal DNA.

PubMed

Teixeira, M M; Campaner, M; Camargo, E P

1994-01-01

To improve the diagnosis of Phytomonas infections in plants, we developed a polymerase chain reaction (PCR) assay using synthetic oligonucleotides complementary to conserved sequences of the 18S small subunit ribosomal (SSU) gene. From 10 ng upward of DNA of cultures of Phytomonas isolated from plants, fruits, and insects, PCR amplified an 800-bp DNA band that, after restriction analysis and probe hybridization, proved to be of 18S rDNA Phytomonas origin. PCR was also done with sap samples of tomatoes experimentally infected with Phytomonas, yielding amplified 800-bp ribosomal DNA bands before any flagellate could be detected by microscopic examination of the fruit sap.

Myxobolus cerebralis internal transcribed spacer 1 (ITS-1) sequences support recent spread of the parasite to North America and within Europe

USGS Publications Warehouse

Whipps, Christopher M.; El-Matbouli, M.; Hedrick, R.P.; Blazer, V.; Kent, M.L.

2004-01-01

Molecular approaches for resolving relationships among the Myxozoa have relied mainly on small subunit (SSU) ribosomal DNA (rDNA) sequence analysis. This region of the gene is generally used for higher phylogenetic studies, and the conservative nature of this gene may make it inadequate for intraspecific comparisons. Previous intraspecific studies of Myxobolus cerebralis based on molecular analyses reported that the sequence of SSU rDNA and the internal transcribed spacer (ITS) were highly conserved in representatives of the parasite from North America and Europe. Considering that the ITS is usually a more variable region than the SSU, we reanalyzed available sequences on GenBank and obtained sequences from other M. cerebralis representatives from the states of California and West Virginia in the USA and from Germany and Russia. With the exception of 7 base pairs, most of the sequence designated as ITS-1 in GenBank was a highly conserved portion of the rDNA near the 3-prime end of the SSU region. Nonetheless, the additional ITS-1 sequences obtained from the available geographic representatives were well conserved. It is unlikely that we would have observed virtually identical ITS-1 sequences between European and American M. cerebralis samples had it spread naturally over time, particularly when compared to the variation seen between isolates of another myxozoan (Kudoa thyrsites) that has most likely spread naturally. These data further support the hypothesis that the current distribution of M. cerebralis in North America is a result of recent introductions followed by dispersal via anthropogenic means, largely through the stocking of infected trout for sport fishing.

Long-term tracing of Rhizophagus irregularis isolate BEG140 inoculated on Phalaris arundinacea in a coal mine spoil bank, using mitochondrial large subunit rDNA markers.

PubMed

Sýkorová, Zuzana; Börstler, Boris; Zvolenská, Soňa; Fehrer, Judith; Gryndler, Milan; Vosátka, Miroslav; Redecker, Dirk

2012-01-01

During the last decade, the application of arbuscular mycorrhizal fungi (AMF) as bioenhancers has increased significantly. However, until now, it has been difficult to verify the inoculation success in terms of fungal symbiont establishment in roots of inoculated plants because specific fungal strains could not be detected within colonized roots. Using mitochondrial large subunit ribosomal DNA, we show that Rhizophagus irregularis (formerly known as Glomus intraradices) isolate BEG140 consists of two different haplotypes. We developed nested PCR assays to specifically trace each of the two haplotypes in the roots of Phalaris arundinacea from a field experiment in a spoil bank of a former coal mine, where BEG140 was used as inoculant. We revealed that despite the relatively high diversity of native R. irregularis strains, R. irregularis BEG140 survived and proliferated successfully in the field experiment and was found significantly more often in the inoculated than control plots. This work is the first one to show tracing of an inoculated AMF isolate in the roots of target plants and to verify its survival and propagation in the field. These results will have implications for basic research on the ecology of AMF at the intraspecific level as well as for commercial users of mycorrhizal inoculation.

Plunging hands into the mushroom jar: a phylogenetic framework for Lyophyllaceae (Agaricales, Basidiomycota).

PubMed

Bellanger, J-M; Moreau, P-A; Corriol, G; Bidaud, A; Chalange, R; Dudova, Z; Richard, F

2015-04-01

During the last two decades, the unprecedented development of molecular phylogenetic tools has propelled an opportunity to revisit the fungal kingdom under an evolutionary perspective. Mycology has been profoundly changed but a sustained effort to elucidate large sections of the astonishing fungal diversity is still needed. Here we fill this gap in the case of Lyophyllaceae, a species-rich and ecologically diversified family of mushrooms. Assembly and genealogical concordance multigene phylogenetic analysis of a large dataset that includes original, vouchered material from expert field mycologists reveal the phylogenetic topology of the family, from higher (generic) to lower (species) levels. A comparative analysis of the most widely used phylogenetic markers in Fungi indicates that the nuc rDNA region encompassing the internal transcribed spacers 1 and 2, along with the 5.8S rDNA (ITS) and portions of the genes for RNA polymerase II second largest subunit (RPB2) is the most performing combination to resolve the broadest range of taxa within Lyophyllaceae. Eleven distinct evolutionary lineages are identified, that display partial overlap with traditional genera as well as with the phylogenetic framework previously proposed for the family. Eighty phylogenetic species are delineated, which shed light on a large number of morphological concepts, including rare and poorly documented ones. Probing these novel phylogenetic species to the barcoding method of species limit delineation, indicates that the latter method fully resolves Lyophyllaceae species, except in one clade. This case study provides the first comprehensive phylogenetic overview of Lyophyllaceae, a necessary step towards a taxonomical, ecological and nomenclatural revision of this family of mushrooms. It also proposes a set of methodological guidelines that may be of relevance for future taxonomic works in other groups of Fungi.

A Suspected Parasite Spill-Back of Two Novel Myxidium spp. (Myxosporea) Causing Disease in Australian Endemic Frogs Found in the Invasive Cane Toad

PubMed Central

Hartigan, Ashlie; Fiala, Ivan; Dyková, Iva; Jirků, Miloslav; Okimoto, Ben; Rose, Karrie; Phalen, David N.; Šlapeta, Jan

2011-01-01

Infectious diseases are contributing to the decline of endangered amphibians. We identified myxosporean parasites, Myxidium spp. (Myxosporea: Myxozoa), in the brain and liver of declining native frogs, the Green and Golden Bell frog (Litoria aurea) and the Southern Bell frog (Litoria raniformis). We unequivocally identified two Myxidium spp. (both generalist) affecting Australian native frogs and the invasive Cane toad (Bufo marinus, syn. Rhinella marina) and demonstrated their association with disease. Our study tested the identity of Myxidium spp. within native frogs and the invasive Cane toad (brought to Australia in 1935, via Hawaii) to resolve the question whether the Cane toad introduced them to Australia. We showed that the Australian brain and liver Myxidium spp. differed 9%, 7%, 34% and 37% at the small subunit rDNA, large subunit rDNA, internal transcribed spacers 1 and 2, but were distinct from Myxidium cf. immersum from Cane toads in Brazil. Plotting minimum within-group distance against maximum intra-group distance confirmed their independent evolutionary trajectory. Transmission electron microscopy revealed that the brain stages localize inside axons. Myxospores were morphologically indistinguishable, therefore genetic characterisation was necessary to recognise these cryptic species. It is unlikely that the Cane toad brought the myxosporean parasites to Australia, because the parasites were not found in 261 Hawaiian Cane toads. Instead, these data support the enemy-release hypothesis predicting that not all parasites are translocated with their hosts and suggest that the Cane toad may have played an important spill-back role in their emergence and facilitated their dissemination. This work emphasizes the importance of accurate species identification of pathogens relevant to wildlife management and disease control. In our case it is paving the road for the spill-back role of the Cane toad and the parasite emergence. PMID:21541340

Ultrastructural morphology of the reproductive swarmers of Sphaerozoum punctatum (Huxley) from the East China Sea.

PubMed

Yuasa, Tomoko; Takahashi, Osamu

2014-04-01

Reproductive swarmers of the polycystine radiolarian Sphaerozoum punctatum (Huxley) collected from the East China Sea were examined using light, scanning and transmission electron microscopy. The swarmer cells were about 8-10 μm in length with a pear-like shape and a conical end with two flagella. A nucleus, mitochondria, Golgi body, lipid droplets and, characteristically, a single, large, vacuole-bound SrSO₄ crystal were present in the cytoplasm. Centering on the crystal inclusion, swarmers swam in a rapid rotational movement both clockwise and anticlockwise. Small subunit (SSU) rDNA sequences obtained for the reproductive swarmer cells from S. punctatum show a monophyletic group together with colonial spumellarians and grouped with S. punctatum from Bermuda in the clade. The morphological features and molecular phylogeny of the reproductive swarmers of S. punctatum show evidence of ancestral traits of radiolarians; acantharians and polycystines have a common ancestry. In addition, SrSO₄ inclusion of the swarmer cell may be a form of ballast deposited by the swarmer to allow proper positioning in the water column. We hypothesize that radiolarian-affiliated sequences from SSU rDNA clone libraries of marine picoeukaryotes may be derived from the picoplanktonic cells of radiolarians; i.e., small flagellated life stages such as reproductive swarmers or gametes. Copyright © 2013 Elsevier GmbH. All rights reserved.

Evaluation of Novel Broad-Range Real-Time PCR Assay for Rapid Detection of Human Pathogenic Fungi in Various Clinical Specimens▿

PubMed Central

Vollmer, Tanja; Störmer, Melanie; Kleesiek, Knut; Dreier, Jens

2008-01-01

In the present study, a novel broad-range real-time PCR was developed for the rapid detection of human pathogenic fungi. The assay targets a part of the 28S large-subunit ribosomal RNA (rDNA) gene. We investigated its application for the most important human pathogenic fungal genera, including Aspergillus, Candida, Cryptococcus, Mucor, Penicillium, Pichia, Microsporum, Trichophyton, and Scopulariopsis. Species were identified in PCR-positive reactions by direct DNA sequencing. A noncompetitive internal control was applied to prevent false-negative results due to PCR inhibition. The minimum detection limit for the PCR was determined to be one 28S rDNA copy per PCR, and the 95% detection limit was calculated to 15 copies per PCR. To assess the clinical applicability of the PCR method, intensive-care patients with artificial respiration and patients with infective endocarditis were investigated. For this purpose, 76 tracheal secretion samples and 70 heart valve tissues were analyzed in parallel by real-time PCR and cultivation. No discrepancies in results were observed between PCR analysis and cultivation methods. Furthermore, the application of the PCR method was investigated for other clinical specimens, including cervical swabs, nail and horny skin scrapings, and serum, blood, and urine samples. The combination of a broad-range real-time PCR and direct sequencing facilitates rapid screening for fungal infection in various clinical specimens. PMID:18385440

Evaluation of novel broad-range real-time PCR assay for rapid detection of human pathogenic fungi in various clinical specimens.

PubMed

Vollmer, Tanja; Störmer, Melanie; Kleesiek, Knut; Dreier, Jens

2008-06-01

In the present study, a novel broad-range real-time PCR was developed for the rapid detection of human pathogenic fungi. The assay targets a part of the 28S large-subunit ribosomal RNA (rDNA) gene. We investigated its application for the most important human pathogenic fungal genera, including Aspergillus, Candida, Cryptococcus, Mucor, Penicillium, Pichia, Microsporum, Trichophyton, and Scopulariopsis. Species were identified in PCR-positive reactions by direct DNA sequencing. A noncompetitive internal control was applied to prevent false-negative results due to PCR inhibition. The minimum detection limit for the PCR was determined to be one 28S rDNA copy per PCR, and the 95% detection limit was calculated to 15 copies per PCR. To assess the clinical applicability of the PCR method, intensive-care patients with artificial respiration and patients with infective endocarditis were investigated. For this purpose, 76 tracheal secretion samples and 70 heart valve tissues were analyzed in parallel by real-time PCR and cultivation. No discrepancies in results were observed between PCR analysis and cultivation methods. Furthermore, the application of the PCR method was investigated for other clinical specimens, including cervical swabs, nail and horny skin scrapings, and serum, blood, and urine samples. The combination of a broad-range real-time PCR and direct sequencing facilitates rapid screening for fungal infection in various clinical specimens.

Babesia vesperuginis, a neglected piroplasmid: new host and geographical records, and phylogenetic relations.

PubMed

Corduneanu, Alexandra; Hrazdilová, Kristýna; Sándor, Attila D; Matei, Ioana Adriana; Ionică, Angela Monica; Barti, Levente; Ciocănău, Marius-Alexandru; Măntoiu, Dragoş Ștefan; Coroiu, Ioan; Hornok, Sándor; Fuehrer, Hans-Peter; Leitner, Natascha; Bagó, Zoltán; Stefke, Katharina; Modrý, David; Mihalca, Andrei Daniel

2017-12-06

Babesia spp. are hemoparasites which infect the red blood cells of a large variety of mammals. In bats, the only known species of the genus is Babesia vesperuginis. However, except a few old reports, the host range and geographical distribution of this bat parasite have been poorly studied. This study aimed to investigate the presence of piroplasms in tissues of bats collected in four different countries from eastern and central Europe: Austria, Czech Republic, Hungary and Romania. A total of 461 bat carcasses (24 species) were collected between 2001 and 2016 from caves, mines and buildings. PCR was performed using specific primers targeting a portion of the 18S rDNA nuclear gene and cytochrome c oxidase subunit 1 mitochondrial gene, followed by sequencing. The results of this study show for the first time the presence of B. vesperuginis in bats in central and eastern Europe. The phylogenetic analysis of the 18S rDNA nuclear gene revealed no variability between the sequences and the phylogenetic analysis of the cox1 mitochondrial gene proved that B. vesperuginis could be divided into two subclades. Our study showed a broad geographical distribution of B. vesperuginis in European bats, reporting its presence in five new host species (M. cf. alcathoe, M. bechsteinii, M. myotis, Pi. nathusii and V. murinus) and three new countries.

Phosphorylation by casein kinase 2 facilitates rRNA gene transcription by promoting dissociation of TIF-IA from elongating RNA polymerase I.

PubMed

Bierhoff, Holger; Dundr, Miroslav; Michels, Annemieke A; Grummt, Ingrid

2008-08-01

The protein kinase casein kinase 2 (CK2) phosphorylates different components of the RNA polymerase I (Pol I) transcription machinery and exerts a positive effect on rRNA gene (rDNA) transcription. Here we show that CK2 phosphorylates the transcription initiation factor TIF-IA at serines 170 and 172 (Ser170/172), and this phosphorylation triggers the release of TIF-IA from Pol I after transcription initiation. Inhibition of Ser170/172 phosphorylation or covalent tethering of TIF-IA to the RPA43 subunit of Pol I inhibits rDNA transcription, leading to perturbation of nucleolar structure and cell cycle arrest. Fluorescence recovery after photobleaching and chromatin immunoprecipitation experiments demonstrate that dissociation of TIF-IA from Pol I is a prerequisite for proper transcription elongation. In support of phosphorylation of TIF-IA switching from the initiation into the elongation phase, dephosphorylation of Ser170/172 by FCP1 facilitates the reassociation of TIF-IA with Pol I, allowing a new round of rDNA transcription. The results reveal a mechanism by which the functional interplay between CK2 and FCP1 sustains multiple rounds of Pol I transcription.

Eukaryotic Plankton Species Diversity in the Western Channel of the Korea Strait using 18S rDNA Sequences and its Implications for Water Masses

NASA Astrophysics Data System (ADS)

Lee, Sang-Rae; Song, Eun Hye; Lee, Tongsup

2018-03-01

Organisms entering the East Sea (Sea of Japan) through the Korea Strait, together with water, salt, and energy, affect the East Sea ecosystem. In this study, we report on the biodiversity of eukaryotic plankton found in the Western Channel of the Korea Strait for the first time using small subunit ribosomal RNA gene (18S rDNA) sequences. We also discuss the characteristics of water masses and their physicochemical factors. Diverse taxonomic groups were recovered from 18S rDNA clone libraries, including putative novel, higher taxonomic entities affiliated with Cercozoa, Raphidophyceae, Picozoa, and novel marine Stramenopiles. We also found that there was cryptic genetic variation at both the intraspecific and interspecific levels among arthropods, diatoms, and green algae. Specific plankton assemblages were identified at different sampling depths and they may provide useful information that could be used to interpret the origin and the subsequent mixing history of the water masses that contribute to the Tsushima Warm Current waters. Furthermore, the biological information highlighted in this study may help improve our understanding about the complex water mass interactions that were highlighted in the Korea Strait.

Morphological and molecular characterization and phylogenetic relationships of a new species of trypanosome in Tapirus terrestris (lowland tapir), Trypanosoma terrestris sp. nov., from Atlantic Rainforest of southeastern Brazi.

PubMed

Acosta, Igor da Cunha Lima; da Costa, Andrea Pereira; Nunes, Pablo Henrique; Gondim, Maria Fernanda Naegeli; Gatti, Andressa; Rossi, João Luiz; Gennari, Solange Maria; Marcili, Arlei

2013-12-11

The Lowland tapir (Tapirus terrestris) is the largest Brazilian mammal and despite being distributed in various Brazilian biomes, it is seriously endangered in the Atlantic Rainforest. These hosts were never evaluated for the presence of Trypanosoma parasites. The Lowland tapirs were captured in the Brazilian southeastern Atlantic Rainforest, Espírito Santo state. Trypanosomes were isolated by hemoculture, and the molecular phylogeny based on small subunit rDNA (SSU rDNA) and glycosomal-3-phosphate dehydrogenase (gGAPDH) gene sequences and the ultrastructural features seen via light microscopy and scanning and transmission electron microscopy are described. Phylogenetic trees using combined SSU rDNA and gGAPDH data sets clustered the trypanosomes of Lowland tapirs, which were highly divergent from other trypanosome species. The phylogenetic position and morphological discontinuities, mainly in epimastigote culture forms, made it possible to classify the trypanosomes from Lowland tapirs as a separate species. The isolated trypanosomes from Tapirus terrestris are a new species, Trypanosoma terrestris sp. n., and were positioned in a new Trypanosoma clade, named T. terrestris clade.

Morphological and molecular characterization and phylogenetic relationships of a new species of trypanosome in Tapirus terrestris (lowland tapir), Trypanosoma terrestris sp. nov., from Atlantic Rainforest of southeastern Brazi

PubMed Central

2013-01-01

Background The Lowland tapir (Tapirus terrestris) is the largest Brazilian mammal and despite being distributed in various Brazilian biomes, it is seriously endangered in the Atlantic Rainforest. These hosts were never evaluated for the presence of Trypanosoma parasites. Methods The Lowland tapirs were captured in the Brazilian southeastern Atlantic Rainforest, Espírito Santo state. Trypanosomes were isolated by hemoculture, and the molecular phylogeny based on small subunit rDNA (SSU rDNA) and glycosomal-3-phosphate dehydrogenase (gGAPDH) gene sequences and the ultrastructural features seen via light microscopy and scanning and transmission electron microscopy are described. Results Phylogenetic trees using combined SSU rDNA and gGAPDH data sets clustered the trypanosomes of Lowland tapirs, which were highly divergent from other trypanosome species. The phylogenetic position and morphological discontinuities, mainly in epimastigote culture forms, made it possible to classify the trypanosomes from Lowland tapirs as a separate species. Conclusions The isolated trypanosomes from Tapirus terrestris are a new species, Trypanosoma terrestris sp. n., and were positioned in a new Trypanosoma clade, named T. terrestris clade. PMID:24330660

The Warsaw breakage syndrome-related protein DDX11 is required for ribosomal RNA synthesis and embryonic development.

PubMed

Sun, Xinliang; Chen, Hongbo; Deng, Zaian; Hu, Bo; Luo, Hui; Zeng, Xiaobin; Han, Liqiao; Cai, Guoping; Ma, Lan

2015-09-01

DDX11 was recently identified as a cause of Warsaw breakage syndrome (WABS). However, the functional mechanism of DDX11 and the contribution of clinically described mutations to the pathogenesis of WABS are elusive. Here, we show that DDX11 is a novel nucleolar protein that preferentially binds to hypomethylated active ribosomal DNA (rDNA) gene loci, where it interacts with upstream binding factor (UBF) and the RNA polymerase I (Pol I). DDX11 knockdown changed the epigenetic state of rDNA loci from euchromatic structures to more heterochromatic structures, reduced the activity of UBF, decreased the recruitment of UBF and RPA194 (a subunit of Pol I) to rDNA promoter, suppressed rRNA transcription and thereby inhibited growth and proliferation of HeLa cells. Importantly, two indentified WABS-derived mutants, R263Q and K897del, and a Fe-S deletion construct demonstrated significantly reduced binding abilities to rDNA promoters and lowered DNA-dependent ATPase activities compared with wild-type DDX11. Knockdown of the zebrafish ortholog of human DDX11 by morpholinos resulted in growth retardation and vertebral and craniofacial malformations in zebrafish, concomitant with the changes in histone epigenetic modifications at rDNA loci, the reduction of Pol I recruitment to the rDNA promoter and a significant decrease in nascent pre-RNA levels. These growth disruptions in zebrafish in response to DDX11 reduction showed similarities to the clinically described developmental abnormalities found in WABS patients for the first time in any vertebrate. Thus, our results indicate that DDX11 functions as a positive regulator of rRNA transcription and provides a novel insight into the pathogenesis of WABS. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Candida middelhoveniana sp. nov., a new yeast species found on the rhizoplane of organically cultivated sugarcane.

PubMed

Ribeiro, José R de A; Carvalho, Patrícia M B de; Cabral, Anderson de S; Macrae, Andrew; Mendonça-Hagler, Leda C S; Berbara, Ricardo L L; Hagler, Allen N

2011-10-01

A novel yeast species within the Metschnikowiaceae is described based on a strain from the sugarcane (Saccharum sp.) rhizoplane of an organically managed farm in Rio de Janeiro, Brazil. The D1/D2 domain of the large subunit ribosomal RNA gene sequence analysis showed that the closest related species were Candida tsuchiyae with 86.2% and Candida thailandica with 86.7% of sequence identity. All three are anamorphs in the Clavispora opuntiae clade. The name Candida middelhoveniana sp. nov. is proposed to accommodate this highly divergent organism with the type strain Instituto de Microbiologia, Universidade Federal do Rio de Janeiro (IMUFRJ) 51965(T) (=Centraalbureau voor Schimmelcultures (CBS) 12306(T), Universidade Federal de Minas Gerais (UFMG)-70(T), DBVPG 8031(T)) and the GenBank/EMBL/DDBJ accession number for the D1/D2 domain LSU rDNA sequence is FN428871. The Mycobank deposit number is MB 519801.

Delimitation of Umbelopsis (Mucorales, Umbelopsidaceae fam. nov.) based on ITS sequence and RFLP data.

PubMed

Meyer, Wieland; Gams, Walter

2003-03-01

In a continuation of studies started by de Ruiter et al. (1993), all known species of the Mortierella isabellina-group (Micromucor/Umbelopsis clade of O'Donnell et al. 2001) and a few other Mucorales and species of Mortierella were investigated by RFLP (including ITS1, 5.8S, ITS2 and the 5' end of the large subunit rDNA gene) and ITS1 sequence analyses. This monophyletic group is unrelated to Mortierella and is only distantly related to the core group of the Mucoraceae. M. longicollis falls outside the Umbelopsis clade. Molecular data resolved two subclades within the M. isabellina-group; however, they are not correlated with any differences in sporangial wall and shape, spore pigmentation and shape, or sporangiophore branching. Therefore we subsume all taxa in one genus, Umbelopsis. The new family Umbelopsidaceae and the new combinations U. isabellina, U. ramanniana, and U. autotrophica are proposed.

Detection of Fungi from an Indoor Environment using Loop-mediated Isothermal Amplification (LAMP) Method.

PubMed

Nakayama, Takako; Yamazaki, Takashi; Yo, Ayaka; Tone, Kazuya; Mahdi Alshahni, Mohamed; Fujisaki, Ryuichi; Makimura, Koichi

2017-01-01

　Loop-mediated isothermal amplification (LAMP) is a useful DNA detection method with high specificity and sensitivity. The LAMP reaction is carried out within a short time at a constant temperature without the need for thermal cycling. We developed a LAMP primer set for detecting a wide range of fungi by aligning the sequences of the large subunit ribosomal RNA gene of Candida albicans (Ascomycota), Cryptococcus neoformans (Basidiomycota), and Mucor racemosus (Mucorales). The threshold of C. albicans rDNA as template with our LAMP primer set was in the range of 10-100 copies per a reaction. In this study, we evaluated the correlation between colony forming units (CFU) and LAMP detection rate using the LAMP method for environmental fungi. The LAMP method should be a useful means of detecting fungi in indoor environments, disaster areas, or even in confined manned spacecraft to prevent allergies or infections caused by fungi.

Pichia insulana sp. nov., a novel cactophilic yeast from the Caribbean

PubMed Central

Ganter, Philip F.; Cardinali, Gianluigi; Boundy-Mills, Kyria

2010-01-01

A novel species of ascomycetous yeast, Pichia insulana sp. nov., is described from necrotic tissue of columnar cacti on Caribbean islands. P. insulana is closely related to and phenotypically very similar to Pichia cactophila and Pichia pseudocactophila. There are few distinctions between these taxa besides spore type, host preference and locality. Sporogenous strains of P. insulana that produce asci with four hat-shaped spores have been found only on Curaçao, whereas there was no evidence of sporogenous P. cactophila from that island. In addition, sequences of the D1/D2 fragment of the large-subunit rDNA from 12 Curaçao strains showed consistent differences from the sequences of the type strains of P. cactophila and P. pseudocactophila. The type strain of P. insulana is TSU00-106.5T (=CBS 11169T =UCD-FST 09-160T). PMID:19661524

Marine Fungi: Their Ecology and Molecular Diversity

NASA Astrophysics Data System (ADS)

Richards, Thomas A.; Jones, Meredith D. M.; Leonard, Guy; Bass, David

2012-01-01

Fungi appear to be rare in marine environments. There are relatively few marine isolates in culture, and fungal small subunit ribosomal DNA (SSU rDNA) sequences are rarely recovered in marine clone library experiments (i.e., culture-independent sequence surveys of eukaryotic microbial diversity from environmental DNA samples). To explore the diversity of marine fungi, we took a broad selection of SSU rDNA data sets and calculated a summary phylogeny. Bringing these data together identified a diverse collection of marine fungi, including sequences branching close to chytrids (flagellated fungi), filamentous hypha-forming fungi, and multicellular fungi. However, the majority of the sequences branched with ascomycete and basidiomycete yeasts. We discuss evidence for 36 novel marine lineages, the majority and most divergent of which branch with the chytrids. We then investigate what these data mean for the evolutionary history of the Fungi and specifically marine-terrestrial transitions. Finally, we discuss the roles of fungi in marine ecosystems.

Genetic and morphologic differentiation of Bolbophorus confusus and B. levantinus (Digenea: Diplostomatidae), based on rDNA SSU polymorphism and SEM.

PubMed

Dzikowski, R; Levy, M G; Poore, M F; Flowers, J R; Paperna, I

2003-12-29

Metacercariae of Bolbophorus species are serious pathogens of farmed fish. Molecular diagnostic tools, capable of identifying and differentiating these parasites, may assist in the development of rationale control strategies. The rDNA 18S (small sub-unit: SSU) genes of adult B. confusus and B. levantinus obtained from a pelican, Pelecanus onocrotalus, and a night heron, Nycticorax nycticorax, respectively, were amplified, sequenced, and aligned. Based on this alignment, we developed a genetic differentiation assay between B. confusus and B. levantinus. These 2 species were compared genetically with the North American species B. damnificus and Bolbophorus sp. ('Type 2'). The relationship between species is outlined and discussed. In addition to the molecular study, specimens of B. confusus and B. levantinus were compared morphologically, using scanning electron microscopy. Morphologic analysis revealed interspecific differences in details of the holdfast organ and the position of the acetabulum.

The Nucleolus: In Genome Maintenance and Repair.

PubMed

Tsekrekou, Maria; Stratigi, Kalliopi; Chatzinikolaou, Georgia

2017-07-01

The nucleolus is the subnuclear membrane-less organelle where rRNA is transcribed and processed and ribosomal assembly occurs. During the last 20 years, however, the nucleolus has emerged as a multifunctional organelle, regulating processes that go well beyond its traditional role. Moreover, the unique organization of rDNA in tandem arrays and its unusually high transcription rates make it prone to unscheduled DNA recombination events and frequent RNA:DNA hybrids leading to DNA double strand breaks (DSBs). If not properly repaired, rDNA damage may contribute to premature disease onset and aging. Deregulation of ribosomal synthesis at any level from transcription and processing to ribosomal subunit assembly elicits a stress response and is also associated with disease onset. Here, we discuss how genome integrity is maintained within nucleoli and how such structures are functionally linked to nuclear DNA damage response and repair giving an emphasis on the newly emerging roles of the nucleolus in mammalian physiology and disease.

Ribosomal DNA analysis of tsetse and non-tsetse transmitted Ethiopian Trypanosoma vivax strains in view of improved molecular diagnosis.

PubMed

Fikru, Regassa; Matetovici, Irina; Rogé, Stijn; Merga, Bekana; Goddeeris, Bruno Maria; Büscher, Philippe; Van Reet, Nick

2016-04-15

Animal trypanosomosis caused by Trypanosoma vivax (T. vivax) is a devastating disease causing serious economic losses. Most molecular diagnostics for T. vivax infection target the ribosomal DNA locus (rDNA) but are challenged by the heterogeneity among T. vivax strains. In this study, we investigated the rDNA heterogeneity of Ethiopian T. vivax strains in relation to their presence in tsetse-infested and tsetse-free areas and its effect on molecular diagnosis. We sequenced the rDNA loci of six Ethiopian (three from tsetse-infested and three from tsetse-free areas) and one Nigerian T. vivax strain. We analysed the obtained sequences in silico for primer-mismatches of some commonly used diagnostic PCR assays and for GC content. With these data, we selected some rDNA diagnostic PCR assays for evaluation of their diagnostic accuracy. Furthermore we constructed two phylogenetic networks based on sequences within the smaller subunit (SSU) of 18S and within the 5.8S and internal transcribed spacer 2 (ITS2) to assess the relatedness of Ethiopian T. vivax strains to strains from other African countries and from South America. In silico analysis of the rDNA sequence showed important mismatches of some published diagnostic PCR primers and high GC content of T. vivax rDNA. The evaluation of selected diagnostic PCR assays with specimens from cattle under natural T. vivax challenge showed that this high GC content interferes with the diagnostic accuracy of PCR, especially in cases of mixed infections with T. congolense. Adding betain to the PCR reaction mixture can enhance the amplification of T. vivax rDNA but decreases the sensitivity for T. congolense and Trypanozoon. The networks illustrated that Ethiopian T. vivax strains are considerably heterogeneous and two strains (one from tsetse-infested and one from tsetse-free area) are more related to the West African and South American strains than to the East African strains. The rDNA locus sequence of six Ethiopian T. vivax strains showed important differences and higher GC content compared to other animal trypanosomes but could not be related to their origin from tsetse-infested or tsetse-free area. The high GC content of T. vivax DNA renders accurate diagnosis of all pathogenic animal trypanosomes with one single PCR problematic. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Genetic Diversity of Daphnia pulex in the Middle and Lower Reaches of the Yangtze River

PubMed Central

Wang, Wenping; Zhang, Kun; Deng, Daogui; Zhang, Ya-Nan; Peng, Shuixiu; Xu, Xiaoxue

2016-01-01

Increased human activities and environmental changes may lead to genetic diversity variations of Cladocerans in water. Daphnia pulex are distributed throughout the world and often regarded as a model organism. The 16S rDNA, cytochrome c oxidase subunit I (COI), and 18S genes were used as molecular marks. The genetic diversity and phylogeny of D. pulex obtained from 10 water bodies in the middle and lower reaches of the Yangtze River were studied. For 16S rDNA, COI gene, and 18S gene, the A+T content (65.4%, 58.4%, and 54.6%) was significantly higher than the G+C content (34.6%, 41.6% and 45.4%). This result was consistent with higher A and T contents among invertebrates. Based on the genetic distances of 16S rDNA and COI genes, the genetic differences of D. pulex from 10 water bodies located in the middle and lower reaches of the Yangtze River in China was minimal (0%–0.8% for 16S rDNA and 0%–1.5% for COI gene). However, D. pulex evolved into two branches in the phylogenetic trees, which coincided with its geographical distribution. Compared with D. pulex from other countries, the average genetic distance of D. pulex obtained from 10 water bodies in the middle and lower reaches of the Yangtze River reached 9.1%–10.5%, thereby indicating that D. pulex may have evolved into different subspecies. PMID:27015539

Identification of Giardia species and Giardia duodenalis assemblages by sequence analysis of the 5.8S rDNA gene and internal transcribed spacers.

PubMed

Cacciò, Simone M; Beck, Relja; Almeida, Andre; Bajer, Anna; Pozio, Edoardo

2010-05-01

PCR assays have been developed mainly to assist investigations into the epidemiology of Giardia duodenalis, the only species in the Giardia genus having zoonotic potential. However, a reliable identification of all species is of practical importance, particularly when water samples and samples from wild animals are investigated. The aim of the present work was to genotype Giardia species and G. duodenalis assemblages using as a target the region spanning the 5.8S gene and the 2 flanking internal transcribed spacers (ITS1 and ITS2) of the ribosomal gene. Primers were designed to match strongly conserved regions in the 3' end of the small subunit and in the 5' end of the large subunit ribosomal genes. The corresponding region (about 310 bp) was amplified from 49 isolates of both human and animal origin, representing all G. duodenalis assemblages as well as G. muris and G. microti. Sequence comparison and phylogenetic analysis showed that G. ardeae, G. muris, G. microti as well as the 7 G. duodenalis assemblages can be easily distinguished. Since the major subgroups within the zoonotic assemblages A and B can be identified by sequence analysis, this assay is also informative for molecular epidemiological studies.

High and uneven levels of 45S rDNA site-number variation across wild populations of a diploid plant genus (Anacyclus, Asteraceae)

PubMed Central

Rosato, Marcela; Álvarez, Inés; Nieto Feliner, Gonzalo

2017-01-01

The nuclear genome harbours hundreds to several thousand copies of ribosomal DNA. Despite their essential role in cellular ribogenesis few studies have addressed intrapopulation, interpopulation and interspecific levels of rDNA variability in wild plants. Some studies have assessed the extent of rDNA variation at the sequence and copy-number level with large sampling in several species. However, comparable studies on rDNA site number variation in plants, assessed with extensive hierarchical sampling at several levels (individuals, populations, species) are lacking. In exploring the possible causes for ribosomal loci dynamism, we have used the diploid genus Anacyclus (Asteraceae) as a suitable system to examine the evolution of ribosomal loci. To this end, the number and chromosomal position of 45S rDNA sites have been determined in 196 individuals from 47 populations in all Anacyclus species using FISH. The 45S rDNA site-number has been assessed in a significant sample of seed plants, which usually exhibit rather consistent features, except for polyploid plants. In contrast, the level of rDNA site-number variation detected in Anacyclus is outstanding in the context of angiosperms particularly regarding populations of the same species. The number of 45S rDNA sites ranged from four to 11, accounting for 14 karyological ribosomal phenotypes. Our results are not even across species and geographical areas, and show that there is no clear association between the number of 45S rDNA loci and the life cycle in Anacyclus. A single rDNA phenotype was detected in several species, but a more complex pattern that included intra-specific and intra-population polymorphisms was recorded in A. homogamos, A. clavatus and A. valentinus, three weedy species showing large and overlapping distribution ranges. It is likely that part of the cytogenetic changes and inferred dynamism found in these species have been triggered by genomic rearrangements resulting from contemporary hybridisation. PMID:29088249

High and uneven levels of 45S rDNA site-number variation across wild populations of a diploid plant genus (Anacyclus, Asteraceae).

PubMed

Rosato, Marcela; Álvarez, Inés; Nieto Feliner, Gonzalo; Rosselló, Josep A

2017-01-01

The nuclear genome harbours hundreds to several thousand copies of ribosomal DNA. Despite their essential role in cellular ribogenesis few studies have addressed intrapopulation, interpopulation and interspecific levels of rDNA variability in wild plants. Some studies have assessed the extent of rDNA variation at the sequence and copy-number level with large sampling in several species. However, comparable studies on rDNA site number variation in plants, assessed with extensive hierarchical sampling at several levels (individuals, populations, species) are lacking. In exploring the possible causes for ribosomal loci dynamism, we have used the diploid genus Anacyclus (Asteraceae) as a suitable system to examine the evolution of ribosomal loci. To this end, the number and chromosomal position of 45S rDNA sites have been determined in 196 individuals from 47 populations in all Anacyclus species using FISH. The 45S rDNA site-number has been assessed in a significant sample of seed plants, which usually exhibit rather consistent features, except for polyploid plants. In contrast, the level of rDNA site-number variation detected in Anacyclus is outstanding in the context of angiosperms particularly regarding populations of the same species. The number of 45S rDNA sites ranged from four to 11, accounting for 14 karyological ribosomal phenotypes. Our results are not even across species and geographical areas, and show that there is no clear association between the number of 45S rDNA loci and the life cycle in Anacyclus. A single rDNA phenotype was detected in several species, but a more complex pattern that included intra-specific and intra-population polymorphisms was recorded in A. homogamos, A. clavatus and A. valentinus, three weedy species showing large and overlapping distribution ranges. It is likely that part of the cytogenetic changes and inferred dynamism found in these species have been triggered by genomic rearrangements resulting from contemporary hybridisation.

Soil Bacterial Community Shift Correlated with Change from Forest to Pasture Vegetation in a Tropical Soil

PubMed Central

Nüsslein, Klaus; Tiedje, James M.

1999-01-01

The change in vegetative cover of a Hawaiian soil from forest to pasture led to significant changes in the composition of the soil bacterial community. DNAs were extracted from both soil habitats and compared for the abundance of guanine-plus-cytosine (G+C) content, by analysis of abundance of phylotypes of small-subunit ribosomal DNA (SSU rDNA) amplified from fractions with 63 and 35% G+C contents, and by phylogenetic analysis of the dominant rDNA clones in the 63% G+C content fraction. All three methods showed differences between the forest and pasture habitats, providing evidence that vegetation had a strong influence on microbial community composition at three levels of taxon resolution. The forest soil DNA had a peak in G+C content of 61%, while the DNA of the pasture soil had a peak in G+C content of 67%. None of the dominant phylotypes found in the forest soil were detected in the pasture soil. For the 63% G+C fraction SSU rDNA sequence analysis of the three most dominant members revealed that their phyla changed from Fibrobacter and Syntrophomonas assemblages in the forest soil to Burkholderia and Rhizobium–Agrobacterium assemblages in the pasture soil. PMID:10427058

Rubrobacter-related bacteria associated with rosy discolouration of masonry and lime wall paintings.

PubMed

Schabereiter-Gurtner, C; Piñar, G; Vybiral, D; Lubitz, W; Rölleke, S

2001-11-01

A molecular approach was chosen to analyse the correlation between bacterial colonisation and rosy discolouration of masonry and lime wall paintings of two historically important buildings in Austria and Germany. The applied molecular method included PCR amplification of genes encoding the small subunit rRNA of bacteria (16S rDNA), genetic fingerprinting by denaturing gradient gel electrophoresis (DGGE), construction of 16S rDNA clone libraries, and comparative phylogenetic sequence analyses. The bacterial community of one red-pigmented biofilm sampled in Herberstein (Austria) contained bacteria phylogenetically related to the genera Saccharopolyspora, Nocardioides, Pseudonocardia, Rubrobacter, and to a Kineococcus-like bacterium. The bacterial community of the second red-pigmented biofilm sampled in Herberstein contained bacteria related to Arthrobacter, Comamonas, and to Rubrobacter. Rubrobacter-related 16S rDNA sequences were the most abundant. In the red-pigmented biofilm sampled in Burggen (Germany), only Rubrobacter-related bacteria were identified. No Rubrobacter-related bacteria were detected in non-rosy biofilms. The majority of sequences (70%) obtained from the bacterial communities of the three investigated rosy biofilms were related to sequences of the genus Rubrobacter (red-pigmented bacteria), demonstrating a correlation between Rubrobacter-related bacteria and the phenomenon of rosy discolouration of masonry and lime wall paintings.

New data on two remarkable Antarctic species Amblydorylaimus isokaryon (Loof, 1975) Andrássy, 1998 and Pararhyssocolpus paradoxus (Loof, 1975), gen. n., comb. n. (Nematoda, Dorylaimida)

PubMed Central

Elshishka, Milka; Lazarova, Stela; Radoslavov, Georgi; Hristov, Petar; Peneva, Vlada K.

2015-01-01

Abstract The taxonomic position of two antarctic dorylaimid species Amblydorylaimus isokaryon (Loof, 1975) Andrássy, 1998 and Pararhyssocolpus paradoxus (Loof, 1975), gen. n., comb. n. are discussed on the basis of morphological, including SEM study, morphometric, postembryonic and sequence data of 18S rDNA and the D2-D3 expansion fragments of large subunit rDNA. The evolutionary trees inferred from 18S sequences show insufficient resolution to determine the assignment of the two species to particular families, moreover Pararhyssocolpus paradoxus gen. n., comb. n. (=Rhyssocolpus paradoxus) previously regarded as a member of Nordiidae or Qudsianematidae, showed distant relationship both to Rhyssocolpus vinciguerrae and Eudorylaimus spp. The phylogram inferred from 28S sequences revealed that Amblydorylaimus isokaryon is a member of a well-supported group comprised of several Aporcelaimellus spp., while, no close relationships could be revealed for the Pararhyssocolpus paradoxus gen. n., comb. n. to any nematode genus. On the basis of molecular data and morphological characteristics, some taxonomic changes are proposed. Amblydorylaimus isokaryon is transferred from family Qudsianematidae to family Aporcelaimidae, and a new monotypic genus Pararhyssocolpus gen. n. is proposed, attributed to Pararhyssocolpidae fam. n. The diagnosis of the new family is provided together with emended diagnosis of the genera Amblydorylaimus and Pararhyssocolpus gen. n. Data concerning distribution of these endemic genera in the Antarctic region are also given. PMID:26257550

Molecular phylogenetics of Floridosentis ward, 1953 (Acanthocephala: Neoechinorhynchidae) parasites of mullets (Osteichthyes) from Mexico, using 28S rDNA sequences.

PubMed

Rosas-Valdez, Rogelio; Morrone, Juan J; García-Varela, Martín

2012-08-01

Species of Floridosentis (Acanthocephala) are common parasites of mullets (Mugil spp., Mugilidae) found in tropical marine and brackish water in the Americas. Floridosentis includes 2 species distributed in Mexico, i.e., Floridosentis pacifica, restricted to the Pacific Ocean near Salina Cruz, Oaxaca, and Floridosentis mugilis, distributed along the coast of the Pacific Ocean and the Gulf of Mexico. We sampled 18 populations of F. mugilis and F. pacifica (12 from the Pacific and 6 from the Gulf of Mexico) and sequenced a fragment of the rDNA large subunit to evaluate phylogenetic relationships of populations of Floridosentis spp. from Mexico. Species identification of museum specimens of F. mugilis from the Pacific Ocean was confirmed by examination of morphology traits. Phylogenetic trees inferred with maximum parsimony, maximum likelihood, and Bayesian inference indicate that Floridosentis is monophyletic comprising of 2 major well-supported clades, the first clade corresponding to F. mugilis from the Gulf of Mexico, and the second to F. pacifica from the Pacific Ocean. Genetic divergence between species ranged from 7.68 to 8.60%. Intraspecific divergence ranged from 0.14 to 0.86% for F. mugilis and from 1.72 to 4.49% for F. pacifica. Data obtained from diagnostic characters indicate that specimens from the Pacific Ocean in Mexico have differences in some traits among locations. These results are consistent with the phylogenetic hypothesis, indicating that F. pacifica is distributed in the Pacific Ocean in Mexico with 3 major lineages.

Diversity of bats trypanosomes in hydroeletric area of Belo Monte in Brazilian Amazonia.

PubMed

da Costa, Andréa P; Nunes, Pablo Henrique; Leite, Beatriz Helena Santos; Ferreira, Juliana Isabel G da S; Tonhosolo, Renata; da Rosa, Adriana Ruckert; da Rocha, Patricio Adriano; Aires, Caroline Cotrim; Gennari, Solange Maria; Marcili, Arlei

2016-12-01

The Trypanosoma comprises flagellates able to infect many mammalian species and is transmitted by several groups of invertebrates. The order Chiroptera can be infected by the subgenera Herpetosoma, Schizotrypanum, Megatrypanum and Trypanozoon. In this study, we described the diversity of bats trypanosomes, inferring the phylogenetic relationships among the trypanosomes from bats caught Belo Monte Hydroeletric area (Brazilian Amazonia). Trypanosomes from bats were isolated by haemoculture, and the molecular phylogeny based on small subunit rDNA (SSU rDNA) and glycosomal-3-phosphate dehydrogenase (gGAPDH) gene sequences. Morphological characterization included light and scanning electron microscopy. A total of 157 bats were caught in the area belonging 6 Families (Emballonuridae, Furipteridae, Mormoopidae, Natalidae, Phyllostomidae and Vespertilionidae) and 34 species. The bat trypanosome prevalence, as evaluated through haemoculture, was 5,7%. Phylogenetic trees grouped the isolates in T. cruzi branch (TCI and TCbat lineage), T. cruzi marinkellei and Trypanosoma wauwau from Pteronotus parnellii. This is the first isolate from T. wauwau in Para state. The occurrence of T. cruzi in the ​​ Belo Monte Hydroeletric area (UHE Belo Monte) in Amazon/Brazil attentive to the risk of migration human population required for the works of the dam and new cities that grow in the vicinity of these businesses, but it is a zoonosis already known to the Amazon region, and the presence of unclassified Trypanosoma species, attend to the large parasitic biodiversity still unknown. Copyright © 2016 Elsevier B.V. All rights reserved.

Islandinium minutum subsp. barbatum subsp. nov. (Dinoflagellata), a New Organic-Walled Dinoflagellate Cyst from the Western Arctic: Morphology, Phylogenetic Position Based on SSU rDNA and LSU rDNA, and Distribution.

PubMed

Potvin, Éric; Kim, So-Young; Yang, Eun Jin; Head, Martin J; Kim, Hyun-Cheol; Nam, Seung-Il; Yim, Joung Han; Kang, Sung-Ho

2018-03-25

A study of modern sediment from the Western Arctic has revealed the presence of a distinctive brown-colored cyst with a spherical central body bearing unbranched processes that are usually solid with a small basal pericoel. Distinctive barbs project from some processes, and process tips are usually minutely expanded into conjoined barbs. The archeopyle is apical and saphopylic. This cyst corresponds to Islandinium? cezare morphotype 2 of Head et al. (2001, J. Quat. Sci., 16:621). Phylogenetic analyses based on the small and large subunit rRNA genes infer close relationship with Islandinium minutum, the type of which is that of the genus. Re-examination of specimens of I. minutum reveals the presence of minute barbs on its processes, but differences with Islandinium? cezare morphotype 2 remain based on size, process distribution, and barb development. Furthermore, the internal transcribed spacer shows I. minutum to be distinct from this morphotype. On the basis of these small but discrete differences, we propose the new subspecies Islandinium minutum subsp. barbatum subsp. nov. Molecular sequencing of other cysts encountered, namely Echinidinium karaense, an unidentified flattened cyst, and "Polykrikos quadratus", places them in the Monovela clade, the latter showing greater morphological variability than previously thought. © 2018 The Author(s) Journal of Eukaryotic Microbiology © 2018 International Society of Protistologists.

Phylogenetic position of parabasalid symbionts from the termite Calotermes flavicollis based on small subunit rRNA sequences.

PubMed

Gerbod, D; Edgcomb, V P; Noël, C; Delgado-Viscogliosi, P; Viscogliosi, E

2000-09-01

Small subunit rDNA genes were amplified by polymerase chain reaction using specific primers from mixed-population DNA obtained from the whole hindgut of the termite Calotermes flavicollis. Comparative sequence analysis of the clones revealed two kinds of sequences that were both from parabasalid symbionts. In a molecular tree inferred by distance, parsimony and likelihood methods, and including 27 parabasalid sequences retrieved from the data bases, the sequences of the group II (clones Cf5 and Cf6) were closely related to the Devescovinidae/Calonymphidae species and thus were assigned to the Devescovinidae Foaina. The sequence of the group I (clone Cf1) emerged within the Trichomonadinae and strongly clustered with Tetratrichomonas gallinarum. On the basis of morphological data, the Monocercomonadidae Hexamastix termitis might be the most likely origin of this sequence.

Molecular phylogeny of Pasiphaeidae (Crustacea, Decapoda, Caridea) reveals systematic incongruence of the current classification.

PubMed

Liao, Yunshi; De Grave, Sammy; Ho, Tsz Wai; Ip, Brian H Y; Tsang, Ling Ming; Chan, Tin-Yam; Chu, Ka Hou

2017-10-01

Caridean shrimps constitute one of the most diverse groups of decapod crustaceans, notwithstanding their poorly resolved infraordinal relationships. One of the systematically controversial families in Caridea is the predominantly pelagic Pasiphaeidae, comprises 101 species in seven genera. Pasiphaeidae species exhibit high morphological disparity, as well as ecological niche width, inhabiting shallow to very deep waters (>4000m). The present work presents the first molecular phylogeny of the family, based on a combined dataset of six mitochondrial and nuclear gene markers (12S rDNA, 16S rDNA, histone 3, sodium-potassium ATPase α-subunit, enolase and ATP synthase β-subunit) from 33 species belonged to six genera of Pasiphaeidae with 19 species from 12 other caridean families as outgroup taxa. Maximum likelihood and Bayesian inference analyses conducted on the concatenated dataset of 2265bp suggest the family Pasiphaeidae is not monophyletic, with Psathyrocaris more closely related to other carideans than to the other five pasiphaeid genera included in this analysis. Leptochela occupies a sister position to the remaining genera and is genetically quite distant from them. At the generic level, the analysis supports the monophyly of Pasiphaea, Leptochela and Psathyrocaris, while Eupasiphae is shown to be paraphyletic, closely related to Parapasiphae and Glyphus. The present molecular result strongly implies that certain morphological characters used in the present systematic delineation within Pasiphaeidae may not be synapomorphies and the classification within the family needs to be urgently revised. Copyright © 2017 Elsevier Inc. All rights reserved.

Multigene phylogeny of land plants with special reference to bryophytes and the earliest land plants.

PubMed

Nickrent, D L; Parkinson, C L; Palmer, J D; Duff, R J

2000-12-01

A widely held view of land plant relationships places liverworts as the first branch of the land plant tree, whereas some molecular analyses and a cladistic study of morphological characters indicate that hornworts are the earliest land plants. To help resolve this conflict, we used parsimony and likelihood methods to analyze a 6, 095-character data set composed of four genes (chloroplast rbcL and small-subunit rDNA from all three plant genomes) from all major land plant lineages. In all analyses, significant support was obtained for the monophyly of vascular plants, lycophytes, ferns (including PSILOTUM: and EQUISETUM:), seed plants, and angiosperms. Relationships among the three bryophyte lineages were unresolved in parsimony analyses in which all positions were included and weighted equally. However, in parsimony and likelihood analyses in which rbcL third-codon-position transitions were either excluded or downweighted (due to apparent saturation), hornworts were placed as sister to all other land plants, with mosses and liverworts jointly forming the second deepest lineage. Decay analyses and Kishino-Hasegawa tests of the third-position-excluded data set showed significant support for the hornwort-basal topology over several alternative topologies, including the commonly cited liverwort-basal topology. Among the four genes used, mitochondrial small-subunit rDNA showed the lowest homoplasy and alone recovered essentially the same topology as the multigene tree. This molecular phylogeny presents new opportunities to assess paleontological evidence and morphological innovations that occurred during the early evolution of terrestrial plants.

Characteristics of the nuclear (18S, 5.8S, 28S and 5S) and mitochondrial (12S and 16S) rRNA genes of Apis mellifera (Insecta: Hymenoptera): structure, organization, and retrotransposable elements

PubMed Central

Gillespie, J J; Johnston, J S; Cannone, J J; Gutell, R R

2006-01-01

As an accompanying manuscript to the release of the honey bee genome, we report the entire sequence of the nuclear (18S, 5.8S, 28S and 5S) and mitochondrial (12S and 16S) ribosomal RNA (rRNA)-encoding gene sequences (rDNA) and related internally and externally transcribed spacer regions of Apis mellifera (Insecta: Hymenoptera: Apocrita). Additionally, we predict secondary structures for the mature rRNA molecules based on comparative sequence analyses with other arthropod taxa and reference to recently published crystal structures of the ribosome. In general, the structures of honey bee rRNAs are in agreement with previously predicted rRNA models from other arthropods in core regions of the rRNA, with little additional expansion in non-conserved regions. Our multiple sequence alignments are made available on several public databases and provide a preliminary establishment of a global structural model of all rRNAs from the insects. Additionally, we provide conserved stretches of sequences flanking the rDNA cistrons that comprise the externally transcribed spacer regions (ETS) and part of the intergenic spacer region (IGS), including several repetitive motifs. Finally, we report the occurrence of retrotransposition in the nuclear large subunit rDNA, as R2 elements are present in the usual insertion points found in other arthropods. Interestingly, functional R1 elements usually present in the genomes of insects were not detected in the honey bee rRNA genes. The reverse transcriptase products of the R2 elements are deduced from their putative open reading frames and structurally aligned with those from another hymenopteran insect, the jewel wasp Nasonia (Pteromalidae). Stretches of conserved amino acids shared between Apis and Nasonia are illustrated and serve as potential sites for primer design, as target amplicons within these R2 elements may serve as novel phylogenetic markers for Hymenoptera. Given the impending completion of the sequencing of the Nasonia genome, we expect our report eventually to shed light on the evolution of the hymenopteran genome within higher insects, particularly regarding the relative maintenance of conserved rDNA genes, related variable spacer regions and retrotransposable elements. PMID:17069639

Diversity of mitochondrial large subunit rDNA haplotypes of Glomus intraradices in two agricultural field experiments and two semi-natural grasslands.

PubMed

Börstler, Boris; Thiéry, Odile; Sýkorová, Zuzana; Berner, Alfred; Redecker, Dirk

2010-04-01

Glomus intraradices, an arbuscular mycorrhizal fungus (AMF), is frequently found in a surprisingly wide range of ecosystems all over the world. It is used as model organism for AMF and its genome is being sequenced. Despite the ecological importance of AMF, little has been known about their population structure, because no adequate molecular markers have been available. In the present study we analyse for the first time the intraspecific genetic structure of an AMF directly from colonized roots in the field. A recently developed PCR-RFLP approach for the mitochondrial rRNA large subunit gene (mtLSU) of these obligate symbionts was used and complemented by sequencing and primers specific for a particularly frequent mtLSU haplotype. We analysed root samples from two agricultural field experiments in Switzerland and two semi-natural grasslands in France and Switzerland. RFLP type composition of G. intraradices (phylogroup GLOM A-1) differed strongly between agricultural and semi-natural sites and the G. intraradices populations of the two agricultural sites were significantly differentiated. RFLP type richness was higher in the agricultural sites compared with the grasslands. Detailed sequence analyses which resolved multiple sequence haplotypes within some RFLP types even revealed that there was no overlap of haplotypes among any of the study sites except between the two grasslands. Our results demonstrate a surprisingly high differentiation among semi-natural and agricultural field sites for G. intraradices. These findings will have major implications on our views of processes of adaptation and specialization in these plant/fungus associations.

Cryphodera sinensis n. sp. (Nematoda: Heteroderidae), a non-cyst-forming parasitic nematode from the root of ramie Boehmeria nivea in China.

PubMed

Zhuo, K; Wang, H H; Ye, W; Peng, D L; Liao, J L

2014-12-01

Cryphodera sinensis n. sp. is described from ramie (Boehmeria nivea) based on the morphology and molecular analyses of rRNA small subunit (SSU), D2D3 expansion domains of large subunit (LSU D2D3) and internal transcribed spacer (ITS). This new species is characterized by oval females with a distinct subcrystalline layer and pronounced and protruding vulval lip, distinctly concave vulva-anus profile and a vulva-anus distance of 29.5-35.8 μm. Males possess two annuli in the lip region, a stylet 27-32.5 μm in length with round knobs sloping slightly posteriorly, lateral fields with three lines, spicules 20-28 μm long and the presence of a short cloacal tube. Second-stage juveniles possess three lip annuli, a stylet 28-31 μm in length with well-developed knobs projected anteriorly and three lines along the lateral field. The pointed tail, 52-65 μm long, possesses a mucro-like tip and a hyaline region, 24.5-35 μm long. Large phasmids with a lens-like structure are located 2-6 annuli posterior to the anus. Phylogenetic analysis shows that the species has unique SSU, LSU D2D3 and ITS rRNA sequences. Phylogenetic relationships of the three rDNA sequences of C. sinensis n. sp. and other cystoid/cyst nematodes are analysed together with a comparison of other species within the genus Cryphodera.

Novel division level bacterial diversity in a Yellowstone hot spring.

PubMed

Hugenholtz, P; Pitulle, C; Hershberger, K L; Pace, N R

1998-01-01

A culture-independent molecular phylogenetic survey was carried out for the bacterial community in Obsidian Pool (OP), a Yellowstone National Park hot spring previously shown to contain remarkable archaeal diversity (S. M. Barns, R. E. Fundyga, M. W. Jeffries, and N. R. Page, Proc. Natl. Acad. Sci. USA 91:1609-1613, 1994). Small-subunit rRNA genes (rDNA) were amplified directly from OP sediment DNA by PCR with universally conserved or Bacteria-specific rDNA primers and cloned. Unique rDNA types among > 300 clones were identified by restriction fragment length polymorphism, and 122 representative rDNA sequences were determined. These were found to represent 54 distinct bacterial sequence types or clusters (> or = 98% identity) of sequences. A majority (70%) of the sequence types were affiliated with 14 previously recognized bacterial divisions (main phyla; kingdoms); 30% were unaffiliated with recognized bacterial divisions. The unaffiliated sequence types (represented by 38 sequences) nominally comprise 12 novel, division level lineages termed candidate divisions. Several OP sequences were nearly identical to those of cultivated chemolithotrophic thermophiles, including the hydrogen-oxidizing Calderobacterium and the sulfate reducers Thermodesulfovibrio and Thermodesulfobacterium, or belonged to monophyletic assemblages recognized for a particular type of metabolism, such as the hydrogen-oxidizing Aquificales and the sulfate-reducing delta-Proteobacteria. The occurrence of such organisms is consistent with the chemical composition of OP (high in reduced iron and sulfur) and suggests a lithotrophic base for primary productivity in this hot spring, through hydrogen oxidation and sulfate reduction. Unexpectedly, no archaeal sequences were encountered in OP clone libraries made with universal primers. Hybridization analysis of amplified OP DNA with domain-specific probes confirmed that the analyzed community rDNA from OP sediment was predominantly bacterial. These results expand substantially our knowledge of the extent of bacterial diversity and call into question the commonly held notion that Archaea dominate hydrothermal environments. Finally, the currently known extent of division level bacterial phylogenetic diversity is collated and summarized.

Introducing W.A.T.E.R.S.: a workflow for the alignment, taxonomy, and ecology of ribosomal sequences.

PubMed

Hartman, Amber L; Riddle, Sean; McPhillips, Timothy; Ludäscher, Bertram; Eisen, Jonathan A

2010-06-12

For more than two decades microbiologists have used a highly conserved microbial gene as a phylogenetic marker for bacteria and archaea. The small-subunit ribosomal RNA gene, also known as 16 S rRNA, is encoded by ribosomal DNA, 16 S rDNA, and has provided a powerful comparative tool to microbial ecologists. Over time, the microbial ecology field has matured from small-scale studies in a select number of environments to massive collections of sequence data that are paired with dozens of corresponding collection variables. As the complexity of data and tool sets have grown, the need for flexible automation and maintenance of the core processes of 16 S rDNA sequence analysis has increased correspondingly. We present WATERS, an integrated approach for 16 S rDNA analysis that bundles a suite of publicly available 16 S rDNA analysis software tools into a single software package. The "toolkit" includes sequence alignment, chimera removal, OTU determination, taxonomy assignment, phylogentic tree construction as well as a host of ecological analysis and visualization tools. WATERS employs a flexible, collection-oriented 'workflow' approach using the open-source Kepler system as a platform. By packaging available software tools into a single automated workflow, WATERS simplifies 16 S rDNA analyses, especially for those without specialized bioinformatics, programming expertise. In addition, WATERS, like some of the newer comprehensive rRNA analysis tools, allows researchers to minimize the time dedicated to carrying out tedious informatics steps and to focus their attention instead on the biological interpretation of the results. One advantage of WATERS over other comprehensive tools is that the use of the Kepler workflow system facilitates result interpretation and reproducibility via a data provenance sub-system. Furthermore, new "actors" can be added to the workflow as desired and we see WATERS as an initial seed for a sizeable and growing repository of interoperable, easy-to-combine tools for asking increasingly complex microbial ecology questions.

The Nucleolus: In Genome Maintenance and Repair

PubMed Central

Tsekrekou, Maria; Stratigi, Kalliopi; Chatzinikolaou, Georgia

2017-01-01

The nucleolus is the subnuclear membrane-less organelle where rRNA is transcribed and processed and ribosomal assembly occurs. During the last 20 years, however, the nucleolus has emerged as a multifunctional organelle, regulating processes that go well beyond its traditional role. Moreover, the unique organization of rDNA in tandem arrays and its unusually high transcription rates make it prone to unscheduled DNA recombination events and frequent RNA:DNA hybrids leading to DNA double strand breaks (DSBs). If not properly repaired, rDNA damage may contribute to premature disease onset and aging. Deregulation of ribosomal synthesis at any level from transcription and processing to ribosomal subunit assembly elicits a stress response and is also associated with disease onset. Here, we discuss how genome integrity is maintained within nucleoli and how such structures are functionally linked to nuclear DNA damage response and repair giving an emphasis on the newly emerging roles of the nucleolus in mammalian physiology and disease. PMID:28671574

Paratylenchus shenzhenensis n. sp. (Nematoda: Paratylenchinae) from the rhizosphere soil of Anthurium andraeanum in China.

PubMed

Wang, Ke; Xie, Hui; Li, Yu; Xu, Chun-Ling; Yu, Lu; Wang, Dong-Wei

2013-12-18

Paratylenchus shenzhenensis n. sp. was collected from the rhizosphere soil of Anthurium andraeanum in Shenzhen, Guangdong Province, China. The new species is characterized by having a female with a small body (249-302 μm), well developed stylet (17-21 μm), rounded head with four submedian lobes and lip-region with a slight depression at the oral area, small post-vulval uterine sac with a few vestigial cells; male with body dorsally curved behind the cloacal opening, stylet absent, pharynx degenerate, prominent penial sheath; and juveniles with a stylet. It is morphologically similar to P. minutus. The internal transcribed spacer sequences of ribosomal DNA (ITS-rDNA) of the new species only have 72-73% identity with P. minutus, confirming its status as a separate species. The D2/D3 region of 28S ribosomal DNA (28S rDNA) and 18S small subunit ribosomal DNA (18S rDNA) from P. shenzhenensis n. sp. were also amplified and sequenced in this study. 

Aquatic tetrasporoblastic microsporidia from caddis flies (Insecta, Trichoptera): characterisation, phylogeny and taxonomic reevaluation of the genera Episeptum Larsson, 1986, Pyrotheca Hesse, 1935 and Cougourdella Hesse, 1935.

PubMed

Hylis, Miroslav; Oborník, Miroslav; Nebesárová, Jana; Vávra, Jirí

2007-08-01

Seven microsporidian species infecting caddis fly larvae, corresponding to conventional genera Episeptum, Pyrotheca and Cougourdella were studied using light and electron microscopy. Parts of their small subunit, ITS and large subunit ribosomal RNA genes were sequenced and compared with sequences of rDNA obtained from syntype slides of Cougourdella polycentropi Weiser 1965 and Pyrotheca sp. from Hydropsyche pellucidula. All studied caddis fly microsporidia form a closely related group. Their developmental stages in trichopteran hosts are restricted to fat body cells and oenocytes and have isolated nuclei. In late merogony, uninucleate meronts and binucleate plasmodia are formed. In sporogony a sporogonial plasmodium with four nuclei gives rise by rosette-like budding to four sporoblasts within a non-persistent sporophorous vesicle. Sporoblasts mature into pyriform to lageniform spores. The shape and size of spores, the number of polar filament coils, the structure of the polaroplast and of the exospore, together with morphometric characters present a set of markers unique for respective species. Four new species are established. The new genus Paraepiseptum is proposed to replace the tetrasporoblastic Pyrotheca and Cougourdella species from caddis flies. The genus Episeptum is redefined. Field and laboratory examinations as well as the phylogenetic position within the aquatic clade of microsporidia suggest that the life cycle of trichopteran microsporidia probably involves an alternate (copepod?) host and (or) transovarial transmission.

A TBP-containing multiprotein complex (TIF-IB) mediates transcription specificity of murine RNA polymerase I.

PubMed

Eberhard, D; Tora, L; Egly, J M; Grummt, I

1993-09-11

TIF-IB is a transcription factor which interacts with the mouse ribosomal gene promoter and nucleates the formation of an initiation complex containing RNA polymerase I (Pol I). We have purified this factor to near homogeneity and demonstrate that TIF-IB is a large complex (< 200 kDa) which contains several polypeptides. One of the subunits present in this protein complex is the TATA-binding protein (TBP) as revealed by copurification of TIF-IB activity and TBP over different chromatographic steps including immunoaffinity purification. In addition to TBP, three tightly associated proteins (TAFs-I) with apparent molecular weights of 95, 68, and 48 kDa are contained in this multimeric complex. This subunit composition is similar--but not identical--to the analogous human factor SL1. Depletion of TBP from TIF-IB-containing fractions by immunoprecipitation eliminates TIF-IB activity. Neither TBP alone nor fractions containing other TBP complexes are capable of substituting for TIF-IB activity. Therefore, TIF-IB is a unique complex with Pol I-specific TAFs distinct from other TBP-containing complexes. The identification of TBP as an integral part of the murine rDNA promoter-specific transcription initiation factor extends the previously noted similarity of transcriptional initiation by the three nuclear RNA polymerases and underscores the importance of TAFs in determining promoter specificity.

A TBP-containing multiprotein complex (TIF-IB) mediates transcription specificity of murine RNA polymerase I.

PubMed Central

Eberhard, D; Tora, L; Egly, J M; Grummt, I

1993-01-01

TIF-IB is a transcription factor which interacts with the mouse ribosomal gene promoter and nucleates the formation of an initiation complex containing RNA polymerase I (Pol I). We have purified this factor to near homogeneity and demonstrate that TIF-IB is a large complex (< 200 kDa) which contains several polypeptides. One of the subunits present in this protein complex is the TATA-binding protein (TBP) as revealed by copurification of TIF-IB activity and TBP over different chromatographic steps including immunoaffinity purification. In addition to TBP, three tightly associated proteins (TAFs-I) with apparent molecular weights of 95, 68, and 48 kDa are contained in this multimeric complex. This subunit composition is similar--but not identical--to the analogous human factor SL1. Depletion of TBP from TIF-IB-containing fractions by immunoprecipitation eliminates TIF-IB activity. Neither TBP alone nor fractions containing other TBP complexes are capable of substituting for TIF-IB activity. Therefore, TIF-IB is a unique complex with Pol I-specific TAFs distinct from other TBP-containing complexes. The identification of TBP as an integral part of the murine rDNA promoter-specific transcription initiation factor extends the previously noted similarity of transcriptional initiation by the three nuclear RNA polymerases and underscores the importance of TAFs in determining promoter specificity. Images PMID:8414971

Novel Curvularia species from clinical specimens.

PubMed

Madrid, H; da Cunha, K C; Gené, J; Dijksterhuis, J; Cano, J; Sutton, D A; Guarro, J; Crous, P W

2014-12-01

The fungal genus Curvularia includes numerous plant pathogens and some emerging opportunistic pathogens of humans. In a previous study we used morphology and sequences of the nuclear ribosomal internal transcribed spacer region (ITS) and the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene to identify species within a set of 99 clinical Curvularia isolates from the USA. Seventy-two isolates could be identified while the remaining 27 isolates belonged in three unclassified clades that were tentatively labelled Curvularia sp. I, II and III. In the present study, we further assess the taxonomic placement of these isolates using sequences of ITS, gpd, the large subunit rDNA, and the second largest subunit of RNA polymerase II. DNA sequence comparisons with a set of 87 isolates representing 33 Curvularia spp. and members of the closely-related genera Bipolaris and Exserohilum revealed that Curvularia sp. I, II and III represent novel lineages in Curvularia. These lineages are morphologically different from the currently accepted species. In the phylogenetic tree, Curvularia sp. I and sp. III were each split into two distinct lineages. Morphology and phylogeny supported the proposal of five new species, to be named C. americana, C. chlamydospora, C. hominis, C. muehlenbeckiae and C. pseudolunata. The concatenated 4-locus phylogeny revealed the existence of six clades in Curvularia, which are associated with particular morphological features. They were named after representative species, namely americana, eragrostidis, hominis, lunata, spicifera and trifolii.

Diverse tulasnelloid fungi form mycorrhizas with epiphytic orchids in an Andean cloud forest.

PubMed

Suárez, Juan Pablo; Weiss, Michael; Abele, Andrea; Garnica, Sigisfredo; Oberwinkler, Franz; Kottke, Ingrid

2006-11-01

The mycorrhizal state of epiphytic orchids has been controversially discussed, and the state and mycobionts of the pleurothallid orchids, occurring abundantly and with a high number of species on stems of trees in the Andean cloud forest, were unknown. Root samples of 77 adult individuals of the epiphytic orchids Stelis hallii, S. superbiens, S. concinna and Pleurothallis lilijae were collected in a tropical mountain rainforest of southern Ecuador. Ultrastructural evidence of symbiotic interaction was combined with molecular sequencing of fungi directly from the mycorrhizas and isolation of mycobionts. Ultrastructural analyses displayed vital orchid mycorrhizas formed by fungi with an imperforate parenthesome and cell wall slime bodies typical for the genus Tulasnella. Three different Tulasnella isolates were obtained in pure culture. Phylogenetic analysis of nuclear rDNA sequences from coding regions of the ribosomal large subunit (nucLSU) and the 5.8S subunit, including parts of the internal transcribed spacers, obtained directly from the roots and from the fungal isolates, yielded seven distinct Tulasnella clades. Tulasnella mycobionts in Stelis concinna were restricted to two Tulasnella sequence types while the other orchids were associated with up to six Tulasnella sequence types. All Tulasnella sequences are new to science and distinct from known sequences of mycobionts of terrestrial orchids. The results indicate that tulasnelloid fungi, adapted to the conditions on tree stems, might be important for orchid growth and maintenance in the Andean cloud forest.

Characterization of three different clusters of 18S-26S ribosomal DNA genes in the sea urchin P. lividus: Genetic and epigenetic regulation synchronous to 5S rDNA.

PubMed

Bellavia, Daniele; Dimarco, Eufrosina; Caradonna, Fabio

2016-04-15

We previously reported the characterization 5S ribosomal DNA (rDNA) clusters in the common sea urchin Paracentrotus lividus and demonstrated the presence of DNA methylation-dependent silencing of embryo specific 5S rDNA cluster in adult tissue. In this work, we show genetic and epigenetic characterization of 18S-26S rDNA clusters in this specie. The results indicate the presence of three different 18S-26S rDNA clusters with different Non-Transcribed Spacer (NTS) regions that have different chromosomal localizations. Moreover, we show that the two largest clusters are hyper-methylated in the promoter-containing NTS regions in adult tissues, as in the 5S rDNA. These findings demonstrate an analogous epigenetic regulation in small and large rDNA clusters and support the logical synchronism in building ribosomes. In fact, all the ribosomal RNA genes must be synchronously and equally transcribed to perform their unique final product. Copyright © 2016 Elsevier B.V. All rights reserved.

Second-generation ethanol from non-detoxified sugarcane hydrolysate by a rotting wood isolated yeast strain.

PubMed

Bazoti, Suzana F; Golunski, Simone; Pereira Siqueira, Diego; Scapini, Thamarys; Barrilli, Évelyn T; Alex Mayer, Diego; Barros, Katharina O; Rosa, Carlos A; Stambuk, Boris U; Alves, Sérgio L; Valério, Alexsandra; de Oliveira, Débora; Treichel, Helen

2017-11-01

This work aims to evaluate the production of second-generation ethanol from sugarcane bagasse hydrolysate without acetic acid (inhibitor) detoxification. Three isolated yeast strains from lignocellulosic materials were evaluated, and one strain (UFFS-CE-3.1.2), identified using large subunit rDNA sequences as Wickerhamomyces sp., showed satisfactory results in terms of ethanol production without acetic acid removal. A Plackett-Burman design was used to evaluate the influence of hydrolysate composition and nutrients supplementation in the fermentation medium for the second-generation ethanol production. Two fermentation kinetics were performed, with controlled pH at 5.5, or keeping the initial pH at 4.88. The fermentation conducted without pH adjustment and supplementation of nutrients reported the best result in terms of second-generation ethanol production. Wickerhamomyces sp., isolated as UFFS-CE-3.1.2, was considered promising in the production of second-generation ethanol by using crude (non-detoxified) sugarcane hydrolysate. Copyright © 2017 Elsevier Ltd. All rights reserved.

New Entamoeba group in howler monkeys (Alouatta spp.) associated with parasites of reptiles.

PubMed

Villanueva-García, Claudia; Gordillo-Chávez, Elías José; Baños-Ojeda, Carlos; Rendón-Franco, Emilio; Muñoz-García, Claudia Irais; Carrero, Julio César; Córdoba-Aguilar, Alex; Maravilla, Pablo; Galian, José; Martínez-Hernández, Fernando; Villalobos, Guiehdani

2017-08-01

Our knowledge of the parasite species present in wildlife hosts is incomplete. Protozoans such as amoebae of the genus Entamoeba infect a large variety of vertebrate species, including NHPs. However, traditionally, their identification has been accomplished through microscopic evaluation; therefore, amoeba species have not always been identified correctly. We searched for Entamoeba spp. using a fragment of the small subunit rDNA in free-ranging howler monkeys (Alouatta palliata and A. pigra) from southeast Mexico. One hundred fifty five samples were collected, with 46 from A. palliata and 109 from A. pigra and 8 of the total samples were positive. We detected a new clade of Entamoeba, which was separated from other described species but closer to E. insolita, as well as an unnamed sequence typically found in iguana species with low shared identity values (<90%). We designated this new clade as conditional lineage 8 (CL8) and we have shown that members of this group are not exclusive to reptiles.

Development of PCR primers specific for the amplification and direct sequencing of gyrB genes from microbacteria, order Actinomycetales.

PubMed

Richert, Kathrin; Brambilla, Evelyne; Stackebrandt, Erko

2005-01-01

PCR primer sets were developed for the specific amplification and sequence analyses encoding the gyrase subunit B (gyrB) of members of the family Microbacteriaceae, class Actinobacteria. The family contains species highly related by 16S rRNA gene sequence analyses. In order to test if the gene sequence analysis of gyrB is appropriate to discriminate between closely related species, we evaluate the 16S rRNA gene phylogeny of its members. As the published universal primer set for gyrB failed to amplify the responding gene of the majority of the 80 type strains of the family, three new primer sets were identified that generated fragments with a composite sequence length of about 900 nt. However, the amplification of all three fragments was successful only in 25% of the 80 type strains. In this study, the substitution frequencies in genes encoding gyrase and 16S rDNA were compared for 10 strains of nine genera. The frequency of gyrB nucleotide substitution is significantly higher than that of the 16S rDNA, and no linear correlation exists between the similarities of both molecules among members of the Microbacteriaceae. The phylogenetic analyses using the gyrB sequences provide higher resolution than using 16S rDNA sequences and seem able to discriminate between closely related species.

Physical mapping of 5S and 18S ribosomal DNA in three species of Agave (Asparagales, Asparagaceae).

PubMed

Gomez-Rodriguez, Victor Manuel; Rodriguez-Garay, Benjamin; Palomino, Guadalupe; Martínez, Javier; Barba-Gonzalez, Rodrigo

2013-01-01

Agave Linnaeus, 1753 is endemic of America and is considered one of the most important crops in Mexico due to its key role in the country's economy. Cytogenetic analysis was carried out in Agave tequilana Weber, 1902 'Azul', Agave cupreata Trelease et Berger, 1915 and Agave angustifolia Haworth, 1812. The analysis showed that in all species the diploid chromosome number was 2n = 60, with bimodal karyotypes composed of five pairs of large chromosomes and 25 pairs of small chromosomes. Furthermore, different karyotypical formulae as well as a secondary constriction in a large chromosome pair were found in all species. Fluorescent in situ hybridization (FISH) was used for physical mapping of 5S and 18S ribosomal DNA (rDNA). All species analyzed showed that 5S rDNA was located in both arms of a small chromosome pair, while 18S rDNA was associated with the secondary constriction of a large chromosome pair. Data of FISH analysis provides new information about the position and number of rDNA loci and helps for detection of hybrids in breeding programs as well as evolutionary studies.

Physical mapping of 5S and 18S ribosomal DNA in three species of Agave (Asparagales, Asparagaceae)

PubMed Central

Gomez-Rodriguez, Victor Manuel; Rodriguez-Garay, Benjamin; Palomino, Guadalupe; Martínez, Javier; Barba-Gonzalez, Rodrigo

2013-01-01

Abstract Agave Linnaeus, 1753 is endemic of America and is considered one of the most important crops in Mexico due to its key role in the country’s economy. Cytogenetic analysis was carried out in Agave tequilana Weber, 1902 ‘Azul’, Agave cupreata Trelease et Berger, 1915 and Agave angustifolia Haworth, 1812. The analysis showed that in all species the diploid chromosome number was 2n = 60, with bimodal karyotypes composed of five pairs of large chromosomes and 25 pairs of small chromosomes. Furthermore, different karyotypical formulae as well as a secondary constriction in a large chromosome pair were found in all species. Fluorescent in situ hybridization (FISH) was used for physical mapping of 5S and 18S ribosomal DNA (rDNA). All species analyzed showed that 5S rDNA was located in both arms of a small chromosome pair, while 18S rDNA was associated with the secondary constriction of a large chromosome pair. Data of FISH analysis provides new information about the position and number of rDNA loci and helps for detection of hybrids in breeding programs as well as evolutionary studies. PMID:24260700

Conserved Organisation of 45S rDNA Sites and rDNA Gene Copy Number among Major Clades of Early Land Plants

PubMed Central

Rosato, Marcela; Kovařík, Aleš; Garilleti, Ricardo; Rosselló, Josep A.

2016-01-01

Genes encoding ribosomal RNA (rDNA) are universal key constituents of eukaryotic genomes, and the nuclear genome harbours hundreds to several thousand copies of each species. Knowledge about the number of rDNA loci and gene copy number provides information for comparative studies of organismal and molecular evolution at various phylogenetic levels. With the exception of seed plants, the range of 45S rDNA locus (encoding 18S, 5.8S and 26S rRNA) and gene copy number variation within key evolutionary plant groups is largely unknown. This is especially true for the three earliest land plant lineages Marchantiophyta (liverworts), Bryophyta (mosses), and Anthocerotophyta (hornworts). In this work, we report the extent of rDNA variation in early land plants, assessing the number of 45S rDNA loci and gene copy number in 106 species and 25 species, respectively, of mosses, liverworts and hornworts. Unexpectedly, the results show a narrow range of ribosomal locus variation (one or two 45S rDNA loci) and gene copies not present in vascular plant lineages, where a wide spectrum is recorded. Mutation analysis of whole genomic reads showed higher (3-fold) intragenomic heterogeneity of Marchantia polymorpha (Marchantiophyta) rDNA compared to Physcomitrella patens (Bryophyta) and two angiosperms (Arabidopsis thaliana and Nicotiana tomentosifomis) suggesting the presence of rDNA pseudogenes in its genome. No association between phylogenetic position, taxonomic adscription and the number of rDNA loci and gene copy number was found. Our results suggest a likely evolutionary rDNA stasis during land colonisation and diversification across 480 myr of bryophyte evolution. We hypothesise that strong selection forces may be acting against ribosomal gene locus amplification. Despite showing a predominant haploid phase and infrequent meiosis, overall rDNA homogeneity is not severely compromised in bryophytes. PMID:27622766

Prevalence and Genotype Distribution of Pneumocystis jirovecii in Cuban Infants and Toddlers with Whooping Cough

PubMed Central

Monroy-Vaca, Ernesto X.; de Armas, Yaxsier; Illnait-Zaragozí, María T.; Toraño, Gilda; Diaz, Raúl; Vega, Dania; Alvarez-Lam, Ileana; Calderón, Enrique J.

2014-01-01

This study describes the prevalence and genotype distribution of Pneumocystis jirovecii obtained from nasopharyngeal (NP) swabs from immunocompetent Cuban infants and toddlers with whooping cough (WC). A total of 163 NP swabs from 163 young Cuban children with WC who were admitted to the respiratory care units at two pediatric centers were studied. The prevalence of the organism was determined by a quantitative PCR (qPCR) assay targeting the P. jirovecii mitochondrial large subunit (mtLSU) rRNA gene. Genotypes were identified by direct sequencing of mtLSU ribosomal DNA (rDNA) and restriction fragment length polymorphism (RFLP) analysis of the dihydropteroate synthase (DHPS) gene amplicons. qPCR detected P. jirovecii DNA in 48/163 (29.4%) samples. mtLSU rDNA sequence analysis revealed the presence of three different genotypes in the population. Genotype 2 was most common (48%), followed in prevalence by genotypes 1 (23%) and 3 (19%); mixed-genotype infections were seen in 10% of the cases. RFLP analysis of DHPS PCR products revealed four genotypes, 18% of which were associated with resistance to sulfa drugs. Only contact with coughers (prevalence ratio [PR], 3.51 [95% confidence interval {CI}, 1.79 to 6.87]; P = 0.000) and exposure to tobacco smoke (PR, 1.82 [95% CI, 1.14 to 2.92]; P = 0.009) were statistically associated with being colonized by P. jirovecii. The prevalence of P. jirovecii in infants and toddlers with WC and the genotyping results provide evidence that this population represents a potential reservoir and transmission source of P. jirovecii. PMID:24131683

Prevalence and genotype distribution of Pneumocystis jirovecii in Cuban infants and toddlers with whooping cough.

PubMed

Monroy-Vaca, Ernesto X; de Armas, Yaxsier; Illnait-Zaragozí, María T; Toraño, Gilda; Diaz, Raúl; Vega, Dania; Alvarez-Lam, Ileana; Calderón, Enrique J; Stensvold, Christen R

2014-01-01

This study describes the prevalence and genotype distribution of Pneumocystis jirovecii obtained from nasopharyngeal (NP) swabs from immunocompetent Cuban infants and toddlers with whooping cough (WC). A total of 163 NP swabs from 163 young Cuban children with WC who were admitted to the respiratory care units at two pediatric centers were studied. The prevalence of the organism was determined by a quantitative PCR (qPCR) assay targeting the P. jirovecii mitochondrial large subunit (mtLSU) rRNA gene. Genotypes were identified by direct sequencing of mtLSU ribosomal DNA (rDNA) and restriction fragment length polymorphism (RFLP) analysis of the dihydropteroate synthase (DHPS) gene amplicons. qPCR detected P. jirovecii DNA in 48/163 (29.4%) samples. mtLSU rDNA sequence analysis revealed the presence of three different genotypes in the population. Genotype 2 was most common (48%), followed in prevalence by genotypes 1 (23%) and 3 (19%); mixed-genotype infections were seen in 10% of the cases. RFLP analysis of DHPS PCR products revealed four genotypes, 18% of which were associated with resistance to sulfa drugs. Only contact with coughers (prevalence ratio [PR], 3.51 [95% confidence interval {CI}, 1.79 to 6.87]; P = 0.000) and exposure to tobacco smoke (PR, 1.82 [95% CI, 1.14 to 2.92]; P = 0.009) were statistically associated with being colonized by P. jirovecii. The prevalence of P. jirovecii in infants and toddlers with WC and the genotyping results provide evidence that this population represents a potential reservoir and transmission source of P. jirovecii.

Molecular characterization and histopathology of Myxobolus koi infecting the gills of a koi, Cyprinus carpio, with an amended morphological description of the agent.

PubMed

Camus, Alvin C; Griffin, Matt J

2010-02-01

A Myxobolus sp., morphologically resembling M. toyamai, M. longisporus, and M. koi, was isolated from the gills of a koi, Cyprinus carpio, that died in an ornamental pond. Large plasmodia were localized within lamellae, causing severe disruption of the normal branchial architecture, sufficient to compromise respiration. Although the case isolate shared several features with the aforementioned species, several key characteristics were most compatible with M. koi. In valvular view, spores were elongate and pyriform with a rounded posterior, 15.4 (14.5-16.5) microm long and 8.3 (7.1-9.0) microm wide. Polar capsules were pyriform and elongate, 10.1 (9.0-10.9) microm long and 3.1 (2.5-3.5) microm wide. Polar filaments were coiled perpendicular to the long axis of the spore making 10 turns (9-11). A BLAST search using a generated 18S SSU rDNA sequence resulted in no direct matches. Phylogenetic analysis of the sequence indicates a close relationship to M. longisporus and M. toyamai from C. carpio, within the clade of Myxobolus species infecting gills of cyprinid fishes (89% or greater bootstrap support by maximum parsimony and minimum evolution distance analysis). Although published descriptions are inconsistent, morphometric similarities to this isolate suggest a morphotype of M. koi. The description of M. koi is supplemented here with new data on pathogenicity and spore morphology and is characterized at the molecular level using 18S small subunit rDNA sequence data.

Molecular gene organisation and secondary structure of the mitochondrial large subunit ribosomal RNA from the cultivated Basidiomycota Agrocybe aegerita: a 13 kb gene possessing six unusual nucleotide extensions and eight introns.

PubMed

Gonzalez, P; Barroso, G; Labarère, J

1999-04-01

The complete gene sequence and secondary structure of the mitochondrial LSU rRNA from the cultivated Basidiomycota Agrocybe aegerita was derived by chromosome walking. The A.aegerita LSU rRNA gene (13 526 nt) represents, to date, the longest described, due to the highest number of introns (eight) and the occurrence of six long nucleotidic extensions. Seven introns belong to group I, while the intronic sequence i5 constitutes the first typical group II intron reported in a fungal mitochondrial LSU rDNA. As with most fungal LSU rDNA introns reported to date, four introns (i5-i8) are distributed in domain V associated with the peptidyl-transferase activity. One intron (i1) is located in domain I, and three (i2-i4) in domain II. The introns i2-i8 possess homologies with other fungal, algal or protozoan introns located at the same position in LSU rDNAs. One of them (i6) is located at the same insertion site as most Ascomycota or algae LSU introns, suggesting a possible inheritance from a common ancestor. On the contrary, intron i1 is located at a so-far unreported insertion site. Among the six unusual nucleotide extensions, five are located in domain I and one in domain V. This is the first report of a mitochondrial LSU rRNA gene sequence and secondary structure for the whole Basidiomycota division.

Biomphalaria straminea (Mollusca: Planorbidae) as an intermediate host of Drepanocephalus spp. (Trematoda: Echinostomatidae) in Brazil: a morphological and molecular study.

PubMed

Pinto, Hudson A; Griffin, Matt J; Quiniou, Sylvie M; Ware, Cynthia; Melo, Alan L

2016-01-01

Species of trematodes belonging to the genus Drepanocephalus are intestinal parasites of piscivorous birds, primarily cormorants (Phalachrocorax spp.), and are widely reported in the Americas. During a 4-year malacological study conducted on an urban lake in Brazil, 27-collar-spined echinostome cercariae were found in 1665/15,459 (10.7 %) specimens of Biomphalaria straminea collected. The cercariae were identified as Drepanocephalus spp. by sequencing the 18S (SSU) rDNA, ITS1/5.8S rDNA/ITS2 (ITS), 28S (LSU) rDNA region, cytochrome oxidase subunit 1 (CO1), and nicotinamide adenine dinucleotide dehydrogenase subunit 1 (ND1) markers. In experimental life cycle studies, metacercariae developed in laboratory-reared guppies (Poecilia reticulata); however, attempts to infect birds and rodents were unsuccessful. Two closely related morphotypes of cercariae were characterized. One species, identified by molecular markers as a genetic variant of Drepanocephalus auritus (99.9 % similarity at SSU, ITS, LSU; 97.2 % at CO1; 95.8 % at ND1), differs slightly from an archived North American isolate of this species also sequenced as part of this study. A second species, putatively identified as Drepanocephalus sp., has smaller cercariae and demonstrates significant differences from D. auritus at the CO1 (11.0 %) and ND1 (13.6 %) markers. Aspects related to the morphological taxonomic identification of 27-collar-spined echinostome metacercariae are briefly discussed. This is the first report of the involvement of molluscs of the genus Biomphalaria in the transmission of Drepanocephalus and the first report of D. auritus in South America.

Descolea quercina (Bolbitiaceae), a new species from moist temperate forests in Pakistan

PubMed Central

Khan, Junaid; Sher, Hassan; Naseer, Arooj; Khalid, Abdul Nasir

2017-01-01

Abstract A new species, Descolea quercina, is described and illustrated from Northern parts of Khyber Pakhtunkhwa, Pakistan. It is characterized by medium to large basidiomata, squamose to squamose-granulose hygrophanous pileus, and limoniform, verrucose basidiospores with partly concrescent verrucae. Phylogenetic analyses of nuc rDNA region encompassing the internal transcribed spacers 1 and 2 along with 5.8S rDNA (ITS) and nuc 28S rDNA D1-D2 domains (28S) also confirmed it as a new species. A comparison with similar taxa is provided. PMID:29559818

Genetic sequence data identifies the cercaria of Drepanocephalus spathans (Digenea: Echinostomatidae), a parasite of the double-crested cormorant (Phalacrocorax auritus), with notes on its pathology in juvenile channel catfish (Ictalurus punctatus).

PubMed

Griffin, Matt J; Khoo, Lester H; Quiniou, Sylvie M; O'Hear, Mary M; Pote, Linda M; Greenway, Terrence E; Wise, David J

2012-10-01

An unidentified xiphidio-type cercaria, previously thought inconsequential to catfish health, was found to be released from marsh rams-horn snails (Planorbella trivolvis) inhabiting ponds on a commercial catfish operation in the Mississippi Delta. A preliminary challenge of cohabiting channel catfish ( Ictalurus punctatus ) with snails actively shedding the unidentified cercariae resulted in death of some fish. A second cohabitation trial yielded similar results, as did a third challenge of 250 cercariae/fish. Histopathology revealed developing metacercariae concentrated in the cranial region, especially within the branchial chamber, with several metacercariae at the base of the branchial arches within, or adjacent to, blood vessels, possibly the proximate cause of death. Genetic sequence analysis of the 18S small subunit ribosomal DNA (ssDNA), 28S large subunit rDNA (lsDNA), and cytochrome oxidase (Cox1) genes all matched the cercariae to Drepanocephalus spathans (Digenea: Echinostomatidae), a parasite of the double-crested cormorant (Phalacrocorax auritus), a piscivorous bird endemic on most catfish farms. This is the first commentary regarding pathology of D. spathans in juvenile channel catfish as well as the first report of the marsh rams-horn snail as an intermediate host in the D. spathans life cycle. The data presented here suggest this parasite could have limiting effects on catfish production, further supporting the need for adequate snail control programs to reduce trematode prevalence on commercial catfish operations.

Species boundaries of Gulf of Mexico vestimentiferans (Polychaeta, Siboglinidae) inferred from mitochondrial genes

NASA Astrophysics Data System (ADS)

Pia Miglietta, Maria; Hourdez, Stephane; Cowart, Dominique A.; Schaeffer, Stephen W.; Fisher, Charles

2010-11-01

At least six morphospecies of vestimentiferan tubeworms are associated with cold seeps in the Gulf of Mexico (GOM). The physiology and ecology of the two best-studied species from depths above 1000 m in the upper Louisiana slope (Lamellibrachia luymesi and Seepiophila jonesi) are relatively well understood. The biology of one rare species from the upper slope (escarpiid sp. nov.) and three morphospecies found at greater depths in the GOM (Lamellibrachia sp. 1, L. sp. 2, and Escarpia laminata) are not as well understood. Here we address species distributions and boundaries of cold-seep tubeworms using phylogenetic hypotheses based on two mitochondrial genes. Fragments of the mitochondrial large ribosomal subunit rDNA (16S) and cytochrome oxidase subunit I (COI) genes were sequenced for 167 vestimentiferans collected from the GOM and analyzed in the context of other seep vestimentiferans for which sequence data were available. The analysis supported five monophyletic clades of vestimentiferans in the GOM. Intra-clade variation in both genes was very low, and there was no apparent correlation between the within-clade diversity and collection depth or location. Two of the morphospecies of Lamellibrachia from different depths in the GOM could not be distinguished by either mitochondrial gene. Similarly, E. laminata could not be distinguished from other described species of Escarpia from either the west coast of Africa or the eastern Pacific using COI. We suggest that the mitochondrial COI and 16S genes have little utility as barcoding markers for seep vestimentiferan tubeworms.

A Common Eurasian Fish Tapeworm, Caryophyllaeides fennica (Cestoda), in Western North America: Further Evidence of 'Amphi-Pacific' Vicariance in Freshwater Fish Parasites.

PubMed

Oros, Mikuláš; Choudhury, Anindo; Scholz, Tomáš

2017-10-01

The freshwater fish faunas of the Palaearctic and Nearctic regions show similarities but also notable differences, resulting in diverse distributions of their parasites. Relatively few parasite species occur in both regions and fewer still have been examined using molecular data. We report a rare example of 'amphi-Pacific' distribution, involving the common Palaearctic parasite, the caryophyllidean cestode Caryophyllaeides fennica (Schneider, 1902), in the chiselmouth Acrocheilus alutaceus Agassiz and Pickering, 1855, an endemic cyprinid in northwestern Nearctic, Oregon. Available information on nonnative fish species in Oregon suggests that the parasite is native to the area and not introduced. Molecular data (18S ribosomal [r]DNA, 28S rDNA, internal transcribed spacer 2, and cytochrome c oxidase subunit 1 gene) indicate very little genetic divergence between representatives from the Palaearctic and Nearctic, and possibly a relatively more recent colonization of the Nearctic region by this cestode via the Beringian land bridge. This is remarkable considering that Acrocheilus has reportedly been in Oregon since the Miocene.

Identification of Arcanobacterium pyogenes isolated by post mortem examinations of a bearded dragon and a gecko by phenotypic and genotypic properties.

PubMed

Ulbegi-Mohyla, H; Hijazin, M; Alber, J; Lämmler, C; Hassan, A A; Abdulmawjood, A; Prenger-Berninghoff, E; Weiss, R; Zschöck, M

2010-09-01

The present study was designed to identify phenotypically and genotypically two Arcanobacterium (A.) pyogenes strains isolated by post mortem examinations of a bearded dragon and a gecko. The A. pyogenes strains showed the typical biochemical properties and displayed CAMP-like synergistic hemolytic activities with various indicator strains. The species identity could be confirmed genotypically by amplification and sequencing of the 16S rDNA gene and, as novel target gene, by sequencing of the beta subunit of RNA polymerase encoding gene rpoB, of both strains and of reference strains representing nine species of the genus Arcanobacterium. The species identity of the two A. pyogenes strains could additionally be confirmed by PCR mediated amplification of species specific parts of the 16S-23S rDNA intergenic spacer region, the pyolysin encoding gene plo and by amplification of the collagen-binding protein encoding gene cbpA. All these molecular targets might help to improve the future identification and further characterization of A. pyogenes which, as demonstrated in the present study, could also be isolated from reptile specimens.

Specific PCR for Myxobolus arcticus SSU rDNA in juvenile sockeye salmon Oncorhynchus nerka from British Columbia, Canada.

PubMed

Mahony, Amelia; Fraser, Sarah; Groman, David B; Jones, Simon R M

2015-06-29

A PCR for the specific detection of the salmon brain parasite Myxobolus arcticus (Pugachev and Khokhlov, 1979) was developed using primers designed to amplify a 1363 base pair fragment of the small subunit rDNA. The assay did not amplify DNA from 5 other Myxobolus species or from 7 other myxozoan species belonging to 5 other genera. For juvenile sockeye salmon Oncorhynchus nerka (Walbaum) collected from Chilko Lake, British Columbia (BC), Canada, in 2011, the prevalence by PCR was 96%, in contrast to 71% by histological examination of brain tissue. In 2010, the histological prevalence was 52.5%. Sequence identity between M. arcticus from Chilko Lake and other sites in BC ranged from 99.7 to 99.8% and was 99.6% for a Japanese sequence. In contrast, an M. arcticus sequence from Norway shared 95.3% identity with the Chilko Lake sequence, suggesting misidentification of the parasite. Chilko Lake sockeye salmon were previously reported free of infection with M. arcticus, and more research is required to understand the processes involved in the local and global dispersion of this parasite.

Evidence for Interspecies Gene Transfer in the Evolution of 2,4-Dichlorophenoxyacetic Acid Degraders

PubMed Central

McGowan, Catherine; Fulthorpe, Roberta; Wright, Alice; Tiedje, J. M.

1998-01-01

Small-subunit ribosomal DNA (SSU rDNA) from 20 phenotypically distinct strains of 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria was partially sequenced, yielding 18 unique strains belonging to members of the alpha, beta, and gamma subgroups of the class Proteobacteria. To understand the origin of 2,4-D degradation in this diverse collection, the first gene in the 2,4-D pathway, tfdA, was sequenced. The sequences fell into three unique classes found in various members of the beta and gamma subgroups of Proteobacteria. None of the α-Proteobacteria yielded tfdA PCR products. A comparison of the dendrogram of the tfdA genes with that of the SSU rDNA genes demonstrated incongruency in phylogenies, and hence 2,4-D degradation must have originated from gene transfer between species. Only those strains with tfdA sequences highly similar to the tfdA sequence of strain JMP134 (tfdA class I) transferred all the 2,4-D genes and conferred the 2,4-D degradation phenotype to a Burkholderia cepacia recipient. PMID:9758850

Morphology and 18S rDNA of Henneguya gurlei (Myxosporea) from Ameiurus nebulosus (Siluriformes) in North Carolina.

PubMed

Iwanowicz, Luke R; Iwanowicz, Deborah D; Pote, Linda M; Blazer, Vicki S; Schill, William B

2008-02-01

Henneguya gurlei was isolated from Ameiurus nebulosus captured in North Carolina and redescribed using critical morphological features and 18S small-subunit ribosomal RNA (SSU rDNA) gene sequence. Plasmodia are white, spherical, or subspherical, occur in clusters, measure up to 1.8 mm in length, and are located on the dorsal, pectoral, and anal fins. Histologically, plasmodia are located in the dermis and subdermally, and the larger cysts disrupt the melanocyte pigment layer. The spore body is lanceolate, 18.2 +/- 0.3 microm (range 15.7-20.3) in length, and 5.4 +/- 0.1 microm (range 3.8-6.1) in width in valvular view. The caudal appendages are 41.1 +/- 1.1 microm (range 34.0-49.7) in length. Polar capsules are pyriform and of unequal size. The longer polar capsule measures 6.2 +/- 0.1 microm (range 5.48-7.06), while the shorter is 5.7 +/- 0.1 microm (range 4.8-6.4) in length. Polar capsule width is 1.2 +/- 0.03 microm (range 1.0-1.54). The total length of the spore is 60.9 +/- 1.2 microm (range 48.7-68.5). Morphologically, this species is similar to other species of Henneguya that are known to infect ictalurids. Based on SSU rDNA sequences, this species is most closely related to H. exilis and H. ictaluri, which infect Ictalurus punctatus.

Morphology and 18S rDNA of Henneguya gurlei (Myxosporea) from Ameiurus nebulosus (Siluriformes) in North Carolina

USGS Publications Warehouse

Iwanowicz, L.R.; Iwanowicz, D.D.; Pote, L.M.; Blazer, V.S.; Schill, W.B.

2008-01-01

Henneguya gurlei was isolated from Ameiurus nebulosus captured in North Carolina and redescribed using critical morphological features and 18S small-subunit ribosomal RNA (SSU rDNA) gene sequence. Plasmodia are white, spherical, or subspherical, occur in clusters, measure up to 1.8 mm in length, and are located on the dorsal, pectoral, and anal fins. Histologically, plasmodia are located in the dermis and subdermally, and the larger cysts disrupt the melanocyte pigment layer. The spore body is lanceolate, 18.2 ?? 0.3 ??m (range 15.7-20.3) in length, and 5.4 ?? 0.1 ??m (range 3.8-6.1) in width in valvular view. The caudal appendages are 41.1 ?? 1.1 ??m (range 34.0-49.7) in length. Polar capsules are pyriform and of unequal size. The longer polar capsule measures 6.2 ?? 0.1 ??m (range 5.48-7.06), while the shorter is 5.7 ?? 0.1 ??m (range 4.8-6.4) in length. Polar capsule width is 1.2 ?? 0.03 ??m (range 1.0-1.54). The total length of the spore is 60.9 ?? 1.2 ??m (range 48.7-68.5). Morphologically, this species is similar to other species of Henneguya that are known to infect ictalurids. Based on SSU rDNA sequences, this species is most closely related to H. exilis and H. ictaluri, which infect Ictalurus punctatus. ?? American Society of Parasitologists 2008.

Life cycle and molecular phylogeny of the dinoflagellates Chytriodinium and Dissodinium, ectoparasites of copepod eggs.

PubMed

Gómez, Fernando; Moreira, David; López-García, Purificación

2009-11-01

The dinoflagellates Chytriodinium affine, C. roseum and Dissodinium pseudolunula are ectoparasites of crustacean eggs. Here, we present new observations regarding their life cycle based on coastal plankton samples and incubations and analyze their molecular phylogeny using the small subunit ribosomal RNA gene (SSU rDNA) as a marker. In contrast to the typical stages already documented for its life cycle, we observed that D. pseudolunula dinospores may exceptionally differentiate inside a globular cyst. Despite its parasitic life style, the cysts and dinospores of D. pseudolunula contain chlorophyll a. We obtained the first SSU rDNA sequences for the genera Chytriodinium (the type C. roseum and C. affine) and Dissodinium (D. pseudolunula). Classical taxonomical schemes have ascribed these genera to the order Blastodiniales. However, our SSU rDNA-based phylogenetic analysis shows that these ectoparasites form a clade in the Gymnodinium sensu stricto group, unarmored dinokaryotic dinoflagellates of the order Gymnodiniales. They branch in a subgroup composed of warnowiids, polykrikoids, the type of Gymnodinium, G. fuscum and G. aureolum. Although Chytriodinium and Dissodinium appear to be relatives based on SSU rDNA phylogeny, feeding and host specificity, their life cycles are substantially different. Based on these data we consider that the type of life cycle is a poor criterion for classification at the family level. We suggest that the morphology of the infective cell is probably the most reliable phenotypic characteristic to determine the systematic position of parasitic dinoflagellates.

The famous cultivated mushroom Bailinggu is a separate species of the Pleurotus eryngii species complex.

PubMed

Zhao, Mengran; Zhang, Jinxia; Chen, Qiang; Wu, Xiangli; Gao, Wei; Deng, Wangqiu; Huang, Chenyang

2016-09-15

The mushroom of the genus Pleurotus in western China, called Bailinggu, is a precious edible fungus with high economic value. However, its taxonomical position is unclear. Some researchers regard it as a variety of P. eryngii, namely P. eryngii var. tuoliensis, whereas others consider it to be a subspecies of P. eryngii, viz. P. eryngii subsp. tuoliensis. A total of 51 samples representing seven genetic groups of the genus Pleurotus were subjected to a phylogenetic analysis of partial sequences of the translation elongation factor 1 alpha gene (ef1a), the RNA polymerase II largest subunit gene (rpb1), the RNA polymerase II second largest subunit gene (rpb2) and nuc rDNA internal transcribed spacers (ITS). Our data indicate that the mushroom Bailinggu is a lineage independent of P. eryngii and should be lifted as its own species, namely P. tuoliensis. In addition, its known distribution range consists of both western China and Iran.

Fungal dermatitis, glossitis and disseminated visceral mycosis caused by different Metarhizium granulomatis genotypes in veiled chameleons (Chamaeleo calyptratus) and first isolation in healthy lizards.

PubMed

Schmidt, Volker; Klasen, Linus; Schneider, Juliane; Hübel, Jens; Pees, Michael

2017-08-01

Metarhizium (M.) granulomatis (formerly Chamaeleomyces granulomatis) invariably causes fatal fungal glossitis and systemic mycosis in veiled chameleons (Chamaeleo calyptratus). Isolation of M. granulomatis in other lizards thus far has not been described. The aim of this study therefore was to obtain information on the presence of M. granulomatis in reptiles kept as pets, and to examine whether there was an association between specific genotypes and clinical/pathological outcomes. Besides 18S ribosomal (r) DNA (SSU) and internal transcribed spacer1-5.8S (ITS1-5.8S) rDNA, a fragment of the large subunit of the 28S rDNA (LSU), including the domains 1 (D1) and D2, were sequenced for identification of the fungus and phylogenetic analysis. Metarhizium granulomatis was isolated from 23 veiled chameleons, two panther chameleons (Furcifer pardalis) and one central bearded dragon (Pogona vitticeps). Only the veiled chameleons revealed corresponding pathological findings in the form of glossal hemorrhage, granulomatous glossitis, pharyngitis, dermatitis and/or visceral mycosis. The infection site correlated to survival times of infected veiled chameleons. Combined long-term treatment with terbinafine and nystatin based on susceptibility testing may be helpful for prevention of disease and visceral spreading of the fungus, but elimination of the fungal pathogen or successful treatment of diseased veiled chameleons have not been achieved yet. Sequencing of the ribosomal genes yielded five different genotypes, with genotype A being strongly correlated with dermatitis, and remaining genotypes with pharyngitis and glossitis. However, disseminated visceral mycosis developed irrespective of the genotypes. Copyright © 2017 Elsevier B.V. All rights reserved.

First record of two ectoparasitic ciliates of the genus Trichodina (Ciliophora: Trichodinidae) parasitizing gills of an invasive freshwater fish, Micropercops swinhonis, in Tibet.

PubMed

Wang, Zhe; Deng, Qiong; Zhou, Tong; Yang, Hao; Gu, Zemao

2018-07-01

Although high diversity of parasitic ciliates has been reported in China, little is known about the species from high altitude areas, especially in Tibet. To investigate the species of parasitic ciliates in Tibet, a project was initiated in the Chabalang wetland in 2013. Two Trichodina species, namely, Trichodina sp. and T. reticulata Hirschmann & Partsch, 1955, were isolated from gills of an invasive fish, Micropercops swinhonis for the first time. In the present study, we provided the morphological, morphometrical, and molecular characterizations of the two species and conducted the phylogenetic analyses of mobilids based on the small subunit ribosomal RNA gene (SSU rDNA) sequences. Both morphological characters and morphometric data of the T. reticulata agreed well with previous studies. Although two partial SSU rDNA sequences were obtained in the present study, only the sequence of T. reticulata population in the present study was thought to be reliable. The other sequence may not belong to the other species. Thus, we regarded the other species isolated in the present study as Trichodina sp. to avoid the wrong or confused species identification. Morphologically, Trichodina sp. is distinguished mainly by its large body shape with a broad adhesive disk, robust and obliquely quadrilateral blades, and well-developed rays. T. reticulata is mainly characterized with the 8-12 spherical or elliptical granules in the central zone of adhesive disk. Phylogenetic analyses consistently showed the two ectoparasites clustered with freshwater species of the genus Trichodina within the order Mobilida. Our study extended the host range of T. reticulata and supplemented the molecular data. Also, results reveal that invasion of exotic fish may cause a potential threat to native fish by introducing or dispersing parasitic ciliates.

Phylogeography of ostreopsis along west Pacific coast, with special reference to a novel clade from Japan.

PubMed

Sato, Shinya; Nishimura, Tomohiro; Uehara, Keita; Sakanari, Hiroshi; Tawong, Wittaya; Hariganeya, Naohito; Smith, Kirsty; Rhodes, Lesley; Yasumoto, Takeshi; Taira, Yosuke; Suda, Shoichiro; Yamaguchi, Haruo; Adachi, Masao

2011-01-01

A dinoflagellate genus Ostreopsis is known as a potential producer of Palytoxin derivatives. Palytoxin is the most potent non-proteinaceous compound reported so far. There has been a growing number of reports on palytoxin-like poisonings in southern areas of Japan; however, the distribution of Ostreopsis has not been investigated so far. Morphological plasticity of Ostreopsis makes reliable microscopic identification difficult so the employment of molecular tools was desirable. In total 223 clones were examined from samples mainly collected from southern areas of Japan. The D8-D10 region of the nuclear large subunit rDNA (D8-D10) was selected as a genetic marker and phylogenetic analyses were conducted. Although most of the clones were unable to be identified, there potentially 8 putative species established during this study. Among them, Ostreopsis sp. 1-5 did not belong to any known clade, and each of them formed its own clade. The dominant species was Ostreopsis sp. 1, which accounted for more than half of the clones and which was highly toxic and only distributed along the Japanese coast. Comparisons between the D8-D10 and the Internal Transcribed Spacer (ITS) region of the nuclear rDNA, which has widely been used for phylogenetic/phylogeographic studies in Ostreopsis, revealed that the D8-D10 was less variable than the ITS, making consistent and reliable phylogenetic reconstruction possible. This study unveiled a surprisingly diverse and widespread distribution of Japanese Ostreopsis. Further study will be required to better understand the phylogeography of the genus. Our results posed the urgent need for the development of the early detection/warning systems for Ostreopsis, particularly for the widely distributed and strongly toxic Ostreopsis sp. 1. The D8-D10 marker will be suitable for these purposes.

Monoblepharidomycetes diversity includes new parasitic and saprotrophic species with highly intronized rDNA.

PubMed

Karpov, Sergey A; Mamanazarova, Karomat S; Popova, Olga V; Aleoshin, Vladimir V; James, Timothy Y; Mamkaeva, Maria A; Tcvetkova, Victoria S; Vishnyakov, Andrey E; Longcore, Joyce E

2017-08-01

The Monoblepharidomycetes is the sister class to the Chytridiomycetes in the phylum Chytridiomycota. The six known genera have thalli that are either monocentric and without rhizoids or produce hyphae with an independent evolutionary origin from the hyphae of higher fungi. On the basis of morphological characters and phylogenetic evidence from the small and large subunits of nuclear ribosomal RNA, we established two new genera, Sanchytrium and Telasphaerula, each with a single species. We re-analyzed intergeneric relationships within the monoblephs, and established two new families. The new genera significantly expand the known morphological and ecological diversity of the Monoblepharidomycetes by adding a monocentric, epibiotic, algal parasitic species and a rhizomycelial, saprotrophic species. Based on the presence of environmental sequences related to Sanchytrium strains, the Monoblepharidomycetes contain previously unsuspected diversity. The ribosomal DNA of the new genera contains an unusually high density of group I introns. We found 20 intron insertion positions including six that are new for rRNA genes (S1053, L803, L829, L961, L1844, and L2281). Copyright © 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

One fungus, which genes? Development and assessment of universal primers for potential secondary fungal DNA barcodes.

PubMed

Stielow, J B; Lévesque, C A; Seifert, K A; Meyer, W; Iriny, L; Smits, D; Renfurm, R; Verkley, G J M; Groenewald, M; Chaduli, D; Lomascolo, A; Welti, S; Lesage-Meessen, L; Favel, A; Al-Hatmi, A M S; Damm, U; Yilmaz, N; Houbraken, J; Lombard, L; Quaedvlieg, W; Binder, M; Vaas, L A I; Vu, D; Yurkov, A; Begerow, D; Roehl, O; Guerreiro, M; Fonseca, A; Samerpitak, K; van Diepeningen, A D; Dolatabadi, S; Moreno, L F; Casaregola, S; Mallet, S; Jacques, N; Roscini, L; Egidi, E; Bizet, C; Garcia-Hermoso, D; Martín, M P; Deng, S; Groenewald, J Z; Boekhout, T; de Beer, Z W; Barnes, I; Duong, T A; Wingfield, M J; de Hoog, G S; Crous, P W; Lewis, C T; Hambleton, S; Moussa, T A A; Al-Zahrani, H S; Almaghrabi, O A; Louis-Seize, G; Assabgui, R; McCormick, W; Omer, G; Dukik, K; Cardinali, G; Eberhardt, U; de Vries, M; Robert, V

2015-12-01

The aim of this study was to assess potential candidate gene regions and corresponding universal primer pairs as secondary DNA barcodes for the fungal kingdom, additional to ITS rDNA as primary barcode. Amplification efficiencies of 14 (partially) universal primer pairs targeting eight genetic markers were tested across > 1 500 species (1 931 strains or specimens) and the outcomes of almost twenty thousand (19 577) polymerase chain reactions were evaluated. We tested several well-known primer pairs that amplify: i) sections of the nuclear ribosomal RNA gene large subunit (D1-D2 domains of 26/28S); ii) the complete internal transcribed spacer region (ITS1/2); iii) partial β -tubulin II (TUB2); iv) γ-actin (ACT); v) translation elongation factor 1-α (TEF1α); and vi) the second largest subunit of RNA-polymerase II (partial RPB2, section 5-6). Their PCR efficiencies were compared with novel candidate primers corresponding to: i) the fungal-specific translation elongation factor 3 (TEF3); ii) a small ribosomal protein necessary for t-RNA docking; iii) the 60S L10 (L1) RP; iv) DNA topoisomerase I (TOPI); v) phosphoglycerate kinase (PGK); vi) hypothetical protein LNS2; and vii) alternative sections of TEF1α. Results showed that several gene sections are accessible to universal primers (or primers universal for phyla) yielding a single PCR-product. Barcode gap and multi-dimensional scaling analyses revealed that some of the tested candidate markers have universal properties providing adequate infra- and inter-specific variation that make them attractive barcodes for species identification. Among these gene sections, a novel high fidelity primer pair for TEF1α, already widely used as a phylogenetic marker in mycology, has potential as a supplementary DNA barcode with superior resolution to ITS. Both TOPI and PGK show promise for the Ascomycota, while TOPI and LNS2 are attractive for the Pucciniomycotina, for which universal primers for ribosomal subunits often fail.

Talaromyces rubrifaciens, a new species discovered from heating, ventilation and air conditioning systems in China.

PubMed

Luo, Yi; Lu, Xiaohong; Bi, Wu; Liu, Fan; Gao, Weiwei

2016-01-01

A new Talaromyces species, T. rubrifaciens, was isolated from supply air outlets of heating, ventilation and air conditioning (HVAC) systems in three kinds of public building in Beijing and Nanjing, China. Morphologically it exhibits many characters of section Trachyspermi but is distinguished from other species of this section by restricted growth and broad and strictly biverticillate conidiophores. Phylogenetic analyses based on the internal transcribed spacer rDNA (ITS), β-tubulin (BenA), calmodulin (CaM) and RNA polymerase second largest subunit (RPB2) genes reveal that T. rubrifaciens is a distinct species in section Trachyspermi. © 2016 by The Mycological Society of America.

Subunit association of gamma-glutamyltranspeptidase of Escherichia coli K-12.

PubMed

Hashimoto, W; Suzuki, H; Nohara, S; Tachi, H; Yamamoto, K; Kumagai, H

1995-12-01

gamma-Glutamyltranspeptidase [EC 2.3.2.2] of Escherichia coli K-12 consists of one large subunit and one small subunit, which can be separated from each other by high-performance liquid chromatography. Using ion spray mass spectrometry, the masses of the large and the small subunit were determined to be 39,207 and 20,015, respectively. The large subunit exhibited no gamma-glutamyltranspeptidase activity and the small subunit had little enzymatic activity, but a mixture of the two subunits showed partial recovery of the enzymatic activity. The results of native-polyacrylamide gel electrophoresis suggested that they could partially recombine, and that the recombined dimer exhibited enzymatic activity. The gene of gamma-glutamyltranspeptidase encoded a signal peptide, and the large and small subunits in a single open reading frame in that order. Two kinds of plasmid were constructed encoding the signal peptide and either the large or the small subunit. A gamma-glutamyltranspeptidase-less mutant of E. coli K-12 was transformed with each plasmid or with both of them. The strain harboring the plasmid encoding each subunit produced a small amount of the corresponding subunit protein in the periplasmic space but exhibited no enzymatic activity. The strain transformed with both plasmids together exhibited the enzymatic activity, but its specific activity was approximately 3% of that of a strain harboring a plasmid encoding the intact structural gene. These results indicate that a portion of the separated large and small subunits can be reconstituted in vitro and exhibit the enzymatic activity, and that the expressed large and small subunits independently are able to associate in vivo and be folded into an active structure, though the specific activity of the associated subunits was much lower than that of native enzyme. This suggests that the synthesis of gamma-glutamyltranspeptidase in a single precursor polypeptide and subsequent processing are more effective to construct the intact structure of gamma-glutamyltranspeptidase than the association of the separated large and small subunits.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jack Preiss

Conversion of the Potato tuber ADP-glucose Pyrophopshorylase Regulatory Subunit into a Catalytic Subunit. ADP-glucose synthesis, a rate-limiting reaction in starch synthesis, is catalyzed by ADP-glucose pyrophosphorylase (ADPGlc PPase). The enzyme in plants is allosterically activated by 3-phosphoglycerate (3PGA) and inhibited by inorganic phosphate (Pi) and is composed of two subunits as a heterotetramer, a2b2. Subunit a is the catalytic subunit and subunit b is designated as the regulatory subunit.The b subunit increases the affinty of the activator for the catalytic subunit. Recent results have shown that the subunits are derived from the same ancestor subunit as the regulatory subunit canmore » be converted to a catalytically subunit via mutation of just two amino acids. Lys44 and Thr54 in the large subunit from potato tuber were converted to the homologous catalytic subunit residues, Arg33 and Lys43. The activity of the large subunit mutants cannot be readily tested with a co-expressed wild-type small (catalytic) subunit because of the intrinsic activity of the latter. We co-expressed the regulatory-subunit mutants with SmallD145N, an inactive S subunit in which the catalytic Asp145 was mutated. The activity of the small (catalytic) subunit was reduced more than three orders of magnitude. Coexpression of the L subunit double mutant LargeK44R/T54K with SmallD145N generated an enzyme with considerable activity, 10% and 18% of the wildtype enzyme, in the ADP-glucose synthetic and pyrophosphorolytic direction, respectively. Replacement of those two residues in the small subunit by the homologous amino acids in the L subunits (mutations R33K and K43T) decreased the activity one and two orders of magnitude. The wild-type enzyme and SmallD145NLargeK44R/T54K had very similar kinetic properties indicating that the substrate site has been conserved. The fact that only two mutations in the L subunit restored enzyme activity is very strong evidence that the large subunit is derived from the catalytic ancestor. Previous results showed that Asp145 in the small subunit of the wild-type is essential for catalysis, whereas the homologous Asp160 in the Large WT subunit is not. However, in this study, mutation D160N or D160E in the LK44R/T54K subunit abolished the activity, which shows the ancestral essential role of this residue and confirms that the catalysis of SmallD145NLarge K44R/T54K occurs in the L(b) subunit. A phylogenetic tree of the ADP-Glc PPases present in photosynthetic eukaryotes also sheds information about the origin of the subunits. The tree showed that plant Small and Large subunits can be divided into two and four distinct groups, respectively. The two main groups of S subunits are from dicot and monocot plants, whereas Large subunit groups correlate better with their documented tissue expression. The first Large-subunit group is generally expressed in photosynthetic tissues and comprises Large subunits from dicots and monocots. Group II displays a broader expression pattern, whereas groups III and IV are expressed in storage organs (roots, stems, tubers, seeds). Subunits from group III are only from dicot plants, whereas group IV are seed-specific subunits from monocots. These last two groups stem from the same branch of the phylogenetic tree and split before monocot and dicot separation. Thus few as two mutations turned the L subunit from Solanum tuberosum catalytic, showing that L and S subunits share a common catalytic ancestor, rather than a non-catalytic one. The L subunit evolved to have a regulatory role, lost catalytic residues more than 130 million years ago before monocots and dicots diverged, and preserved, possibly as a byproduct, the active site domain.« less

Nucleolar Association and Transcriptional Inhibition through 5S rDNA in Mammals

PubMed Central

Fedoriw, Andrew M.; Starmer, Joshua; Yee, Della; Magnuson, Terry

2012-01-01

Changes in the spatial positioning of genes within the mammalian nucleus have been associated with transcriptional differences and thus have been hypothesized as a mode of regulation. In particular, the localization of genes to the nuclear and nucleolar peripheries is associated with transcriptional repression. However, the mechanistic basis, including the pertinent cis- elements, for such associations remains largely unknown. Here, we provide evidence that demonstrates a 119 bp 5S rDNA can influence nucleolar association in mammals. We found that integration of transgenes with 5S rDNA significantly increases the association of the host region with the nucleolus, and their degree of association correlates strongly with repression of a linked reporter gene. We further show that this mechanism may be functional in endogenous contexts: pseudogenes derived from 5S rDNA show biased conservation of their internal transcription factor binding sites and, in some cases, are frequently associated with the nucleolus. These results demonstrate that 5S rDNA sequence can significantly contribute to the positioning of a locus and suggest a novel, endogenous mechanism for nuclear organization in mammals. PMID:22275877

Comparison of sequencing the D2 region of the large subunit ribosomal RNA gene (MicroSEQ®) versus the internal transcribed spacer (ITS) regions using two public databases for identification of common and uncommon clinically relevant fungal species.

PubMed

Arbefeville, S; Harris, A; Ferrieri, P

2017-09-01

Fungal infections cause considerable morbidity and mortality in immunocompromised patients. Rapid and accurate identification of fungi is essential to guide accurately targeted antifungal therapy. With the advent of molecular methods, clinical laboratories can use new technologies to supplement traditional phenotypic identification of fungi. The aims of the study were to evaluate the sole commercially available MicroSEQ® D2 LSU rDNA Fungal Identification Kit compared to the in-house developed internal transcribed spacer (ITS) regions assay in identifying moulds, using two well-known online public databases to analyze sequenced data. 85 common and uncommon clinically relevant fungi isolated from clinical specimens were sequenced for the D2 region of the large subunit (LSU) of ribosomal RNA (rRNA) gene with the MicroSEQ® Kit and the ITS regions with the in house developed assay. The generated sequenced data were analyzed with the online GenBank and MycoBank public databases. The D2 region of the LSU rRNA gene identified 89.4% or 92.9% of the 85 isolates to the genus level and the full ITS region (f-ITS) 96.5% or 100%, using GenBank or MycoBank, respectively, when compared to the consensus ID. When comparing species-level designations to the consensus ID, D2 region of the LSU rRNA gene aligned with 44.7% (38/85) or 52.9% (45/85) of these isolates in GenBank or MycoBank, respectively. By comparison, f-ITS possessed greater specificity, followed by ITS1, then ITS2 regions using GenBank or MycoBank. Using GenBank or MycoBank, D2 region of the LSU rRNA gene outperformed phenotypic based ID at the genus level. Comparing rates of ID between D2 region of the LSU rRNA gene and the ITS regions in GenBank or MycoBank at the species level against the consensus ID, f-ITS and ITS2 exceeded performance of the D2 region of the LSU rRNA gene, but ITS1 had similar performance to the D2 region of the LSU rRNA gene using MycoBank. Our results indicated that the MicroSEQ® D2 LSU rDNA Fungal Identification Kit was equivalent to the in-house developed ITS regions assay to identify fungi at the genus level. The MycoBank database gave a better curated database and thus allowed a better genus and species identification for both D2 region of the LSU rRNA gene and ITS regions. Copyright © 2017 Elsevier B.V. All rights reserved.

Intragenomic variation in the ITS rDNA region obscures phylogenetic relationships and inflates estimates of operational taxonomic units in genus Laetiporus

Treesearch

Daniel L. Lindner; Mark T. Banik

2011-01-01

Regions of rDNA are commonly used to infer phylogenetic relationships among fungal species and as DNA barcodes for identification. These regions occur in large tandem arrays, and concerted evolution is believed to reduce intragenomic variation among copies within these arrays, although some variation still might exist. Phylogenetic studies typically use consensus...

Dasytricha dominance in Surti buffalo rumen revealed by 18S rRNA sequences and real-time PCR assay.

PubMed

Singh, K M; Tripathi, A K; Pandya, P R; Rank, D N; Kothari, R K; Joshi, C G

2011-09-01

The genetic diversity of protozoa in Surti buffalo rumen was studied by amplified ribosomal DNA restriction analysis, 18S rDNA sequence homology and phylogenetic and Real-time PCR analysis methods. Three animals were fed diet comprised green fodder Napier bajra 21 (Pennisetum purpureum), mature pasture grass (Dicanthium annulatum) and concentrate mixture (20% crude protein, 65% total digestible nutrients). A protozoa-specific primer (P-SSU-342f) and a eukarya-specific primer (Medlin B) were used to amplify a 1,360 bp fragment of DNA encoding protozoal small subunit (SSU) ribosomal RNA from rumen fluid. A total of 91 clones were examined and identified 14 different 18S RNA sequences based on PCR-RFLP pattern. These 14 phylotypes were distributed into four genera-based 18S rDNA database sequences and identified as Dasytricha (57 clones), Isotricha (14 clones), Ostracodinium (11 clones) and Polyplastron (9 clones). Phylogenetic analyses were also used to infer the makeup of protozoa communities in the rumen of Surti buffalo. Out of 14 sequences, 8 sequences (69 clones) clustered with the Dasytricha ruminantium-like clone and 4 sequences (13 clones) were also phylogenetically placed with the Isotricha prostoma-like clone. Moreover, 2 phylotypes (9 clones) were related to Polyplastron multivesiculatum-like clone. In addition, the number of 18S rDNA gene copies of Dasytricha ruminantium (0.05% to ciliate protozoa) was higher than Entodinium sp. (2.0 × 10(5) vs. 1.3 × 10(4)) in per ml ruminal fluid.

First report of fatal systemic Halicephalobus gingivalis infection in two Lipizzaner horses from Romania: clinical, pathological, and molecular characterization.

PubMed

Taulescu, Marian A; Ionicã, Angela M; Diugan, Eva; Pavaloiu, Alexandra; Cora, Roxana; Amorim, Irina; Catoi, Cornel; Roccabianca, Paola

2016-03-01

Halicephalobus gingivalis (H. gingivalis) causes a rare and fatal infection in horses and humans. Despite the zoonotic potential and severity of the disease, the epidemiology and pathogenesis of halicephalobiasis are still poorly understood. Several European cases of equine halicephalobiasis have been documented; however, in South-Eastern European countries, including Romania, equine neurohelminthiasis caused by H. gingivalis has not been previously described. Two Lipizzaner horses with a clinical history of progressive neurological signs were referred to the Pathology Department of the Cluj-Napoca (Romania) for necropsy. Both horses died with severe neurological signs. Gross examination and cytological, histological, and molecular analyses were performed. The stallions came from two different breeding farms. No history of traveling outside Romania was recorded. At necropsy, granulomatous and necrotizing lesions were observed in the kidneys, lymph nodes, brain, retroperitoneal adipose tissue, and lungs, indicating a systemic infection. Parasitological and histopathological analyses evidenced larval and adult forms of rhabditiform nematodes consistent with Halicephalobus species. Parasites were observed in both lymph and blood vessels of different organs and were also identified in urine samples. A subunit of the large-subunit ribosomal RNA gene (LSU rDNA) of H. gingivalis (673 bp) was amplified from lesions in both horses.To the authors' knowledge, this is the first report of equine systemic H. gingivalis infection in Romania and in South-Eastern Europe. Our findings provide new insights into the geographic distribution of specific genetic lineages of H. gingivalis, while also raising public health awareness, as the parasite is zoonotic.

Multigene-based analyses on evolutionary phylogeny of two controversial ciliate orders: Pleuronematida and Loxocephalida (Protista, Ciliophora, Oligohymenophorea).

PubMed

Gao, Feng; Katz, Laura A; Song, Weibo

2013-07-01

Relationships among members of the ciliate subclass Scuticociliatia (Ciliophora, Oligohymenophorea) are largely unresolved. Phylogenetic studies of its orders Pleuronematida and Loxocephalida were initially based on small subunit ribosomal RNA gene (SSU-rDNA) analyses of a limited number of taxa. Here we characterized 37 sequences (SSU-rDNA, ITS-5.8S and LSU-rDNA) from 21 taxonomically controversial members of these orders. Phylogenetic trees constructed to assess the inter- and intra-generic relationships of pleuronematids and loxocephalids reveal the following: (1) the order Loxocephalida and its two families Loxocephalidae and Cinetochilidae are not monophyletic when more taxa are added; (2) the core pleuronematids are divided into two fully supported clades, however, the order Pleuronematida is not monophyletic because Cyclidium glaucoma is closer to Thigmotrichida; (3) the family Pleuronematidae and the genus Schizocalyptra are monophyletic, though rDNA sequences of Pleuronema species are highly variable; (4) Pseudoplatynematum and Sathrophilus are closely related to the subclass Astomatia, while Cinetochilum forms a monophyletic group with the subclass Apostomatia; and (5) Hippocomos falls in the order Pleuronematida and is closely related to Eurystomatellidae and Cyclidium plouneouri. Further, in an effort to provide a better resolution of evolutionary relationships, the secondary structures of ITS2 transcripts and the variable region 4 (V4) of the small subunit ribosomal RNA (SSU-rRNA) are predicted, revealing that ITS2 structures are conserved at the order level while V4 region structures are more variable than ITS2 structures. Copyright © 2013 Elsevier Inc. All rights reserved.

Focal accumulation of preribosomes outside the nucleolus during metaphase–anaphase in budding yeast

PubMed Central

Moriggi, Giulia; Gaspar, Sonia G.; Nieto, Blanca; Bustelo, Xosé R.

2017-01-01

Saccharomyces cerevisiae contains one nucleolus that remains intact in the mother-cell side of the nucleus throughout most of mitosis. Based on this, it is assumed that the bulk of ribosome production during cell division occurs in the mother cell. Here, we show that the ribosome synthesis machinery localizes not only in the nucleolus but also at a center that is present in the bud side of the nucleus after the initiation of mitosis. This center can be visualized by live microscopy as a punctate body located in close proximity to the nuclear envelope and opposite to the nucleolus. It contains ribosomal DNA (rDNA) and precursors of both 40S and 60S ribosomal subunits. Proteins that actively participate in ribosome synthesis, but not functionally defective variants, accumulate in that site. The formation of this body occurs in the metaphase-to-anaphase transition when discrete regions of rDNA occasionally exit the nucleolus and move into the bud. Collectively, our data unveil the existence of a previously unknown mechanism for preribosome accumulation at the nuclear periphery in budding yeast. We propose that this might be a strategy to expedite the delivery of ribosomes to the growing bud. PMID:28588079

Identification of Arcanobacterium pyogenes isolated by post mortem examinations of a bearded dragon and a gecko by phenotypic and genotypic properties

PubMed Central

Ülbegi-Mohyla, H.; Hijazin, M.; Alber, J.; Hassan, A. A.; Abdulmawjood, A.; Prenger-Berninghoff, E.; Weiß, R.; Zschöck, M.

2010-01-01

The present study was designed to identify phenotypically and genotypically two Arcanobacterium (A.) pyogenes strains isolated by post mortem examinations of a bearded dragon and a gecko. The A. pyogenes strains showed the typical biochemical properties and displayed CAMP-like synergistic hemolytic activities with various indicator strains. The species identity could be confirmed genotypically by amplification and sequencing of the 16S rDNA gene and, as novel target gene, by sequencing of the beta subunit of RNA polymerase encoding gene rpoB, of both strains and of reference strains representing nine species of the genus Arcanobacterium. The species identity of the two A. pyogenes strains could additionally be confirmed by PCR mediated amplification of species specific parts of the 16S-23S rDNA intergenic spacer region, the pyolysin encoding gene plo and by amplification of the collagen-binding protein encoding gene cbpA. All these molecular targets might help to improve the future identification and further characterization of A. pyogenes which, as demonstrated in the present study, could also be isolated from reptile specimens. PMID:20706035

The LSH/HELLS homolog Irc5 contributes to cohesin association with chromatin in yeast

PubMed Central

Bakowski, Tomasz; Maciaszczyk-Dziubinska, Ewa; Wysocki, Robert

2017-01-01

Abstract Accurate chromosome segregation is essential for every living cell as unequal distribution of chromosomes during cell division may result in genome instability that manifests in carcinogenesis and developmental disorders. Irc5 from Saccharomyces cerevisiae is a member of the conserved Snf2 family of ATP-dependent DNA translocases and its function is poorly understood. Here, we identify Irc5 as a novel interactor of the cohesin complex. Irc5 associates with Scc1 cohesin subunit and contributes to cohesin binding to chromatin. Disruption of IRC5 decreases cohesin levels at centromeres and chromosome arms, causing premature sister chromatid separation. Moreover, reduced cohesin occupancy at the rDNA region in cells lacking IRC5 leads to the loss of rDNA repeats. We also show that the translocase activity of Irc5 is required for its function in cohesion pathway. Finally, we demonstrate that in the absence of Irc5 both the level of chromatin-bound Scc2, a member of cohesin loading complex, and physical interaction between Scc1 and Scc2 are reduced. Our results suggest that Irc5 is an auxiliary factor that is involved in cohesin association with chromatin. PMID:28383696

Potential of DNA sequences to identify zoanthids (Cnidaria: Zoantharia).

PubMed

Sinniger, Frederic; Reimer, James D; Pawlowski, Jan

2008-12-01

The order Zoantharia is known for its chaotic taxonomy and difficult morphological identification. One method that potentially could help for examining such troublesome taxa is DNA barcoding, which identifies species using standard molecular markers. The mitochondrial cytochrome oxidase subunit I (COI) has been utilized to great success in groups such as birds and insects; however, its applicability in many other groups is controversial. Recently, some studies have suggested that barcoding is not applicable to anthozoans. Here, we examine the use of COI and mitochondrial 16S ribosomal DNA for zoanthid identification. Despite the absence of a clear barcoding gap, our results show that for most of 54 zoanthid samples, both markers could separate samples to the species, or species group, level, particularly when easily accessible ecological or distributional data were included. Additionally, we have used the short V5 region of mt 16S rDNA to identify eight old (13 to 50 years old) museum samples. We discuss advantages and disadvantages of COI and mt 16S rDNA as barcodes for Zoantharia, and recommend that either one or both of these markers be considered for zoanthid identification in the future.

Geranyl diphosphate synthase large subunit, and methods of use

DOEpatents

Croteau, Rodney B.; Burke, Charles C.; Wildung, Mark R.

2001-10-16

A cDNA encoding geranyl diphosphate synthase large subunit from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase large subunit). In another aspect, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase large subunit. In yet another aspect, the present invention provides isolated, recombinant geranyl diphosphate synthase protein comprising an isolated, recombinant geranyl diphosphate synthase large subunit protein and an isolated, recombinant geranyl diphosphate synthase small subunit protein. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase.

Complete mitochondrial genome sequences from five Eimeria species (Apicomplexa; Coccidia; Eimeriidae) infecting domestic turkeys

PubMed Central

2014-01-01

Background Clinical and subclinical coccidiosis is cosmopolitan and inflicts significant losses to the poultry industry globally. Seven named Eimeria species are responsible for coccidiosis in turkeys: Eimeria dispersa; Eimeria meleagrimitis; Eimeria gallopavonis; Eimeria meleagridis; Eimeria adenoeides; Eimeria innocua; and, Eimeria subrotunda. Although attempts have been made to characterize these parasites molecularly at the nuclear 18S rDNA and ITS loci, the maternally-derived and mitotically replicating mitochondrial genome may be more suited for species level molecular work; however, only limited sequence data are available for Eimeria spp. infecting turkeys. The purpose of this study was to sequence and annotate the complete mitochondrial genomes from 5 Eimeria species that commonly infect the domestic turkey (Meleagris gallopavo). Methods Six single-oocyst derived cultures of five Eimeria species infecting turkeys were PCR-amplified and sequenced completely prior to detailed annotation. Resulting sequences were aligned and used in phylogenetic analyses (BI, ML, and MP) that included complete mitochondrial genomes from 16 Eimeria species or concatenated CDS sequences from each genome. Results Complete mitochondrial genome sequences were obtained for Eimeria adenoeides Guelph, 6211 bp; Eimeria dispersa Briston, 6238 bp; Eimeria meleagridis USAR97-01, 6212 bp; Eimeria meleagrimitis USMN08-01, 6165 bp; Eimeria gallopavonis Weybridge, 6215 bp; and Eimeria gallopavonis USKS06-01, 6215 bp). The order, orientation and CDS lengths of the three protein coding genes (COI, COIII and CytB) as well as rDNA fragments encoding ribosomal large and small subunit rRNA were conserved among all sequences. Pairwise sequence identities between species ranged from 88.1% to 98.2%; sequence variability was concentrated within CDS or between rDNA fragments (where indels were common). No phylogenetic reconstruction supported monophyly of Eimeria species infecting turkeys; Eimeria dispersa may have arisen via host switching from another avian host. Phylogenetic analyses suggest E. necatrix and E. tenella are related distantly to other Eimeria of chickens. Conclusions Mitochondrial genomes of Eimeria species sequenced to date are highly conserved with regard to gene content and structure. Nonetheless, complete mitochondrial genome sequences and, particularly the three CDS, possess sufficient sequence variability for differentiating Eimeria species of poultry. The mitochondrial genome sequences are highly suited for molecular diagnostics and phylogenetics of coccidia and, potentially, genetic markers for molecular epidemiology. PMID:25034633

Complete mitochondrial genome sequences from five Eimeria species (Apicomplexa; Coccidia; Eimeriidae) infecting domestic turkeys.

PubMed

Ogedengbe, Mosun E; El-Sherry, Shiem; Whale, Julia; Barta, John R

2014-07-17

Clinical and subclinical coccidiosis is cosmopolitan and inflicts significant losses to the poultry industry globally. Seven named Eimeria species are responsible for coccidiosis in turkeys: Eimeria dispersa; Eimeria meleagrimitis; Eimeria gallopavonis; Eimeria meleagridis; Eimeria adenoeides; Eimeria innocua; and, Eimeria subrotunda. Although attempts have been made to characterize these parasites molecularly at the nuclear 18S rDNA and ITS loci, the maternally-derived and mitotically replicating mitochondrial genome may be more suited for species level molecular work; however, only limited sequence data are available for Eimeria spp. infecting turkeys. The purpose of this study was to sequence and annotate the complete mitochondrial genomes from 5 Eimeria species that commonly infect the domestic turkey (Meleagris gallopavo). Six single-oocyst derived cultures of five Eimeria species infecting turkeys were PCR-amplified and sequenced completely prior to detailed annotation. Resulting sequences were aligned and used in phylogenetic analyses (BI, ML, and MP) that included complete mitochondrial genomes from 16 Eimeria species or concatenated CDS sequences from each genome. Complete mitochondrial genome sequences were obtained for Eimeria adenoeides Guelph, 6211 bp; Eimeria dispersa Briston, 6238 bp; Eimeria meleagridis USAR97-01, 6212 bp; Eimeria meleagrimitis USMN08-01, 6165 bp; Eimeria gallopavonis Weybridge, 6215 bp; and Eimeria gallopavonis USKS06-01, 6215 bp). The order, orientation and CDS lengths of the three protein coding genes (COI, COIII and CytB) as well as rDNA fragments encoding ribosomal large and small subunit rRNA were conserved among all sequences. Pairwise sequence identities between species ranged from 88.1% to 98.2%; sequence variability was concentrated within CDS or between rDNA fragments (where indels were common). No phylogenetic reconstruction supported monophyly of Eimeria species infecting turkeys; Eimeria dispersa may have arisen via host switching from another avian host. Phylogenetic analyses suggest E. necatrix and E. tenella are related distantly to other Eimeria of chickens. Mitochondrial genomes of Eimeria species sequenced to date are highly conserved with regard to gene content and structure. Nonetheless, complete mitochondrial genome sequences and, particularly the three CDS, possess sufficient sequence variability for differentiating Eimeria species of poultry. The mitochondrial genome sequences are highly suited for molecular diagnostics and phylogenetics of coccidia and, potentially, genetic markers for molecular epidemiology.

Chromatin tethering effects of hNopp140 are involved in the spatial organization of nucleolus and the rRNA gene transcription

PubMed Central

Tsai, Yi-Tzang; Lin, Chen-I; Chen, Hung-Kai; Lee, Kuo-Ming; Hsu, Chia-Yi; Yang, Shun-Jen

2008-01-01

The short arms of five human acrocentric chromosomes contain ribosomal gene (rDNA) clusters where numerous mini-nucleoli arise at the exit of mitosis. These small nucleoli tend to coalesce into one or a few large nucleoli during interphase by unknown mechanisms. Here, we demonstrate that the N- and C-terminal domains of a nucleolar protein, hNopp140, bound respectively to α-satellite arrays and rDNA clusters of acrocentric chromosomes for nucleolar formation. The central acidic-and-basic repeated domain of hNopp140, possessing a weak self-self interacting ability, was indispensable for hNopp140 to build up a nucleolar round-shaped structure. The N- or the C-terminally truncated hNopp140 caused nucleolar segregation and was able to alter locations of the rDNA transcription, as mediated by detaching the rDNA repeats from the acrocentric α-satellite arrays. Interestingly, an hNopp140 mutant, made by joining the N- and C-terminal domains but excluding the entire central repeated region, induced nucleolar disruption and global chromatin condensation. Furthermore, RNAi knockdown of hNopp140 resulted in dispersion of the rDNA and acrocentric α-satellite sequences away from nucleolus that was accompanied by rDNA transcriptional silence. Our findings indicate that hNopp140, a scaffold protein, is involved in the nucleolar assembly, fusion, and maintenance. PMID:18253863

Evolutionary trends in animal ribosomal DNA loci: introduction to a new online database.

PubMed

Sochorová, Jana; Garcia, Sònia; Gálvez, Francisco; Symonová, Radka; Kovařík, Aleš

2018-03-01

Ribosomal DNA (rDNA) loci encoding 5S and 45S (18S-5.8S-28S) rRNAs are important components of eukaryotic chromosomes. Here, we set up the animal rDNA database containing cytogenetic information about these loci in 1343 animal species (264 families) collected from 542 publications. The data are based on in situ hybridisation studies (both radioactive and fluorescent) carried out in major groups of vertebrates (fish, reptiles, amphibians, birds, and mammals) and invertebrates (mostly insects and mollusks). The database is accessible online at www.animalrdnadatabase.com . The median number of 45S and 5S sites was close to two per diploid chromosome set for both rDNAs despite large variation (1-74 for 5S and 1-54 for 45S sites). No significant correlation between the number of 5S and 45S rDNA loci was observed, suggesting that their distribution and amplification across the chromosomes follow independent evolutionary trajectories. Each group, irrespective of taxonomic classification, contained rDNA sites at any chromosome location. However, the distal and pericentromeric positions were the most prevalent (> 75% karyotypes) for 45S loci, while the position of 5S loci was more variable. We also examined potential relationships between molecular attributes of rDNA (homogenisation and expression) and cytogenetic parameters such as rDNA positions, chromosome number, and morphology.

Phylogenetic relationships in the mushroom genus Coprinus and dark-spored allies based on sequence data from the nuclear gene coding for the large ribosomal subunit RNA: divergent domains, outgroups, and monophyly.

PubMed

Hopple, J S; Vilgalys, R

1999-10-01

Phylogenetic relationships were investigated in the mushroom genus Coprinus based on sequence data from the nuclear encoded large-subunit rDNA gene. Forty-seven species of Coprinus and 19 additional species from the families Coprinaceae, Strophariaceae, Bolbitiaceae, Agaricaceae, Podaxaceae, and Montagneaceae were studied. A total of 1360 sites was sequenced across seven divergent domains and intervening sequences. A total of 302 phylogenetically informative characters was found. Ninety-eight percent of the average divergence between taxa was located within the divergent domains, with domains D2 and D8 being most divergent and domains D7 and D10 the least divergent. An empirical test of phylogenetic signal among divergent domains also showed that domains D2 and D3 had the lowest levels of homoplasy. Two equally most parsimonious trees were resolved using Wagner parsimony. A character-state weighted analysis produced 12 equally most parsimonious trees similar to those generated by Wagner parsimony. Phylogenetic analyses employing topological constraints suggest that none of the major taxonomic systems proposed for subgeneric classification is able to completely reflect phylogenetic relationships in Coprinus. A strict consensus integration of the two Wagner trees demonstrates the problematic nature of choosing outgroups within dark-spored mushrooms. The genus Coprinus is found to be polyphyletic and is separated into three distinct clades. Most Coprinus taxa belong to the first two clades, which together form a larger monophyletic group with Lacrymaria and Psathyrella in basal positions. A third clade contains members of Coprinus section Comati as well as the genus Leucocoprinus, Podaxis pistillaris, Montagnea arenaria, and Agaricus pocillator. This third clade is separated from the other species of Coprinus by members of the families Strophariaceae and Bolbitiaceae and the genus Panaeolus. Copyright 1999 Academic Press.

Diverse molecular signatures for ribosomally ‘active’ Perkinsea in marine sediments

PubMed Central

2014-01-01

Background Perkinsea are a parasitic lineage within the eukaryotic superphylum Alveolata. Recent studies making use of environmental small sub-unit ribosomal RNA gene (SSU rDNA) sequencing methodologies have detected a significant diversity and abundance of Perkinsea-like phylotypes in freshwater environments. In contrast only a few Perkinsea environmental sequences have been retrieved from marine samples and only two groups of Perkinsea have been cultured and morphologically described and these are parasites of marine molluscs or marine protists. These two marine groups form separate and distantly related phylogenetic clusters, composed of closely related lineages on SSU rDNA trees. Here, we test the hypothesis that Perkinsea are a hitherto under-sampled group in marine environments. Using 454 diversity ‘tag’ sequencing we investigate the diversity and distribution of these protists in marine sediments and water column samples taken from the Deep Chlorophyll Maximum (DCM) and sub-surface using both DNA and RNA as the source template and sampling four European offshore locations. Results We detected the presence of 265 sequences branching with known Perkinsea, the majority of them recovered from marine sediments. Moreover, 27% of these sequences were sampled from RNA derived cDNA libraries. Phylogenetic analyses classify a large proportion of these sequences into 38 cluster groups (including 30 novel marine cluster groups), which share less than 97% sequence similarity suggesting this diversity encompasses a range of biologically and ecologically distinct organisms. Conclusions These results demonstrate that the Perkinsea lineage is considerably more diverse than previously detected in marine environments. This wide diversity of Perkinsea-like protists is largely retrieved in marine sediment with a significant proportion detected in RNA derived libraries suggesting this diversity represents ribosomally ‘active’ and intact cells. Given the phylogenetic range of hosts infected by known Perkinsea parasites, these data suggest that Perkinsea either play a significant but hitherto unrecognized role as parasites in marine sediments and/or members of this group are present in the marine sediment possibly as part of the ‘seed bank’ microbial community. PMID:24779375

Diversity and Fermentation Products of Xylose-Utilizing Yeasts Isolated from Buffalo Feces in Thailand

PubMed Central

Lorliam, Wanlapa; Akaracharanya, Ancharida; Suzuki, Motofumi; Ohkuma, Moriya; Tanasupawat, Somboon

2013-01-01

Twenty-eight xylose-utilizing yeast strains were isolated by enrichment culture from 11 samples of feces from the rectum of Murrah buffalo and Swamp buffalo in Thailand. On the basis of their morphological and biochemical characteristics, including sequence analysis of the D1/D2 region of the large-subunit ribosomal RNA gene (LSU rDNA), they were identified as Candida tropicalis (designated as Group I, 11 isolates), Candida parasilosis (Group II, 2 isolates), Candida mengyuniae (Group III, 2 isolates), Sporopachydermia lactativora (Group IV, 2 isolates), Geotrichum sp. (Group V, 5 isolates) and Trichosporon asahii (Group VI, 6 isolates). All isolates utilized xylose as the sole carbon source but 27 isolates could ferment xylose to ethanol (0.006–0.602 g L−1) and 21 isolates could ferment xylose to xylitol (0.19–22.84 g L−1). Candida tropicalis isolates produced the highest yield of xylitol (74.80%). Their ability to convert xylose to xylitol and ethanol ranged from 15.06 g L−1 to 22.84 g L−1 xylitol and 0.110 g L−1 to 0.602 g L−1 ethanol, respectively. PMID:24005843

Two Lactarius species associated with a relict Fagus grandifolia var. mexicana population in a Mexican montane cloud forest.

PubMed

Montoya, L; Haug, I; Bandala, V M

2010-01-01

Ectomycorrhizal (EM) fleshy fungi are being monitored in a population of Fagus grandifolia var. mexicana persisting in a montane cloud forest refuge on a volcano in a subtropical region of central Veracruz (eastern Mexico). The population of Fagus studied represents one of the 10 recognized forest fragments still housing this tree genus in Mexico. This is the first attempt to document EM fungi associated with this tree species in Mexico. We present evidence of the ectomycorrhizal symbiosis for Lactarius badiopallescens and L. cinereus with this endemic tree. Species identification of Lactarius on Fagus grandifolia var. mexicana was based on the comparison of DNAsequences (ITS rDNA) of spatiotemporally co-occurring basidiomes and EM root tips. The host of the EM tips was identified by comparison of the large subunit of the ribulose-bisphosphate carboxylase gene (rbcL). The occurrence of Lactarius badiopallescens and L. cinereus populations in the area of study represent the southernmost record known to date of these two species in North America and are new for the Neotropical Lactarius mycota. Descriptions coupled with illustrations of macro- and micromorphological features of basidiomes as well as photographs of ectomycorrhizas are presented.

Non-Random Distribution of 5S rDNA Sites and Its Association with 45S rDNA in Plant Chromosomes.

PubMed

Roa, Fernando; Guerra, Marcelo

2015-01-01

5S and 45S rDNA sites are the best mapped chromosome regions in eukaryotic chromosomes. In this work, a database was built gathering information about the position and number of 5S rDNA sites in 784 plant species, aiming to identify patterns of distribution along the chromosomes and its correlation with the position of 45S rDNA sites. Data revealed that in most karyotypes (54.5%, including polyploids) two 5S rDNA sites (a single pair) are present, with 58.7% of all sites occurring in the short arm, mainly in the proximal region. In karyotypes of angiosperms with only 1 pair of sites (single sites) they are mostly found in the proximal region (52.0%), whereas in karyotypes with multiple sites the location varies according to the average chromosome size. Karyotypes with multiple sites and small chromosomes (<3 µm) often display proximal sites, while medium-sized (between 3 and 6 µm) and large chromosomes (>6 µm) more commonly show terminal or interstitial sites. In species with holokinetic chromosomes, the modal value of sites per karyotype was also 2, but they were found mainly in a terminal position. Adjacent 5S and 45S rDNA sites were often found in the short arm, reflecting the preferential distribution of both sites in this arm. The high frequency of genera with at least 1 species with adjacent 5S and 45S sites reveals that this association appeared several times during angiosperm evolution, but it has been maintained only rarely as the dominant array in plant genera. © 2015 S. Karger AG, Basel.

Detection of Methanotroph Diversity on Roots of Submerged Rice Plants by Molecular Retrieval of pmoA, mmoX, mxaF, and 16S rRNA and Ribosomal DNA, Including pmoA-Based Terminal Restriction Fragment Length Polymorphism Profiling

PubMed Central

Horz, Hans-Peter; Yimga, Merlin Tchawa; Liesack, Werner

2001-01-01

The diversity of methanotrophic bacteria associated with roots of submerged rice plants was assessed using cultivation-independent techniques. The research focused mainly on the retrieval of pmoA, which encodes the α subunit of the particulate methane monooxygenase. A novel methanotroph-specific community-profiling method was established using the terminal restriction fragment length polymorphism (T-RFLP) technique. The T-RFLP profiles clearly revealed a more complex root-associated methanotrophic community than did banding patterns obtained by pmoA-based denaturing gradient gel electrophoresis. The comparison of pmoA-based T-RFLP profiles obtained from rice roots and bulk soil of flooded rice microcosms suggested that there was a substantially higher abundance of type I methanotrophs on rice roots than in the bulk soil. These were affiliated to the genera Methylomonas, Methylobacter, Methylococcus, and to a novel type I methanotroph sublineage. By contrast, type II methanotrophs of the Methylocystis-Methylosinus group could be detected with high relative signal intensity in both soil and root compartments. Phylogenetic treeing analyses and a set of substrate-diagnostic amino acid residues provided evidence that a novel pmoA lineage was detected. This branched distinctly from all currently known methanotrophs. To examine whether the retrieval of pmoA provided a complete view of root-associated methanotroph diversity, we also assessed the diversity detectable by recovery of genes coding for subunits of soluble methane monooxygenase (mmoX) and methanol dehydrogenase (mxaF). In addition, both 16S rRNA and 16S ribosomal DNA (rDNA) were retrieved using a PCR primer set specific to type I methanotrophs. The overall methanotroph diversity detected by recovery of mmoX, mxaF, and 16S rRNA and 16S rDNA corresponded well to the diversity detectable by retrieval of pmoA. PMID:11526021

Cloning, Purification, and Characterization of a Heterodimeric β-Galactosidase from Lactobacillus kefiranofaciens ZW3.

PubMed

He, Xi; Han, Ning; Wang, Yan-Ping

2016-01-01

Lactobacillus kefiranofaciens ZW3 was obtained from kefir grains, which have high lactose hydrolytic activity. In this study, a heterodimeric LacLM-type β-galactosidase gene (lacLM) from ZW3 was isolated, which was composed of two overlapping genes, lacL (1,884 bp) and lacM (960 bp) encoding large and small subunits with calculated molecular masses of 73,620 and 35,682 Da, respectively. LacLM, LacL, and LacM were expressed in Escherichia coli BL21(DE3) and these recombinant proteins were purified and characterized. The results showed that, compared with the recombinant holoenzyme, the recombinant large subunit exhibits obviously lower thermostability and hydrolytic activity. Moreover, the optimal temperature and pH of the holoenzyme and large subunit are 60°C and 7.0, and 50°C and 8.0, respectively. However, the recombinant small subunit alone has no activity. Interestingly, the activity and thermostability of the large subunit were greatly improved after mixing it with the recombinant small subunit. Therefore, the results suggest that the small subunit might play an important role in maintaining the stability of the structure of the catalytic center located in the large subunit.

Revision of torrent mites (Parasitengona, Torrenticolidae, Torrenticola) of the United States and Canada: 90 descriptions, molecular phylogenetics, and a key to species

PubMed Central

Fisher, J. Ray; Fisher, Danielle M.; Skvarla, Michael J.; Nelson, Whitney A.; Dowling, Ashley P.G.

2017-01-01

Abstract The descriptive biology of torrent mites (Parasitengona: Torrenticolidae: Torrenticola) of North America (north of Mexico) is investigated using integrative methods. Material examined includes approximately 2,300 specimens from nearly 500 localities across the United States and Canada, and a few collections in Mexico and Central America. Species hypotheses are derived from a phylogenetic analysis of the barcoding region of cytochrome c oxidase subunit 1 (COI) for 476 specimens and supported with morphology and biogeography. Relationships between species are examined with a combined analysis of COI and two expansion regions (D2–3) of the large ribosomal subunit (28S rDNA) for 57 specimens. All previously described species from the US and Canada are examined. Our results indicate the need to synonymize four species: T. mercedensis (Marshall, 1943) is a junior synonym of T. sierrensis (Marshall, 1943); T. rectiforma Habeeb, 1974 is a junior synonym of T. ellipsoidalis (Marshall, 1943); T. neoconnexa Habeeb, 1957 is a junior synonym of T. magnexa Habeeb, 1955; and T. esbelta Cramer, 1992 is a junior synonym of T. boettgeri KO Viets, 1977. We describe 66 new species and re-describe all previously described regional species. Our findings indicate that total diversity of Torrenticola in the United States and Canada comprises 90 species, 57 known from the east and 33 from the west. We organize these species into four species complexes that include 13 identification groups. An additional 13 species do not fit within an identification group. The southern Appalachians are suspected to contain the highest concentration of remaining undescribed diversity. A key is provided to all known species in the US and Canada. PMID:29118596

Morphological and molecular data for a new species of Pomphorhynchus Monticelli, 1905 (Acanthocephala: Pomphorhynchidae) in the Mexican redhorse Moxostoma austrinum Bean (Cypriniformes: Catostomidae) in central Mexico.

PubMed

García-Varela, Martín; Mendoza-Garfias, Berenit; Choudhury, Anindo; Pérez-Ponce de León, Gerardo

2017-11-01

Pomphorhynchus purhepechus n. sp. is described from the intestine of the Mexican redhorse Moxostoma austrinum Bean (Catostomidae) in central Mexico. The new species can be distinguished from the other seven described species of Pomphorhynchus Monticelli, 1905 in the Americas by a subspherical proboscis and 14 longitudinal rows with 16-18 hooks each; the third and the fourth row of hooks are alternately longest. Sequences of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene and the large subunit (LSU) rDNA (including the domains D2-D3) were used to corroborate the morphological distinction between the new species and Pomphorhynchus bulbocolli Linkins in Van Cleave, 1919, a species widely distributed in several freshwater fish species across Canada, USA, and Mexico. The genetic divergence estimated between the new species and the isolates of P. bulbocolli ranged between 13 and 14% for cox1, and between 0.6 and 0.8% for LSU. Maximum likelihood and Bayesian inference analyses of each dataset showed that the isolates of P. bulbocolli parasitising freshwater fishes from three families, the Catostomidae, Cyprinidae and Centrarchidae, represent a separate lineage, and that the acanthocephalans collected from two localities in central Mexico comprise an independent lineage. In addition, our analysis of the genetic variation of P. bulbocolli demonstrates that individuals of this acanthocephalan from different host species are conspecific. Finally, the distribution, host-association, and phylogenetic relationship of the new species, when placed in the context of the region's geological history, suggest that both host and parasite underwent speciation after their ancestors became isolated in Central Mexico.

Higher-order organisation of extremely amplified, potentially functional and massively methylated 5S rDNA in European pikes (Esox sp.).

PubMed

Symonová, Radka; Ocalewicz, Konrad; Kirtiklis, Lech; Delmastro, Giovanni Battista; Pelikánová, Šárka; Garcia, Sonia; Kovařík, Aleš

2017-05-18

Pikes represent an important genus (Esox) harbouring a pre-duplication karyotype (2n = 2x = 50) of economically important salmonid pseudopolyploids. Here, we have characterized the 5S ribosomal RNA genes (rDNA) in Esox lucius and its closely related E. cisalpinus using cytogenetic, molecular and genomic approaches. Intragenomic homogeneity and copy number estimation was carried out using Illumina reads. The higher-order structure of rDNA arrays was investigated by the analysis of long PacBio reads. Position of loci on chromosomes was determined by FISH. DNA methylation was analysed by methylation-sensitive restriction enzymes. The 5S rDNA loci occupy exclusively (peri)centromeric regions on 30-38 acrocentric chromosomes in both E. lucius and E. cisalpinus. The large number of loci is accompanied by extreme amplification of genes (>20,000 copies), which is to the best of our knowledge one of the highest copy number of rRNA genes in animals ever reported. Conserved secondary structures of predicted 5S rRNAs indicate that most of the amplified genes are potentially functional. Only few SNPs were found in genic regions indicating their high homogeneity while intergenic spacers were more heterogeneous and several families were identified. Analysis of 10-30 kb-long molecules sequenced by the PacBio technology (containing about 40% of total 5S rDNA) revealed that the vast majority (96%) of genes are organised in large several kilobase-long blocks. Dispersed genes or short tandems were less common (4%). The adjacent 5S blocks were directly linked, separated by intervening DNA and even inverted. The 5S units differing in the intergenic spacers formed both homogeneous and heterogeneous (mixed) blocks indicating variable degree of homogenisation between the loci. Both E. lucius and E. cisalpinus 5S rDNA was heavily methylated at CG dinucleotides. Extreme amplification of 5S rRNA genes in the Esox genome occurred in the absence of significant pseudogenisation suggesting its recent origin and/or intensive homogenisation processes. The dense methylation of units indicates that powerful epigenetic mechanisms have evolved in this group of fish to silence amplified genes. We discuss how the higher-order repeat structures impact on homogenisation of 5S rDNA in the genome.

Evolutionary Dynamics of 5S rDNA and Recurrent Association of Transposable Elements in Electric Fish of the Family Gymnotidae (Gymnotiformes): The Case of Gymnotus mamiraua.

PubMed

da Silva, Maelin; Barbosa, Patricia; Artoni, Roberto F; Feldberg, Eliana

2016-01-01

Gymnotidae is a family of electric fish endemic to the Neotropics consisting of 2 genera: Electrophorus and Gymnotus. The genus Gymnotus is widely distributed and is found in all of the major Brazilian river systems. Physical and molecular mapping data for the ribosomal DNA (rDNA) in this genus are still scarce, with its chromosomal location known in only 11 species. As other species of Gymnotus with 2n = 54 chromosomes from the Paraná-Paraguay basin, G. mamiraua was found to have a large number of 5S rDNA sites. Isolation and cloning of the 5S rDNA sequences from G. mamiraua identified a fragment of a transposable element similar to the Tc1/mariner transposon associated with a non-transcribed spacer. Double fluorescence in situ hybridization analysis of this element and the 5S rDNA showed that they were colocalized on several chromosomes, in addition to acting as nonsyntenic markers on others. Our data show the association between these sequences and suggest that the Tc1 retrotransposon may be the agent that drives the spread of these 5S rDNA-like sequences in the G. mamiraua genome. © 2016 S. Karger AG, Basel.

Palmitoylation of the β4-Subunit Regulates Surface Expression of Large Conductance Calcium-activated Potassium Channel Splice Variants*

PubMed Central

Chen, Lie; Bi, Danlei; Tian, Lijun; McClafferty, Heather; Steeb, Franziska; Ruth, Peter; Knaus, Hans Guenther; Shipston, Michael J.

2013-01-01

Regulatory β-subunits of large conductance calcium- and voltage-activated potassium (BK) channels play an important role in generating functional diversity and control of cell surface expression of the pore forming α-subunits. However, in contrast to α-subunits, the role of reversible post-translational modification of intracellular residues on β-subunit function is largely unknown. Here we demonstrate that the human β4-subunit is S-acylated (palmitoylated) on a juxtamembrane cysteine residue (Cys-193) in the intracellular C terminus of the regulatory β-subunit. β4-Subunit palmitoylation is important for cell surface expression and endoplasmic reticulum (ER) exit of the β4-subunit alone. Importantly, palmitoylated β4-subunits promote the ER exit and surface expression of the pore-forming α-subunit, whereas β4-subunits that cannot be palmitoylated do not increase ER exit or surface expression of α-subunits. Strikingly, however, this palmitoylation- and β4-dependent enhancement of α-subunit surface expression was only observed in α-subunits that contain a putative trafficking motif (… REVEDEC) at the very C terminus of the α-subunit. Engineering this trafficking motif to other C-terminal α-subunit splice variants results in α-subunits with reduced surface expression that can be rescued by palmitoylated, but not depalmitoylated, β4-subunits. Our data reveal a novel mechanism by which palmitoylated β4-subunit controls surface expression of BK channels through masking of a trafficking motif in the C terminus of the α-subunit. As palmitoylation is dynamic, this mechanism would allow precise control of specific splice variants to the cell surface. Our data provide new insights into how complex interplay between the repertoire of post-transcriptional and post-translational mechanisms controls cell surface expression of BK channels. PMID:23504458

Host specificity and basic ecology of Mammomonogamus (Nematoda, Syngamidae) from lowland gorillas and forest elephants in Central African Republic.

PubMed

Červená, Barbora; Vallo, Peter; Pafčo, Barbora; Jirků, Kateřina; Jirků, Miloslav; Petrželková, Klára Judita; Todd, Angelique; Turkalo, Andrea K; Modrý, David

2017-07-01

Syngamid strongylids of the genus Mammomonogamus undoubtedly belong among the least known nematodes with apparent zoonotic potential and the real diversity of the genus remains hard to evaluate without extensive molecular data. Eggs of Mammomonogamus sp. are frequently found in feces of African forest elephants (Loxodonta cyclotis) and western lowland gorillas (Gorilla gorilla gorilla) in Dzanga-Sangha Protected Areas. Using sedimentation-based coproscopic techniques, we found the eggs of Mammomonogamus in 19·7% elephant and 54·1% gorilla fecal samples with 8-55 and 1-24 eggs per gram of fecal sediment for elephants and gorillas, respectively. We used a combination of light microscopy, scanning electron microscopy and analysis of cytochrome c oxidase subunit I (cox1) and a partial sequence of 18S rDNA isolated from single eggs to test the hypothesis of possible Mammomonogamus conspecificity in gorillas and elephants. Whereas 18S rDNA sequences were identical in both gorillas and elephants, we distinguished seven different haplotypes within the cox1. Two haplotypes were found in both gorillas and elephants suggesting sharing of Mammomonogamus. Assignment of the parasite to M. loxodontis is proposed. Provided sequences represent the first genomic data on Mammomonogamus spp.

A parvicapsulid (Myxozoa) infecting Sprattus sprattus and Clupea harengus (Clupeidae) in the Northeast Atlantic uses Hydroides norvegicus (Serpulidae) as invertebrate host.

PubMed

Køie, Marianne; Karlsbakk, Egil; Einen, Ann-Cathrine Bårdsgjtere; Nylund, Are

2013-05-01

A myxosporean producing actinospores of the tetractinomyxon type in Hydroides norvegicus Gunnerus (Serpulidae) in Denmark was identified as a member of the family Parvicapsulidae based on small-subunit ribosomal DNA (SSU rDNA) sequences. Myxosporean samples from various Danish and Norwegian marine fishes were examined with primers that detect the novel myxosporean. Sprattus sprattus (Linnaeus) and Clupea harengus Linnaeus (Teleostei, Clupeidae) were found to be infected. The sequences of this parvicapsulid from these hosts were consistently slightly different (0.8% divergence), but both these genotypes were found in H. norvegicus. Disporic trophozoites and minute spores of a novel myxosporean type were observed in the renal tubules of some of the hosts found infected through PCR. The spores appear most similar to those of species of Gadimyxa Køie, Karlsbakk et Nylund, 2007, but are much smaller. The actinospores of the tetractinomyxon type from H. norvegicus have been described previously. In GenBank, the SSU rDNA sequences of Parvicapsulidae gen. sp. show highest identity (82%) with Parvicapsula minibicornis Kent, Whitaker et Dawe, 1997 infecting salmonids (Oncorhynchus spp.) in fresh water in the western North America. A phylogenetic analysis places P. minibicornis and Parvicapsulidae gen. sp. in a sister clade to the other parvicapsulids (Parvicapsula spp. and Gadimyxa spp.).

The LSH/HELLS homolog Irc5 contributes to cohesin association with chromatin in yeast.

PubMed

Litwin, Ireneusz; Bakowski, Tomasz; Maciaszczyk-Dziubinska, Ewa; Wysocki, Robert

2017-06-20

Accurate chromosome segregation is essential for every living cell as unequal distribution of chromosomes during cell division may result in genome instability that manifests in carcinogenesis and developmental disorders. Irc5 from Saccharomyces cerevisiae is a member of the conserved Snf2 family of ATP-dependent DNA translocases and its function is poorly understood. Here, we identify Irc5 as a novel interactor of the cohesin complex. Irc5 associates with Scc1 cohesin subunit and contributes to cohesin binding to chromatin. Disruption of IRC5 decreases cohesin levels at centromeres and chromosome arms, causing premature sister chromatid separation. Moreover, reduced cohesin occupancy at the rDNA region in cells lacking IRC5 leads to the loss of rDNA repeats. We also show that the translocase activity of Irc5 is required for its function in cohesion pathway. Finally, we demonstrate that in the absence of Irc5 both the level of chromatin-bound Scc2, a member of cohesin loading complex, and physical interaction between Scc1 and Scc2 are reduced. Our results suggest that Irc5 is an auxiliary factor that is involved in cohesin association with chromatin. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Focal accumulation of preribosomes outside the nucleolus during metaphase-anaphase in budding yeast.

PubMed

Moriggi, Giulia; Gaspar, Sonia G; Nieto, Blanca; Bustelo, Xosé R; Dosil, Mercedes

2017-09-01

Saccharomyces cerevisiae contains one nucleolus that remains intact in the mother-cell side of the nucleus throughout most of mitosis. Based on this, it is assumed that the bulk of ribosome production during cell division occurs in the mother cell. Here, we show that the ribosome synthesis machinery localizes not only in the nucleolus but also at a center that is present in the bud side of the nucleus after the initiation of mitosis. This center can be visualized by live microscopy as a punctate body located in close proximity to the nuclear envelope and opposite to the nucleolus. It contains ribosomal DNA (rDNA) and precursors of both 40S and 60S ribosomal subunits. Proteins that actively participate in ribosome synthesis, but not functionally defective variants, accumulate in that site. The formation of this body occurs in the metaphase-to-anaphase transition when discrete regions of rDNA occasionally exit the nucleolus and move into the bud. Collectively, our data unveil the existence of a previously unknown mechanism for preribosome accumulation at the nuclear periphery in budding yeast. We propose that this might be a strategy to expedite the delivery of ribosomes to the growing bud. © 2017 Moriggi et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Development of species-specific rDNA probes for Giardia by multiple fluorescent in situ hybridization combined with immunocytochemical identification of cyst wall antigens.

PubMed

Erlandsen, Stanley L; Jarroll, Edward; Wallis, Peter; van Keulen, Harry

2005-08-01

In this study, we describe the development of fluorescent oligonucleotide probes to variable regions in the small subunit of 16S rRNA in three distinct Giardia species. Sense and antisense probes (17-22 mer) to variable regions 1, 3, and 8 were labeled with digoxygenin or selected fluorochomes (FluorX, Cy3, or Cy5). Optimal results were obtained with fluorochome-labeled oligonucleotides for detection of rRNA in Giardia cysts. Specificity of fluorescent in situ hybridization (FISH) was shown using RNase digestion and high stringency to diminish the hybridization signal, and oligonucleotide probes for rRNA in Giardia lamblia, Giardia muris, and Giardia ardeae were shown to specifically stain rRNA only within cysts or trophozoites of those species. The fluorescent oligonucleotide specific for rRNA in human isolates of Giardia was positive for ten different strains. A method for simultaneous FISH detection of cysts using fluorescent antibody (genotype marker) and two oligonucleotide probes (species marker) permitted visualization of G. lamblia and G. muris cysts in the same preparation. Testing of an environmental water sample revealed the presence of FISH-positive G. lamblia cysts with a specific rDNA probe for rRNA, while negative cysts were presumed to be of animal or bird origin.

Candida kashinagacola sp. nov., C. pseudovanderkliftii sp. nov. and C. vanderkliftii sp. nov., three new yeasts from ambrosia beetle-associated sources.

PubMed

Endoh, Rikiya; Suzuki, Motofumi; Benno, Yoshimi; Futai, Kazuyoshi

2008-10-01

Three new yeast species, Candida kashinagacola (JCM 15019(T) = CBS 10903(T)), C. pseudovanderkliftii (JCM 15025(T) = CBS 10904(T)), and C. vanderkliftii (JCM 15029(T) = CBS 10905(T)) are described on the basis of comparison of nucleotide sequences of large subunit ribosomal DNA D1/D2 region (LSU rDNA D1/D2). The nearest assigned species of the three new species was Candida llanquihuensis. Candida kashinagacola and C. pseudovanderkliftii differed from C. llanquihuensis by 3.8% nucleotide substitution of the region, while C. vanderkliftii did by 4.4%. Three new species differed in a number of physiological and growth characteristics from any previously assigned species and from one another. A phylogenetic tree based on the sequences of LSU rDNA D1/D2 showed that these new species together with Candida sp. ST-246, Candida sp. JW01-7-11-1-4-y2, Candida sp. BG02-7-20-001A-2-1 and C. llanquihuensis form a clade near Ambrosiozyma species. The new species did not assimilate methanol as a sole source of carbon, which supported the monophyly of these non methanol-assimilating species which are closely related to the methylotrophic yeasts. Candida kashinagacola was frequently isolated from the beetle galleries of Platypus quercivorus in three different host trees (Quercus serrata, Q. laurifolia and Castanopsis cuspidata) located in the sourthern part of Kyoto, Japan, thus indicating that this species may be a primary ambrosia fungus of P. quercivorus. On the other hand, C. pseudovanderkliftii and C. vanderkliftii were isolated only from beetle galleries in Q. laurifolia. Candida vanderkliftii was isolated from beetle gallery of Platypus lewisi as well as those of P. quercivorus. Candida pseudovanderkliftii and C. vanderkliftii are assumed to be auxiliary ambrosia fungi of P. quercivorus.

Kudoa hypoepicardialis n. sp. (Myxozoa: Kudoidae) and associated lesions from the heart of seven perciform fishes in the northern Gulf of Mexico.

PubMed

Blaylock, Reginald B; Bullard, Stephen A; Whipps, Christopher M

2004-06-01

Kudoa hypoepicardialis n. sp. infects the space between the epicardium and the compact myocardium and, in intense infections, the pericardial chamber of man-of-war fish (Nomeus gronovii) (Nomeidae) (the type host), blue runner (Caranx crysos) (Carangidae), Warsaw grouper (Epinephelus nigritus) (Serranidae), Atlantic tripletail (Lobotes surinamensis) (Lobotidae), northern red snapper (Lutjanus campechanus) (Lutjanidae), black drum (Pogonias cromis) (Sciaenidae), and bluefish (Pomatomus saltatrix) (Pomatomidae) in the northern Gulf of Mexico. This is the first report of a Kudoa sp. from the heart of a fish in the Gulf of Mexico, and of these hosts, only the bluefish was previously identified as a host for a species of Kudoa. Spores of the new species varied slightly in size among these hosts but were regarded as conspecific based on their nearly identical (99.9%) small-subunit (SSU) ribosomal DNA (rDNA) sequence. The new species differs both from the 4 nominal species of Kudoa reported from fishes in the Gulf of Mexico and from K. pericardialis, an allopatric species that infects the pericardial cavity, by the combination of having a large spore, a small polar capsule, and a polar filament with a single coil. The new species is morphologically and genetically most similar to K. shiomitsui, an allopatric species that infects the heart and pericardial cavity, but is distinguished from it based on a 4.2% difference in the SSU rDNA sequence. Heart lesions primarily were restricted to the vicinity of plasmodia and included a layer of fibrinous inflammation characterized by lymphocytes, macrophages, and granulomas as well as epithelioid encapsulations around plasmodia. Heavily infected hosts had melanin-like deposits and adipose cells beneath the epicardium. and the epicardium was discontinuous and apparently breached by plasmodia in some regions. Cardiac muscle, gill, liver, spleen, intestine, and kidney were normal.

Characterization of Paecilomyces variotii and Talaromyces amestolkiae in Korea Based on the Morphological Characteristics and Multigene Phylogenetic Analyses

PubMed Central

Nguyen, Thi Thuong Thuong; Paul, Narayan Chandra

2016-01-01

During fungal diversity surveys of the order Eurotiales in Korea, two fungal strains, EML-DG33-1 and EML-NCP50, were isolated from samples of rat dung and fig tree leaf collected at a garden located in Gwangju in 2014. To complete the National Species List of Korea, it is a prerequisite to verify whether many questionable species, which were previously recorded but not confirmed, indeed present in Korea. Herein, the isolates were confirmed as undescribed species, Paecilomyces variotii and Talaromyces amestolkiae based on the combination of morphological and phylogenetic analyses of multigenes including the rDNA internal transcribed spacer, β-tubulin, and RNA polymerase II subunit 2. PMID:28154482

Protein purification in multicompartment electrolyzers for crystal growth of r-DNA products in microgravity

NASA Technical Reports Server (NTRS)

Righetti, Pier Giorgio; Casale, Elena; Carter, Daniel; Snyder, Robert S.; Wenisch, Elisabeth; Faupel, Michel

1990-01-01

Recombinant-DNA (deoxyribonucleic acid) (r-DNA) proteins, produced in large quantities for human consumption, are now available in sufficient amounts for crystal growth. Crystallographic analysis is the only method now available for defining the atomic arrangements within complex biological molecules and decoding, e.g., the structure of the active site. Growing protein crystals in microgravity has become an important aspect of biology in space, since crystals that are large enough and of sufficient quality to permit complete structure determinations are usually obtained. However even small amounts of impurities in a protein preparation are anathema for the growth of a regular crystal lattice. A multicompartment electrolyzer with isoelectric, immobiline membranes, able to purify large quantities of r-DNA proteins is described. The electrolyzer consists of a stack of flow cells, delimited by membranes of very precise isoelectric point (pI, consisting of polyacrylamide supported by glass fiber filters containing Immobiline buffers and titrants to uniquely define a pI value) and very high buffering power, able to titrate all proteins tangent or crossing such membranes. By properly selecting the pI values of two membranes delimiting a flow chamber, a single protein can be kept isoelectric in a single flow chamber and thus, be purified to homogeneity (by the most stringent criterion, charge homogeneity).

Methylation Pattern of Radish (Raphanus sativus) Nuclear Ribosomal RNA Genes 1

PubMed Central

Delseny, Michel; Laroche, Monique; Penon, Paul

1984-01-01

The methylation pattern of radish Raphanus sativus nuclear rDNA has been investigated using the Hpa II, Msp I, and Hha I restriction enzymes. The presence of numerous target sites for these enzymes has been shown using cloned rDNA fragments. A large fraction of the numerous rDNA units are heavily methylated, being completely resistant to Hpa II and Hpa I. However, specific sites are constantly available in another fraction of the units and are therefore unmethylated. The use of different probes allowed us to demonstrate that hypomethylated sites are present in different regions. Major hypomethylated Hha I sites have been mapped in the 5′ portion of 25S rRNA coding sequence. Among the hypomethylated fraction, different methylation patterns coexist. It has been possible to demonstrate that methylation patterns are specific for particular units. The Hha I pattern of rDNA in tissues of different developmental stages was analyzed. Evidence for possible tissue specific differences in the methylation pattern is reported. Images Fig. 2 Fig. 3 Fig. 5 PMID:16663896

Ribosomal DNA, tri- and bi-partite pericentromeres in the permanent translocation heterozygote Rhoeo spathacea.

PubMed

Golczyk, Hieronim; Hasterok, Robert; Szklarczyk, Marek

2010-12-01

High- and low-stringency FISH and base-specific fluorescence were performed on the permanent translocation heterozygote Rhoeo spathacea (2n = 12). Our results indicate that 45S rDNA arrays, rDNA-related sequences and other GC-rich DNA fraction(s) are located within the pericentromeric regions of all twelve chromosomes, usually colocalizing with the chromomycin A(3)-positive bands. Homogenization of the pericentromeric regions appears to result from the concerted spread of GC-rich sequences, with differential amplification likely. We found new 5S rDNA patterns, which suggest a variability in the breakpoints and in the consequent chromosome reorganizations. It was found that the large 5S rDNA locus residing on each of the 8E and 9E arms consisted of two smaller loci. On each of the two chromosome arms 3b and 4b, in addition to the major subtelomeric 5S rDNA locus, a new minor locus was found interstitially about 40% along the arm length. The arrangement of cytotogenetic landmarks and chromosome arm measurements are discussed with regard to genome repatterning in Rhoeo.

Ontogeny-Driven rDNA Rearrangement, Methylation, and Transcription, and Paternal Influence

PubMed Central

Shiao, Yih-Horng; Leighty, Robert M.; Wang, Cuiju; Ge, Xin; Crawford, Erik B.; Spurrier, Joshua M.; McCann, Sean D.; Fields, Janet R.; Fornwald, Laura; Riffle, Lisa; Driver, Craig; Quiñones, Octavio A.; Wilson, Ralph E.; Kasprzak, Kazimierz S.; Travlos, Gregory S.; Alvord, W. Gregory; Anderson, Lucy M.

2011-01-01

Gene rearrangement occurs during development in some cell types and this genome dynamics is modulated by intrinsic and extrinsic factors, including growth stimulants and nutrients. This raises a possibility that such structural change in the genome and its subsequent epigenetic modifications may also take place during mammalian ontogeny, a process undergoing finely orchestrated cell division and differentiation. We tested this hypothesis by comparing single nucleotide polymorphism-defined haplotype frequencies and DNA methylation of the rDNA multicopy gene between two mouse ontogenic stages and among three adult tissues of individual mice. Possible influences to the genetic and epigenetic dynamics by paternal exposures were also examined for Cr(III) and acid saline extrinsic factors. Variables derived from litters, individuals, and duplicate assays in large mouse populations were examined using linear mixed-effects model. We report here that active rDNA rearrangement, represented by changes of haplotype frequencies, arises during ontogenic progression from day 8 embryos to 6-week adult mice as well as in different tissue lineages and is modifiable by paternal exposures. The rDNA methylation levels were also altered in concordance with this ontogenic progression and were associated with rDNA haplotypes. Sperm showed highest level of methylation, followed by lungs and livers, and preferentially selected haplotypes that are positively associated with methylation. Livers, maintaining lower levels of rDNA methylation compared with lungs, expressed more rRNA transcript. In vitro transcription demonstrated haplotype-dependent rRNA expression. Thus, the genome is also dynamic during mammalian ontogeny and its rearrangement may trigger epigenetic changes and subsequent transcriptional controls, that are further influenced by paternal exposures. PMID:21765958

Bacterial community composition in different sediments from the Eastern Mediterranean Sea: a comparison of four 16S ribosomal DNA clone libraries.

PubMed

Polymenakou, Paraskevi N; Bertilsson, Stefan; Tselepides, Anastasios; Stephanou, Euripides G

2005-10-01

The regional variability of sediment bacterial community composition and diversity was studied by comparative analysis of four large 16S ribosomal DNA (rDNA) clone libraries from sediments in different regions of the Eastern Mediterranean Sea (Thermaikos Gulf, Cretan Sea, and South lonian Sea). Amplified rDNA restriction analysis of 664 clones from the libraries indicate that the rDNA richness and evenness was high: for example, a near-1:1 relationship among screened clones and number of unique restriction patterns when up to 190 clones were screened for each library. Phylogenetic analysis of 207 bacterial 16S rDNA sequences from the sediment libraries demonstrated that Gamma-, Delta-, and Alphaproteobacteria, Holophaga/Acidobacteria, Planctomycetales, Actinobacteria, Bacteroidetes, and Verrucomicrobia were represented in all four libraries. A few clones also grouped with the Betaproteobacteria, Nitrospirae, Spirochaetales, Chlamydiae, Firmicutes, and candidate division OPl 1. The abundance of sequences affiliated with Gammaproteobacteria was higher in libraries from shallow sediments in the Thermaikos Gulf (30 m) and the Cretan Sea (100 m) compared to the deeper South Ionian station (2790 m). Most sequences in the four sediment libraries clustered with uncultured 16S rDNA phylotypes from marine habitats, and many of the closest matches were clones from hydrocarbon seeps, benzene-mineralizing consortia, sulfate reducers, sulk oxidizers, and ammonia oxidizers. LIBSHUFF statistics of 16S rDNA gene sequences from the four libraries revealed major differences, indicating either a very high richness in the sediment bacterial communities or considerable variability in bacterial community composition among regions, or both.

Heterochromatin and rDNA sites distribution in the holocentric chromosomes of Cuscuta approximata Bab. (Convolvulaceae).

PubMed

Guerra, Marcelo; García, Miguel A

2004-02-01

Cuscuta is a widely distributed genus of holoparasitic plants. Holocentric chromosomes have been reported only in species of one of its subgenera (Cuscuta subg. Cuscuta). In this work, a representative of this subgenus, Cuscuta approximata, was investigated looking for its mitotic and meiotic chromosome behaviour and the heterochromatin distribution. The mitotic chromosomes showed neither primary constriction nor Rabl orientation whereas the meiotic ones exhibited the typical quadripartite structure characteristic of holocentrics, supporting the assumption of holocentric chromosomes as a synapomorphy of Cuscuta subg. Cuscuta. Chromosomes and interphase nuclei displayed many heterochromatic blocks that stained deeply with hematoxylin, 4',6-diamidino-2-phenylindole (DAPI), or after C banding. The banded karyotype showed terminal or subterminal bands in all chromosomes and central bands in some of them. The single pair of 45S rDNA sites was observed at the end of the largest chromosome pair, close to a DAPI band and a 5S rDNA site. Two other 5S rDNA site pairs were found, both closely associated with DAPI bands. The noteworthy giant nuclei of glandular cells of petals and ovary wall exhibited large chromocentres typical of polytenic nuclei. The chromosomal location of heterochromatin and rDNA sites and the structure of the endoreplicated nuclei of C. approximata seemed to be similar to those known in monocentric nuclei, suggesting that centromeric organization has little or no effect on chromatin organization.

Gene conversion events and variable degree of homogenization of rDNA loci in cultivars of Brassica napus

PubMed Central

Sochorová, Jana; Coriton, Olivier; Kuderová, Alena; Lunerová, Jana; Chèvre, Anne-Marie; Kovařík, Aleš

2017-01-01

Background and aims Brassica napus (AACC, 2n = 38, oilseed rape) is a relatively recent allotetraploid species derived from the putative progenitor diploid species Brassica rapa (AA, 2n = 20) and Brassica oleracea (CC, 2n = 18). To determine the influence of intensive breeding conditions on the evolution of its genome, we analysed structure and copy number of rDNA in 21 cultivars of B. napus, representative of genetic diversity. Methods We used next-generation sequencing genomic approaches, Southern blot hybridization, expression analysis and fluorescence in situ hybridization (FISH). Subgenome-specific sequences derived from rDNA intergenic spacers (IGS) were used as probes for identification of loci composition on chromosomes. Key Results Most B. napus cultivars (18/21, 86 %) had more A-genome than C-genome rDNA copies. Three cultivars analysed by FISH (‘Darmor’, ‘Yudal’ and ‘Asparagus kale’) harboured the same number (12 per diploid set) of loci. In B. napus ‘Darmor’, the A-genome-specific rDNA probe hybridized to all 12 rDNA loci (eight on the A-genome and four on the C-genome) while the C-genome-specific probe showed weak signals on the C-genome loci only. Deep sequencing revealed high homogeneity of arrays suggesting that the C-genome genes were largely overwritten by the A-genome variants in B. napus ‘Darmor’. In contrast, B. napus ‘Yudal’ showed a lack of gene conversion evidenced by additive inheritance of progenitor rDNA variants and highly localized hybridization signals of subgenome-specific probes on chromosomes. Brassica napus ‘Asparagus kale’ showed an intermediate pattern to ‘Darmor’ and ‘Yudal’. At the expression level, most cultivars (95 %) exhibited stable A-genome nucleolar dominance while one cultivar (‘Norin 9’) showed co-dominance. Conclusions The B. napus cultivars differ in the degree and direction of rDNA homogenization. The prevalent direction of gene conversion (towards the A-genome) correlates with the direction of expression dominance indicating that gene activity may be needed for interlocus gene conversion. PMID:27707747

Ichthyosporidium weissii n. sp. (Microsporidia) Infecting the Arrow Goby (Clevelandia ios)

PubMed Central

SANDERS, JUSTIN; MYERS, MARK S.; TOMANEK, LARS; CALI, ANN; TAKVORIAN, PETER M.; KENT, MICHAEL L.

2012-01-01

Gonadal infections by a novel microsporidium were discovered in 34% (13/38) of arrow gobies, Clevelandia ios, sampled over a three year period from Morro Bay Marina in Morro Bay, California. Gonadal tumors had been reported in arrow gobies from this geographic area. The infected gonads, found primarily in females, typically appeared grossly as large, white-gray firm and lobulated masses. Histological examination revealed large, multilobate xenomas within the ovaries and no evidence of neoplasia. Typical of the genus Ichthyosporidium, the large xenomas were filled with developmental stages and pleomorphic spores. Wet mount preparations showed two general spore types: microspores with mean length of 6.2 (7.0–4.9, SD = 0.6, N = 20) μm and mean width of 4.3 (5.3–2.9, SD = 0.8) μm; and less numerous macrospores with mean length of 8.5 (10.1–7.1, SD = 1.0, N = 10) μm and mean width of 5.5 (6.2–4.8, SD = 0.5) μm. Transmission electron microscopy demonstrated stages consistent with the genus and 35–50 turns of the polar filament. Small subunit (SSU) rDNA gene sequence analysis revealed that the parasite from arrow gobies was most closely related to, but distinct from Ichthyosporidium sp. based on sequences available in GenBank. We conclude that this microsporidium represents a new species of Ichthyosporidium, the first species of this genus described from a member of the family Gobiidae and from the Pacific Ocean. PMID:22486936

Delivery of Iron-Sulfur Clusters to the Hydrogen-Oxidizing [NiFe]-Hydrogenases in Escherichia coli Requires the A-Type Carrier Proteins ErpA and IscA

PubMed Central

Pinske, Constanze; Sawers, R. Gary

2012-01-01

During anaerobic growth Escherichia coli synthesizes two membrane-associated hydrogen-oxidizing [NiFe]-hydrogenases, termed hydrogenase 1 and hydrogenase 2. Each enzyme comprises a catalytic subunit containing the [NiFe] cofactor, an electron-transferring small subunit with a particular complement of [Fe-S] (iron-sulfur) clusters and a membrane-anchor subunit. How the [Fe-S] clusters are delivered to the small subunit of these enzymes is unclear. A-type carrier (ATC) proteins of the Isc (iron-sulfur-cluster) and Suf (sulfur mobilization) [Fe-S] cluster biogenesis pathways are proposed to traffic pre-formed [Fe-S] clusters to apoprotein targets. Mutants that could not synthesize SufA had active hydrogenase 1 and hydrogenase 2 enzymes, thus demonstrating that the Suf machinery is not required for hydrogenase maturation. In contrast, mutants devoid of the IscA, ErpA or IscU proteins of the Isc machinery had no detectable hydrogenase 1 or 2 activities. Lack of activity of both enzymes correlated with the absence of the respective [Fe-S]-cluster-containing small subunit, which was apparently rapidly degraded. During biosynthesis the hydrogenase large subunits receive their [NiFe] cofactor from the Hyp maturation machinery. Subsequent to cofactor insertion a specific C-terminal processing step occurs before association of the large subunit with the small subunit. This processing step is independent of small subunit maturation. Using western blotting experiments it could be shown that although the amount of each hydrogenase large subunit was strongly reduced in the iscA and erpA mutants, some maturation of the large subunit still occurred. Moreover, in contrast to the situation in Isc-proficient strains, these processed large subunits were not membrane-associated. Taken together, our findings demonstrate that both IscA and ErpA are required for [Fe-S] cluster delivery to the small subunits of the hydrogen-oxidizing hydrogenases; however, delivery of the Fe atom to the active site might have different requirements. PMID:22363723

Novel variants of the 5S rRNA genes in Eruca sativa.

PubMed

Singh, K; Bhatia, S; Lakshmikumaran, M

1994-02-01

The 5S ribosomal RNA (rRNA) genes of Eruca sativa were cloned and characterized. They are organized into clusters of tandemly repeated units. Each repeat unit consists of a 119-bp coding region followed by a noncoding spacer region that separates it from the coding region of the next repeat unit. Our study reports novel gene variants of the 5S rRNA genes in plants. Two families of the 5S rDNA, the 0.5-kb size family and the 1-kb size family, coexist in the E. sativa genome. The 0.5-kb size family consists of the 5S rRNA genes (S4) that have coding regions similar to those of other reported plant 5S rDNA sequences, whereas the 1-kb size family consists of the 5S rRNA gene variants (S1) that exist as 1-kb BamHI tandem repeats. S1 is made up of two variant units (V1 and V2) of 5S rDNA where the BamHI site between the two units is mutated. Sequence heterogeneity among S4, V1, and V2 units exists throughout the sequence and is not limited to the noncoding spacer region only. The coding regions of V1 and V2 show approximately 20% dissimilarity to the coding regions of S4 and other reported plant 5S rDNA sequences. Such a large variation in the coding regions of the 5S rDNA units within the same plant species has been observed for the first time. Restriction site variation is observed between the two size classes of 5S rDNA in E. sativa.(ABSTRACT TRUNCATED AT 250 WORDS)

Pyrosequencing assessment of prokaryotic and eukaryotic diversity in biofilm communities from a French river

PubMed Central

Bricheux, Geneviève; Morin, Loïc; Le Moal, Gwenaël; Coffe, Gérard; Balestrino, Damien; Charbonnel, Nicolas; Bohatier, Jacques; Forestier, Christiane

2013-01-01

Despite the recent and significant increase in the study of aquatic microbial communities, little is known about the microbial diversity of complex ecosystems such as running waters. This study investigated the biodiversity of biofilm communities formed in a river with 454 Sequencing™. This river has the particularity of integrating both organic and microbiological pollution, as receiver of agricultural pollution in its upstream catchment area and urban pollution through discharges of the wastewater treatment plant of the town of Billom. Different regions of the small subunit (SSU) ribosomal RNA gene were targeted using nine pairs of primers, either universal or specific for bacteria, eukarya, or archaea. Our aim was to characterize the widest range of rDNA sequences using different sets of polymerase chain reaction (PCR) primers. A first look at reads abundance revealed that a large majority (47–48%) were rare sequences (<5 copies). Prokaryotic phyla represented the species richness, and eukaryotic phyla accounted for a small part. Among the prokaryotic phyla, Proteobacteria (beta and alpha) predominated, followed by Bacteroidetes together with a large number of nonaffiliated bacterial sequences. Bacillariophyta plastids were abundant. The remaining bacterial phyla, Verrucomicrobia and Cyanobacteria, made up the rest of the bulk biodiversity. The most abundant eukaryotic phyla were annelid worms, followed by Diatoms, and Chlorophytes. These latter phyla attest to the abundance of plastids and the importance of photosynthetic activity for the biofilm. These findings highlight the existence and plasticity of multiple trophic levels within these complex biological systems. PMID:23520129

Summary and critique of the new NIH guidelines for recombinant DNA research.

PubMed

Szybalski, W

1979-03-01

New NIH Guidelines for research involving recombinant DNA (R-DNA) molecules were issued on December 15, 1978. These are composed of four main parts, the first defining R-DNA and specifying prohibitions and exemptions, the second describing physical and biological containment, the third assigning the containment levels for many R-DNA experiments, and the fourth detailing the roles and responsibilities of the investigator, research institutions and NIH. Although the new Guidelines reduce restrictions, principally on those R-DNA experiments that use Escherichia coli K-12 host-vector systems, and exempt from the Guidelines several classes of experiments on prokaryotes that naturally exchange their DNA, most of their provisions are unjustified by the present assessment of the absence of any practical risks; many totally innocuous experiments are unnecessarily restricted and even virtually prohibited mainly because no host-vector systems were officially certified. The term Guidelines is a misnomer since they are mandatory regulations, even without any statutory basis. They impose large but unnecessary bureaucratic burdens on scientists, research institutions, research committees and NIH, and represent unwarranted censorship of basic research, which is antithetical to the creativity of human thought, thus posing serious dangers to the traditional freedom of inquiry.

Subunit Conformations and Assembly States of a DNA Translocating Motor: The Terminase of Bacteriophage P22

PubMed Central

Němeček, Daniel; Gilcrease, Eddie B.; Kang, Sebyung; Prevelige, Peter E.; Casjens, Sherwood; Thomas, George J.

2007-01-01

Bacteriophage P22, a podovirus infecting strains of Salmonella typhimurium, packages a 42 kbp genome using a headful mechanism. DNA translocation is accomplished by the phage terminase, a powerful molecular motor consisting of large and small subunits. Although many of the structural proteins of the P22 virion have been well characterized, little is known about the terminase subunits and their molecular mechanism of DNA translocation. We report here structural and assembly properties of ectopically expressed and highly purified terminase large and small subunits. The large subunit (gp2), which contains the nuclease and ATPase activities of terminase, exists as a stable monomer with an α/β fold. The small subunit (gp3), which recognizes DNA for packaging and may regulate gp2 activity, exhibits a highly α-helical secondary structure and self-associates to form a stable oligomeric ring in solution. For wildtype gp3, the ring contains nine subunits, as demonstrated by hydrodynamic measurements, electron microscopy and native mass spectrometry. We have also characterized a gp3 mutant (Ala 112 → Thr) that forms a ten subunit ring, despite a subunit fold indistinguishable from wildtype. Both the nonameric and decameric gp3 rings exhibit nonspecific DNA binding activity, and gp2 is able to bind strongly to the DNA/gp3 complex but not to DNA alone. We propose a scheme for the roles of P22 terminase large and small subunits in the recruitment and packaging of viral DNA and discuss the model in relation to proposals for terminase-driven DNA translocation in other phages. PMID:17945256

Haplosporidium nelsoni (MSX) in Japanese scallops Patinopecten yessoensis (Jay, 1857) from Dalian along the northern coast of the Yellow Sea, China.

PubMed

Wang, Zhongwei; Lu, Xin; Liang, Yubo

2012-04-01

The protozoan parasite Haplosporidium nelsoni (MSX) was identified in Japanese scallops Patinopecten yessoensis (Jay, 1857) from Dalian along the northern coast of the Yellow Sea, China by histopathologic examination, polymerase chain reaction (PCR) amplification, and in situ hybridization (ISH) assay. H. nelsoni plasmodia-like structures were identified in the digestive glands of scallops by histologic examination, but no parasite spores were observed. PCR using the Hap-F2, R2 primer pair produced a sequence with 100% homology with the corresponding small subunit rDNA region of H. nelsoni. An ISH assay using the oligonucleotide probe MSX1347 produced a positive reaction with the Japanese scallop parasite. This is the first report of H. nelsoni in P. yessoensis in China.

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

PubMed

Miao, Wei; Simpson, Alastair G B; Fu, Chengjie; Lobban, Christopher S

2005-01-01

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

A nuptially transmitted ichthyosporean symbiont of Tenebrio molitor (Coleoptera: Tenebrionidae).

PubMed

Lord, Jeffrey C; Hartzer, Kris L; Kambhampati, Srinivas

2012-01-01

The yellow mealworm, Tenebrio molitor, harbors a symbiont that has spores with a thick, laminated wall and infects the fat body and ventral nerve chord of adult and larval beetles. In adult males, there is heavy infection of the epithelial cells of the testes and between testes lobes with occasional penetration of the lobes. Spores are enveloped in the spermatophores when they are formed at the time of mating and transferred to the female's bursa copulatrix. Infection has not been found in the ovaries. The sequence of the nuclear small subunit rDNA indicates that the symbiont is a member of the Ichthyosporea, a class of protists near the animal-fungi divergence. © 2012 The Author(s) Journal of Eukaryotic Microbiology © 2012 International Society of Protistologists.

Dancing together and separate again: gymnosperms exhibit frequent changes of fundamental 5S and 35S rRNA gene (rDNA) organisation

PubMed Central

Garcia, S; Kovařík, A

2013-01-01

In higher eukaryotes, the 5S rRNA genes occur in tandem units and are arranged either separately (S-type arrangement) or linked to other repeated genes, in most cases to rDNA locus encoding 18S–5.8S–26S genes (L-type arrangement). Here we used Southern blot hybridisation, PCR and sequencing approaches to analyse genomic organisation of rRNA genes in all large gymnosperm groups, including Coniferales, Ginkgoales, Gnetales and Cycadales. The data are provided for 27 species (21 genera). The 5S units linked to the 35S rDNA units occur in some but not all Gnetales, Coniferales and in Ginkgo (∼30% of the species analysed), while the remaining exhibit separate organisation. The linked 5S rRNA genes may occur as single-copy insertions or as short tandems embedded in the 26S–18S rDNA intergenic spacer (IGS). The 5S transcript may be encoded by the same (Ginkgo, Ephedra) or opposite (Podocarpus) DNA strand as the 18S–5.8S–26S genes. In addition, pseudogenised 5S copies were also found in some IGS types. Both L- and S-type units have been largely homogenised across the genomes. Phylogenetic relationships based on the comparison of 5S coding sequences suggest that the 5S genes independently inserted IGS at least three times in the course of gymnosperm evolution. Frequent transpositions and rearrangements of basic units indicate relatively relaxed selection pressures imposed on genomic organisation of 5S genes in plants. PMID:23512008

Dancing together and separate again: gymnosperms exhibit frequent changes of fundamental 5S and 35S rRNA gene (rDNA) organisation.

PubMed

Garcia, S; Kovařík, A

2013-07-01

In higher eukaryotes, the 5S rRNA genes occur in tandem units and are arranged either separately (S-type arrangement) or linked to other repeated genes, in most cases to rDNA locus encoding 18S-5.8S-26S genes (L-type arrangement). Here we used Southern blot hybridisation, PCR and sequencing approaches to analyse genomic organisation of rRNA genes in all large gymnosperm groups, including Coniferales, Ginkgoales, Gnetales and Cycadales. The data are provided for 27 species (21 genera). The 5S units linked to the 35S rDNA units occur in some but not all Gnetales, Coniferales and in Ginkgo (∼30% of the species analysed), while the remaining exhibit separate organisation. The linked 5S rRNA genes may occur as single-copy insertions or as short tandems embedded in the 26S-18S rDNA intergenic spacer (IGS). The 5S transcript may be encoded by the same (Ginkgo, Ephedra) or opposite (Podocarpus) DNA strand as the 18S-5.8S-26S genes. In addition, pseudogenised 5S copies were also found in some IGS types. Both L- and S-type units have been largely homogenised across the genomes. Phylogenetic relationships based on the comparison of 5S coding sequences suggest that the 5S genes independently inserted IGS at least three times in the course of gymnosperm evolution. Frequent transpositions and rearrangements of basic units indicate relatively relaxed selection pressures imposed on genomic organisation of 5S genes in plants.

Fluctuations of pol I and fibrillarin contents of the nucleoli.

PubMed

Hornáček, M; Kováčik, L; Mazel, T; Cmarko, D; Bártová, E; Raška, I; Smirnov, E

2017-07-04

Nucleoli are formed on the basis of ribosomal DNA (rDNA) clusters called Nucleolus Organizer Regions (NORs). Each NOR contains multiple genes coding for RNAs of the ribosomal particles. The prominent components of the nucleolar ultrastructure, fibrillar centers (FC) and dense fibrillar components (DFC), together compose FC/DFC units. These units are centers of rDNA transcription by RNA polymerase I (pol I), as well as the early processing events, in which an essential role belongs to fibrillarin. Each FC/DFC unit probably corresponds to a single transcriptionally active gene. In this work, we transfected human-derived cells with GFP-RPA43 (subunit of pol I) and RFP-fibrillarin. Following changes of the fluorescent signals in individual FC/DFC units, we found two kinds of kinetics: 1) the rapid fluctuations with periods of 2-3 min, when the pol I and fibrillarin signals oscillated in anti-phase manner, and the intensities of pol I in the neighboring FC/DFC units did not correlate. 2) fluctuations with periods of 10 to 60 min, in which pol I and fibrillarin signals measured in the same unit did not correlate, but pol I signals in the units belonging to different nucleoli were synchronized. Our data indicate that a complex pulsing activity of transcription as well as early processing is common for ribosomal genes.

Identification of Paramecium bursaria syngens through molecular markers--comparative analysis of three loci in the nuclear and mitochondrial DNA.

PubMed

Greczek-Stachura, Magdalena; Potekhin, Alexey; Przyboś, Ewa; Rautian, Maria; Skoblo, Inna; Tarcz, Sebastian

2012-09-01

This is the first attempt to resolve the phylogenetic relationship between different syngens of Paramecium bursaria and to investigate at a molecular level the intraspecific differentiation of strains originating from very distant geographical locations. Herein we introduce a new collection of five P. bursaria syngens maintained at St Petersburg State University, as the international collection of syngens was lost in the 1960s. To analyze the degree of speciation within Paramecium bursaria, we examined 26 strains belonging to five different syngens from distant and geographically isolated localities using rDNA (ITS1-5.8S-ITS2-5'LSU) fragments, mitochondrial cytochrome c oxidase subunit I (COI), and H4 gene fragments. It was shown that P. bursaria strains of the same syngens cluster together in all three inferred molecular phylogenies. The genetic diversity among the studied P. bursaria strains based on rDNA sequences was rather low. The COI divergence of Paramecium bursaria was also definitely lower than that observed in the Paramecium aurelia complex. The nucleotide sequences of the H4 gene analyzed in the present study indicate the extent of genetic differences between the syngens of Paramecium bursaria. Our study demonstrates the diagnostic value of molecular markers, which are important tools in the identification of Paramecium bursaria syngens. Copyright © 2011 Elsevier GmbH. All rights reserved.

Trypanosoma cruzi and Leishmania infantum chagasi Infection in Wild Mammals from Maranhão State, Brazil.

PubMed

da Costa, Andréa Pereira; Costa, Francisco Borges; Soares, Herbert Sousa; Ramirez, Diego Garcia; Mesquita, Eric Takashi Kamakura de Carvalho; Gennari, Solange Maria; Marcili, Arlei

2015-11-01

Trypanosoma and Leishmania are obligate parasites that cause important diseases in human and domestic animals. Wild mammals are the natural reservoirs of these parasites, which are transmitted by hematophagous arthropods. The present study aimed to detect the natural occurrence of trypanosomatids through serological diagnosis, PCR of whole blood and blood culture (hemoculture), and phylogenetic relationships using small subunit ribosomal DNA (SSU rDNA), cytochrome b, and glycosomal glyceraldehyde 3-phosphate dehydrogenase (gGAPDH) genes. Samples from 131 wild animals, including rodents, marsupials, and bats, were sampled in six areas in the state of Maranhão, in a transition zone of semiarid climates northeast of the equatorial humid Amazon. Serological analysis for Leishmania (Leishmania) infantum chagasi was performed in opossums by indirect fluorescent antibody test (IFAT), and all animals were serologically negative. Nine positive hemocultures (6.77%) were isolated and cryopreserved and from mammals of the Didelphimorphia and Chiroptera orders and positioned in phylogenies on the basis of sequences from different genes with reference strains of Trypanosoma cruzi marinkellei and T. cruzi. From primary samples (blood and tissues) only one bat, Pteronotus parnellii, was positive to SSU rDNA and gGAPDH genes and grouped with the L. infantum chagasi branch. The studies conducted in Maranhão State provide knowledge of parasite diversity. It is important to determine the presence of trypanosomatids in wild mammals with synanthropic habits.

Morphological and molecular identification of Sarcocystis spp. from the sika deer (Cervus nippon), including two new species Sarcocystis frondea and Sarcocystis nipponi.

PubMed

Rudaitytė-Lukošienė, Eglė; Prakas, Petras; Butkauskas, Dalius; Kutkienė, Liuda; Vepštaitė-Monstavičė, Iglė; Servienė, Elena

2018-05-01

Diaphragm muscles of 25 sika deer (Cervus nippon) farmed in Lithuania were examined for sarcocysts of Sarcocystis species. Two new Sarcocystis species, Sarcocystis frondea and Sarcocystis nipponi, were observed using light microscopy (LM) and transmission electron microscopy (TEM) and characterized by 18S ribosomal DNA (rDNA) and subunit I of cytochrome c oxidase (cox1) sequence analyses. By LM, sarcocysts of S. frondea and S. nipponi were ribbon-shaped and had finger-like sarcocyst wall protrusions, respectively. Under TEM, protrusions of S. frondea were about 9 × 1-1.5 μm, filled with clearly visible electron-dense substance and microtubules, type 39-like. Whereas, protrusions (about 9 × 0.2 μm) of S. nipponi arose from dome-shaped bases were filled with microtubules extending to the ground substance layer, type 9o-like. Moreover, three known Sarcocystis spp., Sarcocystis entzerothi, Sarcocystis ovalis, and Sarcocystis truncata previously described in other cervids as intermediate hosts, were characterized in sika deer. The cox1 was more suitable than 18S rDNA delimitating closely related Sarcocystis species from cervids. The phylogenetic results suggest that scavenger birds could be definitive hosts of S. frondea. According to the summarized morphological data on Sarcocystis found in the sika deer, such host should harbor at least nine different Sarcocystis species.

BLM helicase facilitates RNA polymerase I-mediated ribosomal RNA transcription

PubMed Central

Grierson, Patrick M.; Lillard, Kate; Behbehani, Gregory K.; Combs, Kelly A.; Bhattacharyya, Saumitri; Acharya, Samir; Groden, Joanna

2012-01-01

Bloom's syndrome (BS) is an autosomal recessive disorder that is invariably characterized by severe growth retardation and cancer predisposition. The Bloom's syndrome helicase (BLM), mutations of which lead to BS, localizes to promyelocytic leukemia protein bodies and to the nucleolus of the cell, the site of RNA polymerase I-mediated ribosomal RNA (rRNA) transcription. rRNA transcription is fundamental for ribosome biogenesis and therefore protein synthesis, cellular growth and proliferation; its inhibition limits cellular growth and proliferation as well as bodily growth. We report that nucleolar BLM facilitates RNA polymerase I-mediated rRNA transcription. Immunofluorescence studies demonstrate the dependance of BLM nucleolar localization upon ongoing RNA polymerase I-mediated rRNA transcription. In vivo protein co-immunoprecipitation demonstrates that BLM interacts with RPA194, a subunit of RNA polymerase I. 3H-uridine pulse-chase assays demonstrate that BLM expression is required for efficient rRNA transcription. In vitro helicase assays demonstrate that BLM unwinds GC-rich rDNA-like substrates that form in the nucleolus and normally inhibit progression of the RNA polymerase I transcription complex. These studies suggest that nucleolar BLM modulates rDNA structures in association with RNA polymerase I to facilitate RNA polymerase I-mediated rRNA transcription. Given the intricate relationship between rDNA metabolism and growth, our data may help in understanding the etiology of proportional dwarfism in BS. PMID:22106380

BLM helicase facilitates RNA polymerase I-mediated ribosomal RNA transcription.

PubMed

Grierson, Patrick M; Lillard, Kate; Behbehani, Gregory K; Combs, Kelly A; Bhattacharyya, Saumitri; Acharya, Samir; Groden, Joanna

2012-03-01

Bloom's syndrome (BS) is an autosomal recessive disorder that is invariably characterized by severe growth retardation and cancer predisposition. The Bloom's syndrome helicase (BLM), mutations of which lead to BS, localizes to promyelocytic leukemia protein bodies and to the nucleolus of the cell, the site of RNA polymerase I-mediated ribosomal RNA (rRNA) transcription. rRNA transcription is fundamental for ribosome biogenesis and therefore protein synthesis, cellular growth and proliferation; its inhibition limits cellular growth and proliferation as well as bodily growth. We report that nucleolar BLM facilitates RNA polymerase I-mediated rRNA transcription. Immunofluorescence studies demonstrate the dependance of BLM nucleolar localization upon ongoing RNA polymerase I-mediated rRNA transcription. In vivo protein co-immunoprecipitation demonstrates that BLM interacts with RPA194, a subunit of RNA polymerase I. (3)H-uridine pulse-chase assays demonstrate that BLM expression is required for efficient rRNA transcription. In vitro helicase assays demonstrate that BLM unwinds GC-rich rDNA-like substrates that form in the nucleolus and normally inhibit progression of the RNA polymerase I transcription complex. These studies suggest that nucleolar BLM modulates rDNA structures in association with RNA polymerase I to facilitate RNA polymerase I-mediated rRNA transcription. Given the intricate relationship between rDNA metabolism and growth, our data may help in understanding the etiology of proportional dwarfism in BS.

Taxonomic Clarification of A Well-Known Pathogenic Scuticociliate, Miamiensis avidus Thompson & Moewus, 1964 (Ciliophora, Scuticociliatia)

NASA Astrophysics Data System (ADS)

Ma, Mingzhen; Lu, Borong; Fan, Xinpeng; Shi, Yuhong; Chen, Xiangrui

2018-06-01

Miamiensis avidus Thompson & Moewus, 1964, is a cosmopolitan and well-known marine pathogenic ciliated protist. However, the taxonomy of this species up to now has remained controversial, especially with respect to the validity of the morphologically similar species, Philasterides dicentrarchi, which was considered as a junior synonym of M. avidus. In this study, a population of M. avidus was collected from the skin of pharaoh cuttlefish (Sepia pharaonis) cultured near the East China Sea, Ningbo, China and its morphology and phylogeny were investigated in detail based on living characters, infraciliature, small subunit (SSU) rDNA and ITS1-5.8S-ITS2 region sequences. In addition, the morphometrics of a previously reported free-living population, collected from the Bohai Sea, were rechecked and analyzed. We compared the present two isolates with all historic populations of M. avidus and P. dicentrarchi, and found that their morphological characters were either highly similar or exactly identical, indicating that they are the same morphospecies. However, the phylogenetic analyses based on SSU rDNA or ITS1-5.8S-ITS2 region sequences revealed that most M. avidus and P. dicentrarchi populations formed one clade, and the two isolates of M. avidus from Weifang and American Type Culture Collection clustered in another clade, which indicated that there might be cryptic species in Miamiensis avidus.

Morphology and SSU rDNA sequence analysis of two hypotrichous ciliates (Protozoa, Ciliophora, Hypotrichia) including the new species Metaurostylopsis parastruederkypkeae n. sp.

NASA Astrophysics Data System (ADS)

Lu, Borong; Wang, Chundi; Huang, Jie; Shi, Yuhong; Chen, Xiangrui

2016-10-01

The morphology and phylogeny of two hypotrichous ciliates, Metaurostylopsis parastruederkypkeae n. sp. and Neourostylopsis flavicana (Wang et al., 2011) Chen et al., 2013 were investigated based on morphology, infraciliature and the small subunit (SSU) ribosomal RNA gene (rRNA) sequence. The new species, M. parastruederkypkeae n. sp. was identified according to its characteristics: body shape ellipsoidal, size about (165-200) × (45-60) μm in vivo, cell color reddish; two types of cortical granules including wheat grain-like and yellow-greenish larger ones along the marginal cirri rows and dorsal kineties and dot-like and reddish smaller ones, grouped around marginal cirri on ventral side and arranged in short lines on dorsal side; 26-41 adoral membranelles; three frontal and one parabuccal, five to seven frontoterminal, one buccal, and three to six transverse cirri; seven to thirteen midventral pairs; five to nine unpaired ventral cirri, five to seven left and three to five right marginal rows; and three complete dorsal kineties. Phylogenetic analysis based on SSU rDNA sequences showed that both Metaurostylopsis and Neourostylopsis are monophyletic. As the internal relationship between and within both genera are not clear, further studies on the species in these two genera are necessary. The key characteristics of all known twelve Metaurostylopsis-Apourostylopsis-Neourostylopsis species complex were updated.

Phylogenetic analysis with multiple markers indicates repeated loss of the adult medusa stage in Campanulariidae (Hydrozoa, Cnidaria).

PubMed

Govindarajan, Annette F; Boero, Ferdinando; Halanych, Kenneth M

2006-03-01

The Campanulariidae is a group of leptomedusan hydroids (Hydrozoa, Cnidaria) that exhibit a diverse array of life cycles ranging from species with a free medusa stage to those with a reduced or absent medusa stage. Perhaps the best-known member of the taxon is Obelia which is often used as a textbook model of hydrozoan life history. However, Obelia medusae have several unique features leading to a hypothesis that Obelia arose, in a saltational fashion, from an ancestor that lacked a medusa, possibly representing an example of a rare evolutionary reversal. To address the evolution of adult sexual stages in Campanulariidae, a molecular phylogenetic approach was employed using two nuclear (18S rDNA and calmodulin) and two mitochondrial (16S rDNA and cytochrome c oxidase subunit I) genes. Prior to the main analysis, we conducted a preliminary analysis of leptomedusan taxa which suggests that Campanulariidae as presently considered needs to be redefined. Campanulariid analyses are consistent with morphological understanding in that three major clades are recovered. However, several recognized genera are not monophyletic calling into question some "diagnostic" features. Furthermore, ancestral states were reconstructed using parsimony, and a sensitivity analysis was conducted to investigate possible evolutionary transitions in life-history stages. The results indicate that life-cycle transitions have occurred multiple times, and that Obelia might be derived from an ancestor with Clytia-like features.

Epizoanthus spp. associations revealed using DNA markers: a case study from Kochi, Japan.

PubMed

Reimer, James Davis; Hirose, Mamiko; Nishisaka, Taiki; Sinniger, Frederic; Itani, Gyo

2010-09-01

Zoanthids (Cnidaria, Hexacorallia) of the genus Epizoanthus are often found in association with other marine invertebrates, including gastropods and hermit crabs. However, little information exists on the specificity and nature of these associations due to a lack of investigation into Epizoanthus species diversity, and the taxonomy of Epizoanthus is therefore confused. In this study, analyses of morphological data (tentacle number, polyp size, etc) and molecular data (mitochondrial cytochrome oxidase subunit 1 = COI, 16S ribosomal DNA = 16S rDNA) were used to examine Epizoanthus specimens from Tosa Bay, Kochi, Japan. The Epizoanthus specimens were found on both live gastropods (Gemmula unedo) and hermit crabs (Paguristes palythophilus) inhabiting G. unedo and G. cosmoi shells. While morphological analyses did not show clear differences between examined specimens, both COI and mt 16S rDNA clearly divided the specimens into two groups, one associated only with hermit crabs (= Epizoanthus sp. C), and another associated only with living gastropods (= Epizoanthus sp. S). Unexpectedly, DNA sequences from both groups did not match with two previously reported Epizoanthus species from Japan (E. indicus, E. ramosus), indicating they both may be undescribed species. These results highlight the utility of DNA "barcoding" of unknown zoanthids, and will provide a foundation for re-examinations of Epizoanthus species diversity and specificity, which will be critical in understanding the evolution of these unique marine invertebrates.

Quantitative detection method of Enterocytozoon hepatopenaei using TaqMan probe real-time PCR.

PubMed

Liu, Ya-Mei; Qiu, Liang; Sheng, An-Zhi; Wan, Xiao-Yuan; Cheng, Dong-Yuan; Huang, Jie

2018-01-01

A TaqMan probe and a pair of specific primers were selected from the small subunit ribosomal DNA (SSU rDNA) sequence of Enterocytozoon hepatopenaei (EHP); this real-time PCR assay was developed and optimized. It showed a good linearity in detecting standards of EHP SSU rDNA fragments from 4 × 10 2 to 4 × 10 8 copies/reaction using the established method. The detection limit of the qPCR method was as low as 4 × 10 1 copies per reaction, which was higher than the conventional PCR and SYBR Green I-based EHP qPCR reported. Using the qPCR assay, EHP was detected in four batches of slow-growing Penaeus vannamei specimens collected from Tianjin and Zhejiang Province in China was detected using qPCR. The results showed that all the hepatopancreas from the slow-growing P. vannamei specimens were detected as EHP-positive. EHP copies of hepatopancreas in some batches had a negative correlation with the body mass index (BMI) of shrimps; however, not all batches of specimens had this negative correlation between EHP copies of hepatopancreas and BMI. This qPCR technique is sensitive, specific and easy to perform (96 tests in <3 h), which provides technical support for the detection and prevention of EHP. Copyright © 2017 Elsevier Inc. All rights reserved.

Arbuscular Mycorrhizal Fungi Promote the Growth of Ceratocarpus arenarius (Chenopodiaceae) with No Enhancement of Phosphorus Nutrition

PubMed Central

Bai, Dengsha; Chen, Yinglong; Feng, Gu

2012-01-01

The mycorrhizal status of plants in the Chenopodiaceae is not well studied with a few controversial reports. This study examined arbuscular mycorrhizal (AM) colonization and growth response of Ceratocarpus arenarius in the field and a greenhouse inoculation trial. The colonization rate of AM fungi in C. arenarius in in-growth field cores was low (around 15%). Vesicles and intraradical hyphae were present during all growth stages, but no arbuscules were observed. Sequencing analysis of the large ribosomal rDNA subunit detected four culturable Glomus species, G. intraradices, G. mosseae, G. etunicatum and G. microaggregatum together with eight unculturable species belong to the Glomeromycota in the root system of C. arenarius collected from the field. These results establish the mycotrophic status of C. arenarius. Both in the field and in the greenhouse inoculation trial, the growth of C. arenarius was stimulated by the indigenous AM fungal community and the inoculated AM fungal isolates, respectively, but the P uptake and concentration of the mycorrhizal plants did not increase significantly over the controls in both experiments. Furthermore, the AM fungi significantly increased seed production. Our results suggest that an alternative reciprocal benefit to carbon-phosphorus trade-off between AM fungi and the chenopod plant might exist in the extremely arid environment. PMID:22957011

Bacterial diversity in the active stage of a bioremediation system for mineral oil hydrocarbon-contaminated soils.

PubMed

Popp, Nicole; Schlömann, Michael; Mau, Margit

2006-11-01

Soils contaminated with mineral oil hydrocarbons are often cleaned in off-site bioremediation systems. In order to find out which bacteria are active during the degradation phase in such systems, the diversity of the active microflora in a degrading soil remediation system was investigated by small-subunit (SSU) rRNA analysis. Two sequential RNA extracts from one soil sample were generated by a procedure incorporating bead beating. Both extracts were analysed separately by generating individual SSU rDNA clone libraries from cDNA of the two extracts. The sequencing results showed moderate diversity. The two clone libraries were dominated by Gammaproteobacteria, especially Pseudomonas spp. Alphaproteobacteria and Betaproteobacteria were two other large groups in the clone libraries. Actinobacteria, Firmicutes, Bacteroidetes and Epsilonproteobacteria were detected in lower numbers. The obtained sequences were predominantly related to genera for which cultivated representatives have been described, but were often clustered together in the phylogenetic tree, and the sequences that were most similar were originally obtained from soils and not from pure cultures. Most of the dominant genera in the clone libraries, e.g. Pseudomonas, Acinetobacter, Sphingomonas, Acidovorax and Thiobacillus, had already been detected in (mineral oil hydrocarbon) contaminated environmental samples. The occurrence of the genera Zymomonas and Rhodoferax was novel in mineral oil hydrocarbon-contaminated soil.

Molecular diversity of arbuscular mycorrhizal fungi and their distribution patterns related to host-plants and habitats in a hot and arid ecosystem, southwest China.

PubMed

Li, Ling-Fei; Li, Tao; Zhang, Yan; Zhao, Zhi-Wei

2010-03-01

The communities of arbuscular mycorrhizal fungi (AMF) colonizing the roots of Bothriochloa pertusa, Cajanus cajan and Heteropogon contortus in a fallow land (FL) and an undisturbed land (UL) were characterized. The large subunit rDNA genes of AMF from roots were amplified and cloned. A total of 2353 clones were screened by restriction fragment length polymorphism, and 428 clones were subsequently sequenced. A total of 393 AMF sequences, which were grouped into 100 operational taxonomic units, were obtained. Phylogenetic analysis revealed that the AMF sequences belonged to Glomus, Acaulospora and Scutellospora, and that Glomus was the dominant genus. Of the 393 AMF sequences, 81% were novel. The diversity of AMF colonizing the same plant species was higher in the UL than in the FL, which confirmed strongly from the molecular evidence that soil disturbance reduced AMF population and species richness. The results revealed that AMF communities were significantly different among host-plant species and between the two habitats. The similarity of AMF communities colonizing different plant species within a habitat was higher than that of the same plant species from different habitats. The molecular evidence supported our previous hypothesis based on morphological analyses that AMF communities were more influenced by habitats compared with host preference.

Phylogenetic relationships among zooxanthellae (Symbiodinium) associated to excavating sponges (Cliona spp.) reveal an unexpected lineage in the Caribbean.

PubMed

Granados, C; Camargo, C; Zea, S; Sánchez, J A

2008-11-01

Phylogenetic relationships of symbiotic dinoflagellate lineages, distributed in all tropical and subtropical seas, suggest strategies for long distance dispersal but at the same time strong host specialization. Zooxanthellae (Symbiodinium: Dinophyta), which are associated to diverse shallow-water cnidarians, also engage in symbioses with some sponge species of the genus Cliona. In the Caribbean, zooxanthellae-bearing Cliona has recently become abundant due to global warming, overfishing, and algae abundance. Using molecular techniques, the symbionts from five excavating species (Clionacaribbaea, C. tenuis, C. varians, C. aprica and C. laticavicola) from the southern and southwestern Caribbean were surveyed. Several DNA sequence regions were used in order to confirm zooxanthellae identity; 18S rDNA, domain V of chloroplast large subunit (cp23S), internal transcribed spacer 2 (ITS2), and ITS2 secondary structure. Sequence analyses corroborated the presence of three zooxanthellae clades: A, B, and G. Presence of clades A and B in common boring sponges of the Caribbean fit with the general pattern of the province. The discovery of clade G for the first time in any organism of the Atlantic Ocean leads us to consider this unusual finding as a phylogenetic relict through common ancestors of sponge clades or an invasion of the sponge from the Indo-Pacific.

Phylogenetic diversity and biogeography of the Mamiellophyceae lineage of eukaryotic phytoplankton across the oceans.

PubMed

Monier, Adam; Worden, Alexandra Z; Richards, Thomas A

2016-08-01

High-throughput diversity amplicon sequencing of marine microbial samples has revealed that members of the Mamiellophyceae lineage are successful phytoplankton in many oceanic habitats. Indeed, these eukaryotic green algae can dominate the picoplanktonic biomass, however, given the broad expanses of the oceans, their geographical distributions and the phylogenetic diversity of some groups remain poorly characterized. As these algae play a foundational role in marine food webs, it is crucial to assess their global distribution in order to better predict potential changes in abundance and community structure. To this end, we analyzed the V9-18S small subunit rDNA sequences deposited from the Tara Oceans expedition to evaluate the diversity and biogeography of these phytoplankton. Our results show that the phylogenetic composition of Mamiellophyceae communities is in part determined by geographical provenance, and do not appear to be influenced - in the samples recovered - by water depth, at least at the resolution possible with the V9-18S. Phylogenetic classification of Mamiellophyceae sequences revealed that the Dolichomastigales order encompasses more sequence diversity than other orders in this lineage. These results indicate that a large fraction of the Mamiellophyceae diversity has been hitherto overlooked, likely because of a combination of size fraction, sequencing and geographical limitations. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

Proteopedia Entry: The Large Ribosomal Subunit of "Haloarcula Marismortui"

ERIC Educational Resources Information Center

Decatur, Wayne A.

2010-01-01

This article presents a "Proteopedia" page that shows the refined version of the structure of the "Haloarcula" large ribosomal subunit as solved by the laboratories of Thomas Steitz and Peter Moore. The landmark structure is of great impact as it is the first atomic-resolution structure of the highly conserved ribosomal subunit which harbors…

Morphological and molecular characterization of Sarcocystis taeniata and Sarcocystis pilosa n. sp. from the sika deer (Cervus nippon) in Lithuania.

PubMed

Prakas, Petras; Butkauskas, Dalius; Rudaitytė, Eglė; Kutkienė, Liuda; Sruoga, Aniolas; Pūraitė, Irma

2016-08-01

The diaphragm muscles of eight sika deer (Cervus nippon) bred in Lithuania were examined for Sarcocystis cysts. Two Sarcocystis species, Sarcocystis taeniata, which were previously reported in Canadian moose (Alces alces) and Argentinean red deer (Cervus elaphus), and Sarcocystis pilosa n. sp. were described using light microscopy (LM), transmission electron microscopy (TEM), 18S ribosomal DNA (rDNA), and subunit I of cytochrome c oxidase (cox1) sequences analysis. By LM, cysts of S. taeniata were 424.8 × 57.9 (200-837 × 30-100) μm in size and had a thin (up to 1 μm) and smooth cyst wall, while short ribbon-like protrusions arising from broadened cone-shaped bases were seen under TEM. Cysts of S. pilosa (by LM) were ribbon-shaped, measured 848.5 × 63.8 (350-1700 × 30-125) μm and had thin 7-8-μm long hair-like protrusions. By TEM, cyst wall was type 7a-like; protrusions arose from 0.3 μm wide dome-shaped base with minute indentations of the parasitophorous vacuolar membrane near it, the surface of protrusions seemed to be smooth, and the ground substance layer was thin (0.18-0.22 μm). The 18S rDNA, in contrast to the cox1, lacked variability to discriminate S. pilosa from closely related Sarcocystis hjorti from the red deer and moose. S. taeniata, but not S. pilosa, showed a considerable intraspecific variation in both genes analyzed. The phylogenetic analyses based on 18S rDNA and cox1 sequences suggest that canids are definitive hosts of both S. taeniata and S. pilosa. This paper represents the first identification of Sarcocystis species in the sika deer by morphological and molecular methods.

Multigene assessment of the species boundaries and sexual status of the basidiomycetous yeasts Cryptococcus flavescens and C. terrestris (Tremellales).

PubMed

Yurkov, Andrey; Guerreiro, Marco A; Sharma, Lav; Carvalho, Cláudia; Fonseca, Álvaro

2015-01-01

Cryptococcus flavescens and C. terrestris are phenotypically indistinguishable sister species that belong to the order Tremellales (Tremellomycetes, Basidiomycota) and which may be mistaken for C. laurentii based on phenotype. Phylogenetic separation between C. flavescens and C. terrestris was based on rDNA sequence analyses, but very little is known on their intraspecific genetic variability or propensity for sexual reproduction. We studied 59 strains from different substrates and geographic locations, and used a multilocus sequencing (MLS) approach complemented with the sequencing of mating type (MAT) genes to assess genetic variation and reexamine the boundaries of the two species, as well as their sexual status. The following five loci were chosen for MLS: the rDNA ITS-LSU region, the rDNA IGS1 spacer, and fragments of the genes encoding the largest subunit of RNA polymerase II (RPB1), the translation elongation factor 1 alpha (TEF1) and the p21-activated protein kinase (STE20). Phylogenetic network analyses confirmed the genetic separation of the two species and revealed two additional cryptic species, for which the names Cryptococcus baii and C. ruineniae are proposed. Further analyses of the data revealed a high degree of genetic heterogeneity within C. flavescens as well as evidence for recombination between lineages detected for this species. Strains of C. terrestris displayed higher levels of similarity in all analysed genes and appear to make up a single recombining group. The two MAT genes (STE3 and SXI1/SXI2) sequenced for C. flavescens strains confirmed the potential for sexual reproduction and suggest the presence of a tetrapolar mating system with a biallelic pheromone/receptor locus and a multiallelic HD locus. In C. terrestris we could only sequence STE3, which revealed a biallelic P/R locus. In spite of the strong evidence for sexual recombination in the two species, attempts at mating compatible strains of both species on culture media were unsuccessful.

(Cryptic) sex in the microsporidian Nosema granulosis--evidence from parasite rDNA and host mitochondrial DNA.

PubMed

Krebes, Lukas; Zeidler, Lisza; Frankowski, Jens; Bastrop, Ralf

2014-01-01

Microsporidia are single-celled, intracellular eukaryotes that parasitise a wide range of animals. The Nosema/Vairimorpha group includes some putative asexual species, and asexuality is proposed to have originated multiple times from sexual ancestors. Here, we studied the variation in the ribosomal DNA (rDNA) of 14 isolates of the presumed apomictic and vertically transmitted Nosema granulosis to evaluate its sexual status. The analysed DNA fragment contained a part of the small-subunit ribosomal gene (SSU) and the entire intergenic spacer (IGS). The mitochondrial cox1 gene of the host Gammarus duebeni (Crustacea) was analysed to temporally calibrate the system and to test the expectation of cophylogeny of host and parasite genealogies. Genetic variability of the SSU gene was very low within and between the isolates. In contrast, intraisolate (within a single host) variability of the IGS felt in two categories, because 12 isolates possess a very high IGS genetic diversity and two isolates were almost invariable in the IGS. This difference suggests variable models of rDNA evolution involving birth-and-death and unexpectedly concerted evolution. An alternative explanation could be a likewise unattended mixed infection of host individuals by more than one parasite strain. Despite considerable genetic divergence between associated host mitochondrial haplotypes, some N. granulosis 'IGS populations' seem not to belong to different gene pools; the relevant tests failed to show significant differences between populations. A set of recombinant IGS sequences made our data incompatible with the model of a solely maternally inherited, asexual species. In line with recent reports, our study supports the hypothesis that some assumed apomictic Microsporidia did not entirely abstain from the evolutionary advantages of sex. In addition, the presented data indicate that horizontal transmission may occur occasionally. This transmission mode could be a survival strategy of N. granulosis whose host often populates ephemeral habitats. Copyright © 2013 Elsevier B.V. All rights reserved.

Raptor, a positive regulatory subunit of mTOR complex 1, is a novel phosphoprotein of the rDNA transcription machinery in nucleoli and chromosomal nucleolus organizer regions (NORs).

PubMed

Vazquez-Martin, Alejandro; Cufí, Sílvia; Oliveras-Ferraros, Cristina; Menendez, Javier A

2011-09-15

Raptor is the key scaffolding protein that recruits mTOR substrates to rapamycin-sensitive mTOR complex 1 (mTORC1), a molecular integrator of mitogenic and nutrient/energy environmental inputs into protein translation and cell growth. Although Raptor phosphorylation on various sites is pivotal in the regulation of mTORC1 activity, it remains to be elucidated whether site-specific phosphorylation differentially distributes Raptor to unique subcellular compartments. When exploring the spatiotemporal cell cycle dynamics of six different phospho (P)-Raptor isoforms (Thr ( 706) , Ser ( 722) , Ser ( 863) , Ser ( 792) and Ser ( 877) ), a number of remarkable events differentially defined a topological resetting of P-RaptorThr706 on interphasic and mitotic chromosomes. In interphase nuclei, P-Raptor (Thr706) co-localized with fibrillarin, a component of the nucleolar small nuclear ribonucleoprotein particle, as well as with RNA polymerase I, the enzyme that transcribes nucleolar rRNA. Upon Actinomycin D-induced nucleolar segregation and disaggregation, P-RaptorThr706 was excluded from the nucleolus to accumulate at discrete nucleoplasmic bodies. During mitosis, CDK1 inhibition-induced premature assembly of nucleoli relocated fibrillarin to the surrounding regions of chromosomal-associated P-Raptor (Thr706) , suggesting that a subpopulation of mitotic P-Raptor (Thr706) remained targeted at chromosomal loops of rDNA or nuclear organizer regions (NORs). At the end of mitosis and cytokinesis, when reassembly of incipient nucleoli begins upon NORs activation of rDNA transcription, fibrillarin spatially reorganized with P-Raptor (Thr706) to give rise to daughter nucleoli. Treatment with IGF1 exclusively hyperactivated nuclear P-Raptor (Ser706) and concomitantly promoted Ser ( 2481) autophosphorylation of mTOR, which monitors mTORC1-associated catalytic activity. Nucleolar- and NOR-associated P-Raptor (Ser706) may physically link mTORC1 signaling to ever-growing nucleolus plurifunctionality including ribosome biogenesis, cell stress sensor and cell cycle/aging control.

Multigene Assessment of the Species Boundaries and Sexual Status of the Basidiomycetous Yeasts Cryptococcus flavescens and C. terrestris (Tremellales)

PubMed Central

Sharma, Lav; Carvalho, Cláudia; Fonseca, Álvaro

2015-01-01

Cryptococcus flavescens and C. terrestris are phenotypically indistinguishable sister species that belong to the order Tremellales (Tremellomycetes, Basidiomycota) and which may be mistaken for C. laurentii based on phenotype. Phylogenetic separation between C. flavescens and C. terrestris was based on rDNA sequence analyses, but very little is known on their intraspecific genetic variability or propensity for sexual reproduction. We studied 59 strains from different substrates and geographic locations, and used a multilocus sequencing (MLS) approach complemented with the sequencing of mating type (MAT) genes to assess genetic variation and reexamine the boundaries of the two species, as well as their sexual status. The following five loci were chosen for MLS: the rDNA ITS-LSU region, the rDNA IGS1 spacer, and fragments of the genes encoding the largest subunit of RNA polymerase II (RPB1), the translation elongation factor 1 alpha (TEF1) and the p21-activated protein kinase (STE20). Phylogenetic network analyses confirmed the genetic separation of the two species and revealed two additional cryptic species, for which the names Cryptococcus baii and C. ruineniae are proposed. Further analyses of the data revealed a high degree of genetic heterogeneity within C. flavescens as well as evidence for recombination between lineages detected for this species. Strains of C. terrestris displayed higher levels of similarity in all analysed genes and appear to make up a single recombining group. The two MAT genes (STE3 and SXI1/SXI2) sequenced for C. flavescens strains confirmed the potential for sexual reproduction and suggest the presence of a tetrapolar mating system with a biallelic pheromone/receptor locus and a multiallelic HD locus. In C. terrestris we could only sequence STE3, which revealed a biallelic P/R locus. In spite of the strong evidence for sexual recombination in the two species, attempts at mating compatible strains of both species on culture media were unsuccessful. PMID:25811603

rDNA Copy Number Variants Are Frequent Passenger Mutations in Saccharomyces cerevisiae Deletion Collections and de Novo Transformants

PubMed Central

Kwan, Elizabeth X.; Wang, Xiaobin S.; Amemiya, Haley M.; Brewer, Bonita J.; Raghuraman, M. K.

2016-01-01

The Saccharomyces cerevisiae ribosomal DNA (rDNA) locus is known to exhibit greater instability relative to the rest of the genome. However, wild-type cells preferentially maintain a stable number of rDNA copies, suggesting underlying genetic control of the size of this locus. We performed a screen of a subset of the Yeast Knock-Out (YKO) single gene deletion collection to identify genetic regulators of this locus and to determine if rDNA copy number correlates with yeast replicative lifespan. While we found no correlation between replicative lifespan and rDNA size, we identified 64 candidate strains with significant rDNA copy number differences. However, in the process of validating candidate rDNA variants, we observed that independent isolates of our de novo gene deletion strains had unsolicited but significant changes in rDNA copy number. Moreover, we were not able to recapitulate rDNA phenotypes from the YKO yeast deletion collection. Instead, we found that the standard lithium acetate transformation protocol is a significant source of rDNA copy number variation, with lithium acetate exposure being the treatment causing variable rDNA copy number events after transformation. As the effects of variable rDNA copy number are being increasingly reported, our finding that rDNA is affected by lithium acetate exposure suggested that rDNA copy number variants may be influential passenger mutations in standard strain construction in S. cerevisiae. PMID:27449518

rDNA Copy Number Variants Are Frequent Passenger Mutations in Saccharomyces cerevisiae Deletion Collections and de Novo Transformants.

PubMed

Kwan, Elizabeth X; Wang, Xiaobin S; Amemiya, Haley M; Brewer, Bonita J; Raghuraman, M K

2016-09-08

The Saccharomyces cerevisiae ribosomal DNA (rDNA) locus is known to exhibit greater instability relative to the rest of the genome. However, wild-type cells preferentially maintain a stable number of rDNA copies, suggesting underlying genetic control of the size of this locus. We performed a screen of a subset of the Yeast Knock-Out (YKO) single gene deletion collection to identify genetic regulators of this locus and to determine if rDNA copy number correlates with yeast replicative lifespan. While we found no correlation between replicative lifespan and rDNA size, we identified 64 candidate strains with significant rDNA copy number differences. However, in the process of validating candidate rDNA variants, we observed that independent isolates of our de novo gene deletion strains had unsolicited but significant changes in rDNA copy number. Moreover, we were not able to recapitulate rDNA phenotypes from the YKO yeast deletion collection. Instead, we found that the standard lithium acetate transformation protocol is a significant source of rDNA copy number variation, with lithium acetate exposure being the treatment causing variable rDNA copy number events after transformation. As the effects of variable rDNA copy number are being increasingly reported, our finding that rDNA is affected by lithium acetate exposure suggested that rDNA copy number variants may be influential passenger mutations in standard strain construction in S. cerevisiae. Copyright © 2016 Kwan et al.

Highly conserved small subunit residues influence rubisco large subunit catalysis.

PubMed

Genkov, Todor; Spreitzer, Robert J

2009-10-30

The chloroplast enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO(2) fixation. With a deeper understanding of its structure-function relationships and competitive inhibition by O(2), it may be possible to engineer an increase in agricultural productivity and renewable energy. The chloroplast-encoded large subunits form the active site, but the nuclear-encoded small subunits can also influence catalytic efficiency and CO(2)/O(2) specificity. To further define the role of the small subunit in Rubisco function, the 10 most conserved residues in all small subunits were substituted with alanine by transformation of a Chlamydomonas reinhardtii mutant that lacks the small subunit gene family. All the mutant strains were able to grow photosynthetically, indicating that none of the residues is essential for function. Three of the substitutions have little or no effect (S16A, P19A, and E92A), one primarily affects holoenzyme stability (L18A), and the remainder affect catalysis with or without some level of associated structural instability (Y32A, E43A, W73A, L78A, P79A, and F81A). Y32A and E43A cause decreases in CO(2)/O(2) specificity. Based on the x-ray crystal structure of Chlamydomonas Rubisco, all but one (Glu-92) of the conserved residues are in contact with large subunits and cluster near the amino- or carboxyl-terminal ends of large subunit alpha-helix 8, which is a structural element of the alpha/beta-barrel active site. Small subunit residues Glu-43 and Trp-73 identify a possible structural connection between active site alpha-helix 8 and the highly variable small subunit loop between beta-strands A and B, which can also influence Rubisco CO(2)/O(2) specificity.

Development of a Prokaryotic Universal Primer for Simultaneous Analysis of Bacteria and Archaea Using Next-Generation Sequencing

PubMed Central

Takahashi, Shunsuke; Tomita, Junko; Nishioka, Kaori; Hisada, Takayoshi; Nishijima, Miyuki

2014-01-01

For the analysis of microbial community structure based on 16S rDNA sequence diversity, sensitive and robust PCR amplification of 16S rDNA is a critical step. To obtain accurate microbial composition data, PCR amplification must be free of bias; however, amplifying all 16S rDNA species with equal efficiency from a sample containing a large variety of microorganisms remains challenging. Here, we designed a universal primer based on the V3-V4 hypervariable region of prokaryotic 16S rDNA for the simultaneous detection of Bacteria and Archaea in fecal samples from crossbred pigs (Landrace×Large white×Duroc) using an Illumina MiSeq next-generation sequencer. In-silico analysis showed that the newly designed universal prokaryotic primers matched approximately 98.0% of Bacteria and 94.6% of Archaea rRNA gene sequences in the Ribosomal Database Project database. For each sequencing reaction performed with the prokaryotic universal primer, an average of 69,330 (±20,482) reads were obtained, of which archaeal rRNA genes comprised approximately 1.2% to 3.2% of all prokaryotic reads. In addition, the detection frequency of Bacteria belonging to the phylum Verrucomicrobia, including members of the classes Verrucomicrobiae and Opitutae, was higher in the NGS analysis using the prokaryotic universal primer than that performed with the bacterial universal primer. Importantly, this new prokaryotic universal primer set had markedly lower bias than that of most previously designed universal primers. Our findings demonstrate that the prokaryotic universal primer set designed in the present study will permit the simultaneous detection of Bacteria and Archaea, and will therefore allow for a more comprehensive understanding of microbial community structures in environmental samples. PMID:25144201

Detecting Mechanisms of Karyotype Evolution in Heterotaxis (Orchidaceae)

PubMed Central

Olmos Simões, André; Ojeda Alayon, Dario Isidro; de Barros, Fábio; Forni-Martins, Eliana Regina

2016-01-01

The karyotype is shaped by different chromosome rearrangements during species evolution. However, determining which rearrangements are responsible for karyotype changes is a challenging task and the combination of a robust phylogeny with refined karyotype characterization, GS measurements and bioinformatic modelling is necessary. Here, this approach was applied in Heterotaxis to determine what chromosome rearrangements were responsible for the dysploidy variation. We used two datasets (nrDNA and cpDNA, both under MP and BI) to infer the phylogenetic relationships among Heterotaxis species and the closely related genera Nitidobulbon and Ornithidium. Such phylogenies were used as framework to infer how karyotype evolution occurred using statistical methods. The nrDNA recovered Ornithidium, Nitidobulbon and Heterotaxis as monophyletic under both MP and BI; while cpDNA could not completely separate the three genera under both methods. Based on the GS, we recovered two groups within Heterotaxis: (1) "small GS", corresponding to the Sessilis grade, composed of plants with smaller genomes and smaller morphological structure, and (2) "large GS", corresponding to the Discolor clade, composed of plants with large genomes and robust morphological structures. The robust karyotype modeling, using both nrDNA phylogenies, allowed us to infer that the ancestral Heterotaxis karyotype presented 2n = 40, probably with a proximal 45S rDNA on a metacentric chromosome pair. The chromosome number variation was caused by ascending dysploidy (chromosome fission involving the proximal 45S rDNA site resulting in two acrocentric chromosome pairs holding a terminal 45S rDNA), with subsequent descending dysploidy (fusion) in two species, H. maleolens and H. sessilis. However, besides dysploidy, our analysis detected another important chromosome rearrangement in the Orchidaceae: chromosome inversion, that promoted 5S rDNA site duplication and relocation. PMID:27832130

Ribosomal DNA stability is supported by many 'buffer genes'-introduction to the Yeast rDNA Stability Database.

PubMed

Kobayashi, Takehiko; Sasaki, Mariko

2017-01-01

The ribosomal RNA gene (rDNA) is the most abundant gene in yeast and other eukaryotic organisms. Due to its heavy transcription, repetitive structure and programmed replication fork pauses, the rDNA is one of the most unstable regions in the genome. Thus, the rDNA is the best region to study the mechanisms responsible for maintaining genome integrity. Recently, we screened a library of ∼4800 budding yeast gene knockout strains to identify mutants defective in the maintenance of rDNA stability. The results of this screen are summarized in the Yeast rDNA Stability (YRS) Database, in which the stability and copy number of rDNA in each mutant are presented. From this screen, we identified ∼700 genes that may contribute to the maintenance of rDNA stability. In addition, ∼50 mutants had abnormally high or low rDNA copy numbers. Moreover, some mutants with unstable rDNA displayed abnormalities in another chromosome. In this review, we introduce the YRS Database and discuss the roles of newly identified genes that contribute to rDNA maintenance and genome integrity. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Complete sequence analysis of 18S rDNA based on genomic DNA extraction from individual Demodex mites (Acari: Demodicidae).

PubMed

Zhao, Ya-E; Xu, Ji-Ru; Hu, Li; Wu, Li-Ping; Wang, Zheng-Hang

2012-05-01

The study for the first time attempted to accomplish 18S ribosomal DNA (rDNA) complete sequence amplification and analysis for three Demodex species (Demodex folliculorum, Demodex brevis and Demodex canis) based on gDNA extraction from individual mites. The mites were treated by DNA Release Additive and Hot Start II DNA Polymerase so as to promote mite disruption and increase PCR specificity. Determination of D. folliculorum gDNA showed that the gDNA yield reached the highest at 1 mite, tending to descend with the increase of mite number. The individual mite gDNA was successfully used for 18S rDNA fragment (about 900 bp) amplification examination. The alignments of 18S rDNA complete sequences of individual mite samples and those of pooled mite samples ( ≥ 1000mites/sample) showed over 97% identities for each species, indicating that the gDNA extracted from a single individual mite was as satisfactory as that from pooled mites for PCR amplification. Further pairwise sequence analyses showed that average divergence, genetic distance, transition/transversion or phylogenetic tree could not effectively identify the three Demodex species, largely due to the differentiation in the D. canis isolates. It can be concluded that the individual Demodex mite gDNA can satisfy the molecular study of Demodex. 18S rDNA complete sequence is suitable for interfamily identification in Cheyletoidea, but whether it is suitable for intrafamily identification cannot be confirmed until the ascertainment of the types of Demodex mites parasitizing in dogs. Copyright © 2012 Elsevier Inc. All rights reserved.

The Core Subunit of A Chromatin-Remodeling Complex, ZmCHB101, Plays Essential Roles in Maize Growth and Development.

PubMed

Yu, Xiaoming; Jiang, Lili; Wu, Rui; Meng, Xinchao; Zhang, Ai; Li, Ning; Xia, Qiong; Qi, Xin; Pang, Jinsong; Xu, Zheng-Yi; Liu, Bao

2016-12-05

ATP-dependent chromatin remodeling complexes play essential roles in the regulation of diverse biological processes by formulating a DNA template that is accessible to the general transcription apparatus. Although the function of chromatin remodelers in plant development has been studied in A. thaliana, how it affects growth and development of major crops (e.g., maize) remains uninvestigated. Combining genetic, genomic and bioinformatic analyses, we show here that the maize core subunit of chromatin remodeling complex, ZmCHB101, plays essential roles in growth and development of maize at both vegetative and reproductive stages. Independent ZmCHB101 RNA interference plant lines displayed abaxially curling leaf phenotype due to increase of bulliform cell numbers, and showed impaired development of tassel and cob. RNA-seq-based transcriptome profiling revealed that ZmCHB101 dictated transcriptional reprogramming of a significant set of genes involved in plant development, photosynthesis, metabolic regulation, stress response and gene expressional regulation. Intriguingly, we found that ZmCHB101 was required for maintaining normal nucleosome density and 45 S rDNA compaction. Our findings suggest that the SWI3 protein, ZmCHB101, plays pivotal roles in maize normal growth and development via regulation of chromatin structure.

Identification of four squid species by quantitative real-time polymerase chain reaction.

PubMed

Ye, Jian; Feng, Junli; Liu, Shasha; Zhang, Yanping; Jiang, Xiaona; Dai, Zhiyuan

2016-02-01

Squids are distributed worldwide, including many species of commercial importance, and they are often made into varieties of flavor foods. The rapid identification methods for squid species especially their processed products, however, have not been well developed. In this study, quantitative real-time PCR (qPCR) systems based on specific primers and TaqMan probes have been established for rapid and accurate identification of four common squid species (Ommastrephes bartramii, Dosidicus gigas, Illex argentinus, Todarodes pacificus) in Chinese domestic market. After analyzing mitochondrial genes reported in GenBank, the mitochondrial cytochrome b (Cytb) gene was selected for O. bartramii detection, cytochrome c oxidase subunit I (COI) gene for D. gigas and T. Pacificus detection, ATPase subunit 6 (ATPase 6) gene for I. Argentinus detection, and 12S ribosomal RNA (12S rDNA) gene for designing Ommastrephidae-specific primers and probe. As a result, all the TaqMan systems are of good performance, and efficiency of each reaction was calculated by making standard curves. This method could detect target species either in single or mixed squid specimen, and it was applied to identify 12 squid processed products successfully. Thus, it would play an important role in fulfilling labeling regulations and squid fishery control. Copyright © 2016 Elsevier Ltd. All rights reserved.

Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex

PubMed Central

Lyumkis, Dmitry; Oliveira dos Passos, Dario; Tahara, Erich B.; Webb, Kristofor; Bennett, Eric J.; Vinterbo, Staal; Potter, Clinton S.; Carragher, Bridget; Joazeiro, Claudio A. P.

2014-01-01

All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood. Using single-particle cryoelectron microscopy, we have determined the structure of the RQC complex bound to stalled 60S ribosomal subunits. The structure establishes how Ltn1 associates with the large ribosomal subunit and properly positions its E3-catalytic RING domain to mediate nascent chain ubiquitylation. The structure also reveals that a distinguishing feature of stalled 60S particles is an exposed, nascent chain-conjugated tRNA, and that the Tae2 subunit of RQC, which facilitates Ltn1 binding, is responsible for selective recognition of stalled 60S subunits. RQC components are engaged in interactions across a large span of the 60S subunit surface, connecting the tRNA in the peptidyl transferase center to the distally located nascent chain tunnel exit. This work provides insights into a mechanism linking translation and protein degradation that targets defective proteins immediately after synthesis, while ignoring nascent chains in normally translating ribosomes. PMID:25349383

Distribution of a limited Sir2 protein pool regulates the strength of yeast rDNA silencing and is modulated by Sir4p.

PubMed Central

Smith, J S; Brachmann, C B; Pillus, L; Boeke, J D

1998-01-01

Transcriptional silencing in Saccharomyces cerevisiae occurs at the silent mating-type loci HML and HMR, at telomeres, and at the ribosomal DNA (rDNA) locus RDN1. Silencing in the rDNA occurs by a novel mechanism that depends on a single Silent Information Regulator (SIR) gene, SIR2. SIR4, essential for other silenced loci, paradoxically inhibits rDNA silencing. In this study, we elucidate a regulatory mechanism for rDNA silencing based on the finding that rDNA silencing strength directly correlates with cellular Sir2 protein levels. The endogenous level of Sir2p was shown to be limiting for rDNA silencing. Furthermore, small changes in Sir2p levels altered rDNA silencing strength. In rDNA silencing phenotypes, sir2 mutations were shown to be epistatic to sir4 mutations, indicating that SIR4 inhibition of rDNA silencing is mediated through SIR2. Furthermore, rDNA silencing is insensitive to SIR3 overexpression, but is severely reduced by overexpression of full-length Sir4p or a fragment of Sir4p that interacts with Sir2p. This negative effect of SIR4 overexpression was overridden by co-overexpression of SIR2, suggesting that SIR4 directly inhibits the rDNA silencing function of SIR2. Finally, genetic manipulations of SIR4 previously shown to promote extended life span also resulted in enhanced rDNA silencing. We propose a simple model in which telomeres act as regulators of rDNA silencing by competing for limiting amounts of Sir2 protein. PMID:9649515

Co-located hAT transposable element and 5S rDNA in an interstitial telomeric sequence suggest the formation of Robertsonian fusion in armored catfish.

PubMed

Glugoski, Larissa; Giuliano-Caetano, Lucia; Moreira-Filho, Orlando; Vicari, Marcelo R; Nogaroto, Viviane

2018-04-15

Co-located 5S rDNA genes and interstitial telomeric sites (ITS) revealed the involvement of multiple 5S rDNA clusters in chromosome rearrangements of Loricariidae. Interstitial (TTAGGG)n vestiges, in addition to telomeric sites, can coincide with locations of chromosomal rearrangements, and they are considered to be hotspots for chromosome breaks. This study aimed the molecular characterization of 5S rDNA in two Rineloricaria latirostris populations and examination of roles of 5S rDNA in breakpoint sites and its in situ localization. Rineloricaria latirostris from Brazil's Das Pedras river (2n = 46 chromosomes) presented five pairs identified using a 5S rDNA probe, in addition to a pair bearing a co-located ITS/5S rDNA. Rineloricaria latirostris from the Piumhi river (2n = 48 chromosomes) revealed two pairs containing 5S rDNA, without ITS. A 702-bp amplified sequence, using 5S rDNA primers, revealed an insertion of the hAT transposable element (TE), referred to as a degenerate 5S rDNA. Double-FISH (fluorescence in situ hybridization) demonstrated co-localization of 5S rDNA/degenerate 5S rDNA, 5S rDNA/hAT and ITS/5S rDNA from the Das Pedras river population. Piumhi river isolates possessed only 5S rDNA sites. We suggest that the degenerate 5S rDNA was generated by unequal crossing over, which was driven by invasion of hAT, establishing a breakpoint region susceptible to chromosome breakage, non-homologous recombination and Robertsonian (Rb) fusion. Furthermore, the presence of clusters of 5S rDNA at fusion points in other armored catfish species suggests its re-use and that these regions represent hotspots for evolutionary rearrangements within Loricariidae genomes. Copyright © 2018 Elsevier B.V. All rights reserved.

Distinct cellular distributions of Kv4 pore-forming and auxiliary subunits in rat dorsal root ganglion neurons.

PubMed

Matsuyoshi, Hiroko; Takimoto, Koichi; Yunoki, Takakazu; Erickson, Vickie L; Tyagi, Pradeep; Hirao, Yoshihiko; Wanaka, Akio; Yoshimura, Naoki

2012-09-17

Dorsal root ganglia contain heterogeneous populations of primary afferent neurons that transmit various sensory stimuli. This functional diversity may be correlated with differential expression of voltage-gated K(+) (Kv) channels. Here, we examine cellular distributions of Kv4 pore-forming and ancillary subunits that are responsible for fast-inactivating A-type K(+) current. Expression pattern of Kv α-subunit, β-subunit and auxiliary subunit was investigated using immunohistochemistry, in situ hybridization and RT-PCR technique. The two pore-forming subunits Kv4.1 and Kv4.3 show distinct cellular distributions: Kv4.3 is predominantly in small-sized C-fiber neurons, whereas Kv4.1 is seen in DRG neurons in various sizes. Furthermore, the two classes of Kv4 channel auxiliary subunits are also distributed in different-sized cells. KChIP3 is the only significantly expressed Ca(2+)-binding cytosolic ancillary subunit in DRGs and present in medium to large-sized neurons. The membrane-spanning auxiliary subunit DPP6 is seen in a large number of DRG neurons in various sizes, whereas DPP10 is restricted in small-sized neurons. Distinct combinations of Kv4 pore-forming and auxiliary subunits may constitute A-type channels in DRG neurons with different physiological roles. Kv4.1 subunit, in combination with KChIP3 and/or DPP6, form A-type K(+) channels in medium to large-sized A-fiber DRG neurons. In contrast, Kv4.3 and DPP10 may contribute to A-type K(+) current in non-peptidergic, C-fiber somatic afferent neurons. Copyright © 2012 Elsevier Inc. All rights reserved.

Molecular phylogeny of Oncaeidae (Copepoda) using nuclear ribosomal internal transcribed spacer (ITS rDNA)

PubMed Central

Di Capua, Iole; Maffucci, Fulvio; Pannone, Raimondo; Mazzocchi, Maria Grazia

2017-01-01

Copepods belonging to the Oncaeidae family are commonly and abundantly found in marine zooplankton. In the Mediterranean Sea, forty-seven oncaeid species occur, of which eleven in the Gulf of Naples. In this Gulf, several Oncaea species were morphologically analysed and described at the end of the XIX century by W. Giesbrecht. In the same area, oncaeids are being investigated over seasonal and inter-annual scales at the long-term coastal station LTER-MC. In the present work, we identified six oncaeid species using the nuclear ribosomal internal transcribed spacers (ITS rDNA) and the mitochondrial cytochrome c oxidase subunit I (mtCOI). Phylogenetic analyses based on these two genomic regions validated the sisterhood of the genera Triconia and the Oncaea sensu stricto. ITS1 and ITS2 phylogenies produced incongruent results about the position of Oncaea curta, calling for further investigations on this species. We also characterised the ITS2 region by secondary structure predictions and found that all the sequences analysed presented the distinct eukaryotic hallmarks. A Compensatory Base Change search corroborated the close relationship between O. venusta and O. curta and between O. media and O. venusta already identified by ITS phylogenies. The present results, which stem from the integration of molecular and morphological taxonomy, represent an encouraging step towards an improved knowledge of copepod biodiversity: The two complementary approaches, when applied to long-term copepod monitoring, will also help to better understanding their genetic variations and ecological niches of co-occurring species. PMID:28441395

Phylogeny and ecology of the ubiquitous saprobe Cladosporium sphaerospermum, with descriptions of seven new species from hypersaline environments

PubMed Central

Zalar, P.; de Hoog, G.S.; Schroers, H.-J.; Crous, P.W.; Groenewald, J.Z.; Gunde-Cimerman, N.

2007-01-01

Saprobic Cladosporium isolates morphologically similar to C. sphaerospermum are phylogenetically analysed on the basis of DNA sequences of the ribosomal RNA gene cluster, including the internal transcribed spacer regions ITS1 and ITS2, the 5.8S rDNA (ITS) and the small subunit (SSU) rDNA as well as β-tubulin and actin gene introns and exons. Most of the C. sphaerospermum-like species show halotolerance as a recurrent feature. Cladosporium sphaerospermum, which is characterised by almost globose conidia, is redefined on the basis of its ex-neotype culture. Cladosporium dominicanum, C. psychrotolerans, C. velox, C. spinulosum and C. halotolerans, all with globoid conidia, are newly described on the basis of phylogenetic analyses and cryptic morphological and physiological characters. Cladosporium halotolerans was isolated from hypersaline water and bathrooms and detected once on dolphin skin. Cladosporium dominicanum and C. velox were isolated from plant material and hypersaline water. Cladosporium psychrotolerans, which grows well at 4 °C but not at 30 °C, and C. spinulosum, having conspicuously ornamented conidia with long digitate projections, are currently only known from hypersaline water. We also newly describe C. salinae from hypersaline water and C. fusiforme from hypersaline water and animal feed. Both species have ovoid to ellipsoid conidia and are therefore reminiscent of C. herbarum. Cladosporium langeronii (= Hormodendrum langeronii) previously described as a pathogen on human skin, is halotolerant but has not yet been recorded from hypersaline environments. PMID:18490999

Pyrosequencing assessment of prokaryotic and eukaryotic diversity in biofilm communities from a French river.

PubMed

Bricheux, Geneviève; Morin, Loïc; Le Moal, Gwenaël; Coffe, Gérard; Balestrino, Damien; Charbonnel, Nicolas; Bohatier, Jacques; Forestier, Christiane

2013-06-01

Despite the recent and significant increase in the study of aquatic microbial communities, little is known about the microbial diversity of complex ecosystems such as running waters. This study investigated the biodiversity of biofilm communities formed in a river with 454 Sequencing™. This river has the particularity of integrating both organic and microbiological pollution, as receiver of agricultural pollution in its upstream catchment area and urban pollution through discharges of the wastewater treatment plant of the town of Billom. Different regions of the small subunit (SSU) ribosomal RNA gene were targeted using nine pairs of primers, either universal or specific for bacteria, eukarya, or archaea. Our aim was to characterize the widest range of rDNA sequences using different sets of polymerase chain reaction (PCR) primers. A first look at reads abundance revealed that a large majority (47-48%) were rare sequences (<5 copies). Prokaryotic phyla represented the species richness, and eukaryotic phyla accounted for a small part. Among the prokaryotic phyla, Proteobacteria (beta and alpha) predominated, followed by Bacteroidetes together with a large number of nonaffiliated bacterial sequences. Bacillariophyta plastids were abundant. The remaining bacterial phyla, Verrucomicrobia and Cyanobacteria, made up the rest of the bulk biodiversity. The most abundant eukaryotic phyla were annelid worms, followed by Diatoms, and Chlorophytes. These latter phyla attest to the abundance of plastids and the importance of photosynthetic activity for the biofilm. These findings highlight the existence and plasticity of multiple trophic levels within these complex biological systems. © 2013 The Authors. Microbiology Open published by John Wiley & Sons Ltd.

Protection Conferred by recombinant Yersinia pestis Antigens Produced by a Rapid and Highly Scalable Plant Expression System

DTIC Science & Technology

2006-01-24

translational fusions with dsRED (lanes 8), and cytosol-targeted GFP (lanes 9). RbcL, large subunit of Rubisco . 862 ! www.pnas.org"cgi"doi൒.1073...analysis of F1-V expression with SDS"PAGE-Coomassie staining was difficult because the chimeric protein comigrates with the large subunit of Rubisco , a...contaminated by the Rubisco large subunit, which is very similar in size to F1-V. Analysis of Purified Plant-Produced Antigens. Western blots were

Physical locations of 5S and 18S-25S rDNA in Asian and American diploid Hordeum species with the I genome.

PubMed

Taketa, S; Ando, H; Takeda, K; von Bothmer, R

2001-05-01

The physical locations of 5S and 18S-25S rDNA sequences in 15 diploid Hordeum species with the I genome were examined by double-target in situ hybridization with pTa71 (18S-25S rDNA) and pTa794 (5S rDNA) clones as probes. All the three Asian species had a species-specific rDNA pattern. In 12 American species studied, eight different rDNA types were found. The type reported previously in H. chilense (the 'chilense' type) was observed in eight American species. The chilense type had double 5S rDNA sites - two sites on one chromosome arm separated by a short distance - and two pairs of major 18S-25S rDNA sites on two pairs of satellite chromosomes. The other seven types found in American species were similar to the chilense type and could be derived from the chilense type through deletion, reduction or addition of a rDNA site. Intraspecific polymorphisms were observed in three American species. The overall similarity in rDNA patterns among American species indicates the close relationships between North and South American species and their derivation from a single ancestral source. The differences in the distribution patterns of 5S and 18S-25S rDNA between Asian and American species suggest differentiation between the I genomes of Asian and American species. The 5S and 18S-25S rDNA sites are useful chromosome markers for delimiting Asian species, but have limited value as a taxonomic character in American species. On the basis of rDNA patterns, karyotype evolution and phylogeny of the I-genome diploid species are discussed.

Diversity, dynamics, and activity of bacterial communities during production of an artisanal Sicilian cheese as evaluated by 16S rRNA analysis.

PubMed

Randazzo, Cinzia L; Torriani, Sandra; Akkermans, Antoon D L; de Vos, Willem M; Vaughan, Elaine E

2002-04-01

The diversity and dynamics of the microbial communities during the manufacturing of Ragusano cheese, an artisanal cheese produced in Sicily (Italy), were investigated by a combination of classical and culture-independent approaches. The latter included PCR, reverse transcriptase-PCR (RT-PCR), and denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes (rDNA). Bacterial and Lactobacillus group-specific primers were used to amplify the V6 to V8 and V1 to V3 regions of the 16S rRNA gene, respectively. DGGE profiles from samples taken during cheese production indicated dramatic shifts in the microbial community structure. Cloning and sequencing of rDNA amplicons revealed that mesophilic lactic acid bacteria (LAB), including species of Leuconostoc, Lactococcus lactis, and Macrococcus caseolyticus were dominant in the raw milk, while Streptococcus thermophilus prevailed during lactic fermentation. Other thermophilic LAB, especially Lactobacillus delbrueckii and Lactobacillus fermentum, also flourished during ripening. Comparison of the rRNA-derived patterns obtained by RT-PCR to the rDNA DGGE patterns indicated a substantially different degree of metabolic activity for the microbial groups detected. Identification of cultivated LAB isolates by phenotypic characterization and 16S rDNA analysis indicated a variety of species, reflecting to a large extent the results obtained from the 16S rDNA clone libraries, with the significant exception of the Lactobacillus delbrueckii species, which dominated in the ripening cheese but was not detected by cultivation. The present molecular approaches combined with culture can effectively describe the complex ecosystem of natural fermented dairy products, giving useful information for starter culture design and preservation of artisanal fermented food technology.

The site-specific ribosomal insertion element type II of Bombyx mori (R2Bm) contains the coding sequence for a reverse transcriptase-like enzyme.

PubMed Central

Burke, W D; Calalang, C C; Eickbush, T H

1987-01-01

Two classes of DNA elements interrupt a fraction of the rRNA repeats of Bombyx mori. We have analyzed by genomic blotting and sequence analysis one class of these elements which we have named R2. These elements occupy approximately 9% of the rDNA units of B. mori and appear to be homologous to the type II rDNA insertions detected in Drosophila melanogaster. Approximately 25 copies of R2 exist within the B. mori genome, of which at least 20 are located at a precise location within otherwise typical rDNA units. Nucleotide sequence analysis has revealed that the 4.2-kilobase-pair R2 element has a single large open reading frame, occupying over 82% of the total length of the element. The central region of this 1,151-amino-acid open reading frame shows homology to the reverse transcriptase enzymes found in retroviruses and certain transposable elements. Amino acid homology of this region is highest to the mobile line 1 elements of mammals, followed by the mitochondrial type II introns of fungi, and the pol gene of retroviruses. Less homology exists with transposable elements of D. melanogaster and Saccharomyces cerevisiae. Two additional regions of sequence homology between L1 and R2 elements were also found outside the reverse transcriptase region. We suggest that the R2 elements are retrotransposons that are site specific in their insertion into the genome. Such mobility would enable these elements to occupy a small fraction of the rDNA units of B. mori despite their continual elimination from the rDNA locus by sequence turnover. Images PMID:2439905

Mechanism of the modulation of BK potassium channel complexes with different auxiliary subunit compositions by the omega-3 fatty acid DHA.

PubMed

Hoshi, Toshinori; Tian, Yutao; Xu, Rong; Heinemann, Stefan H; Hou, Shangwei

2013-03-19

Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels are well known for their functional versatility, which is bestowed in part by their rich modulatory repertoire. We recently showed that long-chain omega-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA) found in oily fish lower blood pressure by activating vascular BK channels made of Slo1+β1 subunits. Here we examined the action of DHA on BK channels with different auxiliary subunit compositions. Neuronal Slo1+β4 channels were just as well activated by DHA as vascular Slo1+β1 channels. In contrast, the stimulatory effect of DHA was much smaller in Slo1+β2, Slo1+LRRC26 (γ1), and Slo1 channels without auxiliary subunits. Mutagenesis of β1, β2, and β4 showed that the large effect of DHA in Slo1+β1 and Slo1+β4 is conferred by the presence of two residues, one in the N terminus and the other in the first transmembrane segment of the β1 and β4 subunits. Transfer of this amino acid pair from β1 or β4 to β2 introduces a large response to DHA in Slo1+β2. The presence of a pair of oppositely charged residues at the aforementioned positions in β subunits is associated with a large response to DHA. The Slo1 auxiliary subunits are expressed in a highly tissue-dependent fashion. Thus, the subunit composition-dependent stimulation by DHA demonstrates that BK channels are effectors of omega-3 fatty acids with marked tissue specificity.

γ-Aminobutyric Acid Type A α4, β2, and δ Subunits Assemble to Produce More Than One Functionally Distinct Receptor Type

PubMed Central

Eaton, Megan M.; Bracamontes, John; Shu, Hong-Jin; Li, Ping; Mennerick, Steven; Steinbach, Joe Henry

2014-01-01

Native γ-aminobutyric acid (GABA)A receptors consisting of α4, β1–3, and δ subunits mediate responses to the low, tonic concentration of GABA present in the extracellular milieu. Previous studies on heterologously expressed α4βδ receptors have shown a large degree of variability in functional properties, including sensitivity to the transmitter. We studied properties of α4β2δ receptors employing free subunits and concatemeric constructs, expressed in Xenopus oocytes, HEK 293 cells, and cultured hippocampal neurons. The expression system had a strong effect on the properties of receptors containing free subunits. The midpoint of GABA activation curve was 10 nM for receptors in oocytes versus 2300 nM in HEK cells. Receptors activated by the steroid alfaxalone had an estimated maximal open probability of 0.6 in oocytes and 0.01 in HEK cells. Irrespective of the expression system, receptors resulting from combining the tandem construct β2-δ and a free α4 subunit exhibited large steroid responses. We propose that free α4, β2, and δ subunits assemble in different configurations with distinct properties in oocytes and HEK cells, and that subunit linkage can overcome the expression system-dependent preferential assembly of free subunits. Hippocampal neurons transfected with α4 and the picrotoxin-resistant δ(T269Y) subunit showed large responses to alfaxalone in the presence of picrotoxin, suggesting that α4βδ receptors may assemble in a similar configuration in neurons and oocytes. PMID:25238745

Low Genetic Diversity in Wide-Spread Eurasian Liver Fluke Opisthorchis felineus Suggests Special Demographic History of This Trematode Species

PubMed Central

Brusentsov, Ilja I.; Katokhin, Alexey V.; Brusentsova, Irina V.; Shekhovtsov, Sergei V.; Borovikov, Sergei N.; Goncharenko, Grigoriy G.; Lider, Lyudmila A.; Romashov, Boris V.; Rusinek, Olga T.; Shibitov, Samat K.; Suleymanov, Marat M.; Yevtushenko, Andrey V.; Mordvinov, Viatcheslav A.

2013-01-01

Opisthorchis felineus or Siberian liver fluke is a trematode parasite (Opisthorchiidae) that infects the hepato-biliary system of humans and other mammals. Despite its public health significance, this wide-spread Eurasian species is one of the most poorly studied human liver flukes and nothing is known about its population genetic structure and demographic history. In this paper, we attempt to fill this gap for the first time and to explore the genetic diversity in O. felineus populations from Eastern Europe (Ukraine, European part of Russia), Northern Asia (Siberia) and Central Asia (Northern Kazakhstan). Analysis of marker DNA fragments from O. felineus mitochondrial cytochrome c oxidase subunit 1 and 3 (cox1, cox3) and nuclear rDNA internal transcribed spacer 1 (ITS1) sequences revealed that genetic diversity is very low across the large geographic range of this species. Microevolutionary processes in populations of trematodes may well be influenced by their peculiar biology. Nevertheless, we suggest that lack of population genetics structure observed in O. felineus can be primarily explained by the Pleistocene glacial events and subsequent sudden population growth from a very limited group of founders. Rapid range expansion of O. felineus through Asian and European territories after severe bottleneck points to a high dispersal potential of this trematode species. PMID:23634228

The 28S–18S rDNA intergenic spacer from Crithidia fasciculata: repeated sequences, length heterogeneity, putative processing sites and potential interactions between U3 small nucleolar RNA and the ribosomal RNA precursor

PubMed Central

Schnare, Murray N.; Collings, James C.; Spencer, David F.; Gray, Michael W.

2000-01-01

In Crithidia fasciculata, the ribosomal RNA (rRNA) gene repeats range in size from ∼11 to 12 kb. This length heterogeneity is localized to a region of the intergenic spacer (IGS) that contains tandemly repeated copies of a 19mer sequence. The IGS also contains four copies of an ∼55 nt repeat that has an internal inverted repeat and is also present in the IGS of Leishmania species. We have mapped the C.fasciculata transcription initiation site as well as two other reverse transcriptase stop sites that may be analogous to the A0 and A′ pre-rRNA processing sites within the 5′ external transcribed spacer (ETS) of other eukaryotes. Features that could influence processing at these sites include two stretches of conserved primary sequence and three secondary structure elements present in the 5′ ETS. We also characterized the C.fasciculata U3 snoRNA, which has the potential for base-pairing with pre-rRNA sequences. Finally, we demonstrate that biosynthesis of large subunit rRNA in both C.fasciculata and Trypanosoma brucei involves 3′-terminal addition of three A residues that are not present in the corresponding DNA sequences. PMID:10982863

Polyphasic taxonomy of the genus Shewanella and description of Shewanella oneidensis sp. nov

NASA Technical Reports Server (NTRS)

Venkateswaran, K.; Moser, D. P.; Dollhopf, M. E.; Lies, D. P.; Saffarini, D. A.; MacGregor, B. J.; Ringelberg, D. B.; White, D. C.; Nishijima, M.; Sano, H.;

Pelagodinium gen. nov. and P. béii comb. nov., a dinoflagellate symbiont of planktonic foraminifera.

PubMed

Siano, Raffaele; Montresor, Marina; Probert, Ian; Not, Fabrice; de Vargas, Colomban

2010-07-01

The taxonomic status of the free-living stage of the dinoflagellate Gymnodinium béii, symbiont of the foraminifer Orbulina universa, was reassessed on the basis of detailed morpho-genetic analyses. Electron microscopy observations revealed previously undescribed morphological features of the cell that are important for species recognition. The presence of a single elongated apical vesicle (EAV) ornamented with a row of small knobs, absent in species of the genus Gymnodinium, calls into question the current taxonomic position of the symbiont. The presence of a type E extraplastidial eyespot, the arrangement of the amphiesmal vesicles in series and the absence of trichocysts confirm the affiliation with other symbiotic dinoflagellates and certain genetically related non-symbiotic genera, all belonging to the order Suessiales. The arrangement of the series of vesicles of the analyzed strain is unique within the Suessiales, and the ultrastructure of the pyrenoid is different from other symbiotic dinoflagellates. A large subunit (LSU) rDNA phylogenetic analysis confirmed that the analyzed pelagic symbiont clusters in an independent, well-supported clade within the Suessiales with other sequences of symbiotic dinoflagellates extracted from planktonic foraminifera. Hence a novel genus, Pelagodinium gen. nov., is erected for this pelagic, symbiotic dinoflagellate, and Gymnodinium béii is reclassified as Pelagodinium béii. 2010 Elsevier GmbH. All rights reserved.

Diversity and decay ability of basidiomycetes isolated from lodgepole pines killed by the mountain pine beetle.

PubMed

Son, E; Kim, J-J; Lim, Y W; Au-Yeung, T T; Yang, C Y H; Breuil, C

2011-01-01

When lodgepole pines (Pinus contorta Douglas ex Louden var. latifolia Engelm. ex S. Watson) that are killed by the mountain pine beetle (Dendroctonus ponderosae) and its fungal associates are not harvested, fungal decay can affect wood and fibre properties. Ophiostomatoids stain sapwood but do not affect the structural properties of wood. In contrast, white or brown decay basidiomycetes degrade wood. We isolated both staining and decay fungi from 300 lodgepole pine trees killed by mountain pine beetle at green, red, and grey stages at 10 sites across British Columbia. We retained 224 basidiomycete isolates that we classified into 34 species using morphological and physiological characteristics and rDNA large subunit sequences. The number of basidiomycete species varied from 4 to 14 species per site. We assessed the ability of these fungi to degrade both pine sapwood and heartwood using the soil jar decay test. The highest wood mass losses for both sapwood and heartwood were measured for the brown rot species Fomitopsis pinicola and the white rot Metulodontia and Ganoderma species. The sap rot species Trichaptum abietinum was more damaging for sapwood than for heartwood. A number of species caused more than 50% wood mass losses after 12 weeks at room temperature, suggesting that beetle-killed trees can rapidly lose market value due to degradation of wood structural components.

SSU rRNA-based phylogenetic position of the genera Amoeba and Chaos (Lobosea, Gymnamoebia): the origin of gymnamoebae revisited.

PubMed

Bolivar, I; Fahrni, J F; Smirnov, A; Pawlowski, J

2001-12-01

Naked lobose amoebae (gymnamoebae) are among the most abundant group of protists present in all aquatic and terrestrial biotopes. Yet, because of lack of informative morphological characters, the origin and evolutionary history of gymnamoebae are poorly known. The first molecular studies revealed multiple origins for the amoeboid lineages and an extraordinary diversity of amoebae species. Molecular data, however, exist only for a few species of the numerous taxa belonging to this group. Here, we present the small-subunit (SSU) rDNA sequences of four species of typical large gymnamoebae: Amoeba proteus, Amoeba leningradensis, Chaos nobile, and Chaos carolinense. Sequence analysis suggests that the four species are closely related to the species of genera Saccamoeba, Leptomyxa, Rhizamoeba, Paraflabellula, Hartmannella, and Echinamoeba. All of them form a relatively well-supported clade, which corresponds to the subclass Gymnamoebia, in agreement with morphology-based taxonomy. The other gymnamoebae cluster in small groups or branch separately. Their relationships change depending on the type of analysis and the model of nucleotide substitution. All gymnamoebae branch together in Neighbor-Joining analysis with corrections for among-site rate heterogeneity and proportion of invariable sites. This clade, however, is not statistically supported by SSU rRNA gene sequences and further analysis of protein sequence data will be necessary to test the monophyly of gymnamoebae.

Uncovering the molecular organization of unusual highly scattered 5S rDNA: The case of Chariesterus armatus (Heteroptera).

PubMed

Bardella, Vanessa Bellini; Cabral-de-Mello, Diogo Cavalcanti

2018-03-10

One cluster of 5S rDNA per haploid genome is the most common pattern among Heteroptera. However, in Chariesterus armatus, highly scattered signals were noticed. We isolated and characterized the entire 5S rDNA unit of C. armatus aiming to a deeper knowledge of molecular organization of the 5S rDNA among Heteroptera and to understand possible causes and consequences of 5S rDNA chromosomal spreading. For a comparative analysis, we performed the same approach in Holymenia histrio with 5S rDNA restricted to one bivalent. Multiple 5S rDNA variants were observed in both species, though they were more variable in C. armatus, with some of variants corresponding to pseudogenes. These pseudogenes suggest birth-and-death mechanism, though homogenization was also observed (concerted evolution), indicating evolution through mixed model. Association between transposable elements and 5S rDNA was not observed, suggesting spreading of 5S rDNA through other mechanisms, like ectopic recombination. Scattered organization is a rare example for 5S rDNA, and such organization in C. armatus genome could have led to the high diversification of sequences favoring their pseudogenization. Copyright © 2017. Published by Elsevier B.V.

Physical mapping of the 5S and 18S rDNA in ten species of Hypostomus Lacépède 1803 (Siluriformes: Loricariidae): evolutionary tendencies in the genus.

PubMed

Bueno, Vanessa; Venere, Paulo César; Thums Konerat, Jocicléia; Zawadzki, Cláudio Henrique; Vicari, Marcelo Ricardo; Margarido, Vladimir Pavan

2014-01-01

Hypostomus is a diverse group with unclear aspects regarding its biology, including the mechanisms that led to chromosome diversification within the group. Fluorescence in situ hybridization (FISH) with 5S and 18S rDNA probes was performed on ten Hypostomini species. Hypostomus faveolus, H. cochliodon, H. albopunctatus, H. aff. paulinus, and H. topavae had only one chromosome pair with 18S rDNA sites, while H. ancistroides, H. commersoni, H. hermanni, H. regani, and H. strigaticeps had multiple 18S rDNA sites. Regarding the 5S rDNA genes, H. ancistroides, H. regani, H. albopunctatus, H. aff. paulinus, and H. topavae had 5S rDNA sites on only one chromosome pair and H. faveolus, H. cochliodon, H. commersoni, H. hermanni, and H. strigaticeps had multiple 5S rDNA sites. Most species had 18S rDNA sites in the telomeric region of the chromosomes. All species but H. cochliodon had 5S rDNA in the centromeric/pericentromeric region of one metacentric pair. Obtained results are discussed based on existent phylogenies for the genus, with comments on possible dispersion mechanisms to justify the variability of the rDNA sites in Hypostomus.

Evolution of rDNA in Nicotiana Allopolyploids: A Potential Link between rDNA Homogenization and Epigenetics

PubMed Central

Kovarik, Ales; Dadejova, Martina; Lim, Yoong K.; Chase, Mark W.; Clarkson, James J.; Knapp, Sandra; Leitch, Andrew R.

2008-01-01

Background The evolution and biology of rDNA have interested biologists for many years, in part, because of two intriguing processes: (1) nucleolar dominance and (2) sequence homogenization. We review patterns of evolution in rDNA in the angiosperm genus Nicotiana to determine consequences of allopolyploidy on these processes. Scope Allopolyploid species of Nicotiana are ideal for studying rDNA evolution because phylogenetic reconstruction of DNA sequences has revealed patterns of species divergence and their parents. From these studies we also know that polyploids formed over widely different timeframes (thousands to millions of years), enabling comparative and temporal studies of rDNA structure, activity and chromosomal distribution. In addition studies on synthetic polyploids enable the consequences of de novo polyploidy on rDNA activity to be determined. Conclusions We propose that rDNA epigenetic expression patterns established even in F1 hybrids have a material influence on the likely patterns of divergence of rDNA. It is the active rDNA units that are vulnerable to homogenization, which probably acts to reduce mutational load across the active array. Those rDNA units that are epigenetically silenced may be less vulnerable to sequence homogenization. Selection cannot act on these silenced genes, and they are likely to accumulate mutations and eventually be eliminated from the genome. It is likely that whole silenced arrays will be deleted in polyploids of 1 million years of age and older. PMID:18310159

The morphological and chemical characteristics of striatal neurons immunoreactive for the alpha1-subunit of the GABA(A) receptor in the rat.

PubMed

Waldvogel, H J; Kubota, Y; Trevallyan, S C; Kawaguchi, Y; Fritschy, J M; Mohler, H; Faull, R L

1997-10-01

The distribution, morphology and chemical characteristics of neurons immunoreactive for the alpha1-subunit of the GABA(A) receptor in the striatum of the basal ganglia in the rat brain were investigated at the light, confocal and electron microscope levels using single, double and triple immunohistochemical labelling techniques. The results showed that alpha1-subunit immunoreactive neurons were sparsely distributed throughout the rat striatum. Double and triple labelling results showed that all the alpha1-subunit-immunoreactive neurons were positive for glutamate decarboxylase and immunoreactive for the beta2,3 and gamma2 subunits of the GABA(A) receptor. Three types of alpha1-subunit-immunoreactive neurons were identified in the striatum on the basis of cellular morphology and chemical characteristics. The most numerous alpha1-subunit-immunoreactive neurons were medium-sized, aspiny neurons with a widely branching dendritic tree. They were parvalbumin-negative and were located mainly in the dorsolateral regions of the striatum. Electron microscopy showed that these neurons had an indented nuclear membrane, typical of striatal interneurons, and were surrounded by small numbers of axon terminals which established alpha1-subunit-immunoreactive synaptic contacts with the soma and dendrites. These cells were classified as type 1 alpha1-subunit-immunoreactive neurons and comprised 75% of the total population of alpha1-subunit-immunoreactive neurons in the striatum. The remaining alpha1-subunit-immunoreactive neurons comprised of a heterogeneous population of large-sized neurons localized in the ventral and medial regions of the striatum. The most numerous large-sized cells were parvalbumin-negative, had two to three relatively short branching dendrites and were designated type 2 alpha1-subunit-immunoreactive neurons. Electron microscopy showed that the type 2 neurons were characterized by a highly convoluted nuclear membrane and were sparsely covered with small axon terminals. The type 2 neurons comprised 20% of the total population of alpha1-subunit-immunoreactive neurons. The remaining large-sized alpha1-immunoreactive cells were designated type 3 cells; they were positive for parvalbumin and were distinguished by long branching dendrites extending dorsally for 600-800 microm into the striatum. These neurons comprised 5% of the total population of alpha1-subunit-immunoreactive neurons and were surrounded by enkephalin-immunoreactive terminals. Electron microscopy showed that the alpha1-subunit type 3 neurons had an indented nuclear membrane and were densely covered with small axon terminals which established alpha1-subunit-immunoreactive symmetrical synaptic contacts with the soma and dendrites. These results provide a detailed characterization of the distribution, morphology and chemical characteristics of the alpha1-subunit-immunoreactive neurons in the rat striatum and suggest that the type 1 and type 2 neurons comprise of separate populations of striatal interneurons while the type 3 neurons may represent the large striatonigral projection neurons described by Bolam et al. [Bolam J. P., Somogyi P., Totterdell S. and Smith A. D. (1981) Neuroscience 6, 2141-2157.].

Next generation sequencing analysis reveals a relationship between rDNA unit diversity and locus number in Nicotiana diploids

PubMed Central

2012-01-01

Background Tandemly arranged nuclear ribosomal DNA (rDNA), encoding 18S, 5.8S and 26S ribosomal RNA (rRNA), exhibit concerted evolution, a pattern thought to result from the homogenisation of rDNA arrays. However rDNA homogeneity at the single nucleotide polymorphism (SNP) level has not been detailed in organisms with more than a few hundred copies of the rDNA unit. Here we study rDNA complexity in species with arrays consisting of thousands of units. Methods We examined homogeneity of genic (18S) and non-coding internally transcribed spacer (ITS1) regions of rDNA using Roche 454 and/or Illumina platforms in four angiosperm species, Nicotiana sylvestris, N. tomentosiformis, N. otophora and N. kawakamii. We compared the data with Southern blot hybridisation revealing the structure of intergenic spacer (IGS) sequences and with the number and distribution of rDNA loci. Results and Conclusions In all four species the intragenomic homogeneity of the 18S gene was high; a single ribotype makes up over 90% of the genes. However greater variation was observed in the ITS1 region, particularly in species with two or more rDNA loci, where >55% of rDNA units were a single ribotype, with the second most abundant variant accounted for >18% of units. IGS heterogeneity was high in all species. The increased number of ribotypes in ITS1 compared with 18S sequences may reflect rounds of incomplete homogenisation with strong selection for functional genic regions and relaxed selection on ITS1 variants. The relationship between the number of ITS1 ribotypes and the number of rDNA loci leads us to propose that rDNA evolution and complexity is influenced by locus number and/or amplification of orphaned rDNA units at new chromosomal locations. PMID:23259460

Breaks in the 45S rDNA Lead to Recombination-Mediated Loss of Repeats.

PubMed

Warmerdam, Daniël O; van den Berg, Jeroen; Medema, René H

2016-03-22

rDNA repeats constitute the most heavily transcribed region in the human genome. Tumors frequently display elevated levels of recombination in rDNA, indicating that the repeats are a liability to the genomic integrity of a cell. However, little is known about how cells deal with DNA double-stranded breaks in rDNA. Using selective endonucleases, we show that human cells are highly sensitive to breaks in 45S but not the 5S rDNA repeats. We find that homologous recombination inhibits repair of breaks in 45S rDNA, and this results in repeat loss. We identify the structural maintenance of chromosomes protein 5 (SMC5) as contributing to recombination-mediated repair of rDNA breaks. Together, our data demonstrate that SMC5-mediated recombination can lead to error-prone repair of 45S rDNA repeats, resulting in their loss and thereby reducing cellular viability. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spreitzer, Robert Joseph

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of CO 2 fixation in photosynthesis. However, it is a slow enzyme, and O 2 competes with CO 2 at the active site. Oxygenation initiates the photorespiratory pathway, which also results in the loss of CO 2. If carboxylation could be increased or oxygenation decreased, an increase in net CO 2 fixation would be realized. Because Rubisco provides the primary means by which carbon enters all life on earth, there is much interest in engineering Rubisco to increase the production of food and renewable energy. Rubisco is located in the chloroplasts of plants,more » and it is comprised of two subunits. Much is known about the chloroplast-gene-encoded large subunit (rbcL gene), which contains the active site, but much less is known about the role of the nuclear-gene-encoded small subunit in Rubisco function (rbcS gene). Both subunits are coded by multiple genes in plants, which makes genetic engineering difficult. In the eukaryotic, green alga Chlamydomonas reinhardtii, it has been possible to eliminate all the Rubisco genes. These Rubisco-less mutants can be maintained by providing acetate as an alternative carbon source. In this project, focus has been placed on determining whether the small subunit might be a better genetic-engineering target for improving Rubisco. Analysis of a variable-loop structure (βA-βB loop) of the small subunit by genetic selection, directed mutagenesis, and construction of chimeras has shown that the small subunit can influence CO 2/O 2 specificity. X-ray crystal structures of engineered chimeric-loop enzymes have indicated that additional residues and regions of the small subunit may also contribute to Rubisco function. Structural dynamics of the small-subunit carboxyl terminus was also investigated. Alanine-scanning mutagenesis of the most-conserved small-subunit residues has identified a possible structural pathway between the small-subunit βA-βB loop and alpha-helix 8 of the large-subunit α/β-barrel active site. Hybrid enzymes were also created comprised of plant small subunits and Chlamydomonas large subunits, and these enzymes have increases in CO 2/O 2 specificity, further indicating that small subunits may be the key for ultimately engineering an improved Rubisco enzyme.« less

The dynamic assembly of distinct RNA polymerase I complexes modulates rDNA transcription.

PubMed

Torreira, Eva; Louro, Jaime Alegrio; Pazos, Irene; González-Polo, Noelia; Gil-Carton, David; Duran, Ana Garcia; Tosi, Sébastien; Gallego, Oriol; Calvo, Olga; Fernández-Tornero, Carlos

2017-03-06

Cell growth requires synthesis of ribosomal RNA by RNA polymerase I (Pol I). Binding of initiation factor Rrn3 activates Pol I, fostering recruitment to ribosomal DNA promoters. This fundamental process must be precisely regulated to satisfy cell needs at any time. We present in vivo evidence that, when growth is arrested by nutrient deprivation, cells induce rapid clearance of Pol I-Rrn3 complexes, followed by the assembly of inactive Pol I homodimers. This dual repressive mechanism reverts upon nutrient addition, thus restoring cell growth. Moreover, Pol I dimers also form after inhibition of either ribosome biogenesis or protein synthesis. Our mutational analysis, based on the electron cryomicroscopy structures of monomeric Pol I alone and in complex with Rrn3, underscores the central role of subunits A43 and A14 in the regulation of differential Pol I complexes assembly and subsequent promoter association.

First endemic freshwater Gammarus from Crete and its evolutionary history-an integrative taxonomy approach.

PubMed

Hupało, Kamil; Mamos, Tomasz; Wrzesińska, Weronika; Grabowski, Michał

2018-01-01

The Mediterranean islands are known as natural laboratories of evolution with a high level of endemic biodiversity. However, most biodiversity assessments have focused mainly on terrestrial and marine fauna, leaving the freshwater animals aside. Crete is one of the largest islands in the Mediterranean Basin, with a long history of isolation from the continental mainland. Gammarid amphipods are often dominant in macrozoobenthic communities in European inland waters. They are widely used in biomonitoring and exotoxicological studies. Herein, we describe Gammarus plaitisi sp. nov., endemic to Cretan streams, based on morphological characters and a set of molecular species delimitation methods using mitochondrial cytochrome oxidase subunit I and 16S rRNA genes as well as nuclear 28S rDNA, ITS1 and EF1-alpha genes. The divergence of the new species is strongly connected with the geological history of the island supporting its continental origin.

Physical mapping of 18S-25S rDNA and 5S rDNA in Lupinus via fluorescent in situ hybridization.

PubMed

Naganowska, Barbara; Zielińska, Anna

2002-01-01

Double-target fluorescent in situ hybridization (FISH) was used to determine the genomic distribution of ribosomal RNA genes in five Lupinus species: L. cosentinii (2n=32), L. pilosus (2n=42), L. angustifolius (2n=40), L. luteus (2n=52) and L. mutabilis (2n=48). 18S-25S rDNA and 5S rDNA were used as probes. Some interspecific variation was observed in the number and size of the 18S-25S rDNA loci. All the studied species had one chromosome pair carrying 5S rDNA.

Condensin suppresses recombination and regulates double-strand break processing at the repetitive ribosomal DNA array to ensure proper chromosome segregation during meiosis in budding yeast

PubMed Central

Li, Ping; Jin, Hui; Yu, Hong-Guo

2014-01-01

During meiosis, homologues are linked by crossover, which is required for bipolar chromosome orientation before chromosome segregation at anaphase I. The repetitive ribosomal DNA (rDNA) array, however, undergoes little or no meiotic recombination. Hyperrecombination can cause chromosome missegregation and rDNA copy number instability. We report here that condensin, a conserved protein complex required for chromosome organization, regulates double-strand break (DSB) formation and repair at the rDNA gene cluster during meiosis in budding yeast. Condensin is highly enriched at the rDNA region during prophase I, released at the prophase I/metaphase I transition, and reassociates with rDNA before anaphase I onset. We show that condensin plays a dual role in maintaining rDNA stability: it suppresses the formation of Spo11-mediated rDNA breaks, and it promotes DSB processing to ensure proper chromosome segregation. Condensin is unnecessary for the export of rDNA breaks outside the nucleolus but required for timely repair of meiotic DSBs. Our work reveals that condensin coordinates meiotic recombination with chromosome segregation at the repetitive rDNA sequence, thereby maintaining genome integrity. PMID:25103240

A Natural Polymorphism in rDNA Replication Origins Links Origin Activation with Calorie Restriction and Lifespan

PubMed Central

Kwan, Elizabeth X.; Foss, Eric J.; Tsuchiyama, Scott; Alvino, Gina M.; Kruglyak, Leonid; Kaeberlein, Matt; Raghuraman, M. K.; Brewer, Bonita J.; Kennedy, Brian K.; Bedalov, Antonio

2013-01-01

Aging and longevity are complex traits influenced by genetic and environmental factors. To identify quantitative trait loci (QTLs) that control replicative lifespan, we employed an outbred Saccharomyces cerevisiae model, generated by crossing a vineyard and a laboratory strain. The predominant QTL mapped to the rDNA, with the vineyard rDNA conferring a lifespan increase of 41%. The lifespan extension was independent of Sir2 and Fob1, but depended on a polymorphism in the rDNA origin of replication from the vineyard strain that reduced origin activation relative to the laboratory origin. Strains carrying vineyard rDNA origins have increased capacity for replication initiation at weak plasmid and genomic origins, suggesting that inability to complete genome replication presents a major impediment to replicative lifespan. Calorie restriction, a conserved mediator of lifespan extension that is also independent of Sir2 and Fob1, reduces rDNA origin firing in both laboratory and vineyard rDNA. Our results are consistent with the possibility that calorie restriction, similarly to the vineyard rDNA polymorphism, modulates replicative lifespan through control of rDNA origin activation, which in turn affects genome replication dynamics. PMID:23505383

A natural polymorphism in rDNA replication origins links origin activation with calorie restriction and lifespan.

PubMed

Kwan, Elizabeth X; Foss, Eric J; Tsuchiyama, Scott; Alvino, Gina M; Kruglyak, Leonid; Kaeberlein, Matt; Raghuraman, M K; Brewer, Bonita J; Kennedy, Brian K; Bedalov, Antonio

2013-01-01

Aging and longevity are complex traits influenced by genetic and environmental factors. To identify quantitative trait loci (QTLs) that control replicative lifespan, we employed an outbred Saccharomyces cerevisiae model, generated by crossing a vineyard and a laboratory strain. The predominant QTL mapped to the rDNA, with the vineyard rDNA conferring a lifespan increase of 41%. The lifespan extension was independent of Sir2 and Fob1, but depended on a polymorphism in the rDNA origin of replication from the vineyard strain that reduced origin activation relative to the laboratory origin. Strains carrying vineyard rDNA origins have increased capacity for replication initiation at weak plasmid and genomic origins, suggesting that inability to complete genome replication presents a major impediment to replicative lifespan. Calorie restriction, a conserved mediator of lifespan extension that is also independent of Sir2 and Fob1, reduces rDNA origin firing in both laboratory and vineyard rDNA. Our results are consistent with the possibility that calorie restriction, similarly to the vineyard rDNA polymorphism, modulates replicative lifespan through control of rDNA origin activation, which in turn affects genome replication dynamics.

Understanding large multiprotein complexes: applying a multiple allosteric networks model to explain the function of the Mediator transcription complex.

PubMed

Lewis, Brian A

2010-01-15

The regulation of transcription and of many other cellular processes involves large multi-subunit protein complexes. In the context of transcription, it is known that these complexes serve as regulatory platforms that connect activator DNA-binding proteins to a target promoter. However, there is still a lack of understanding regarding the function of these complexes. Why do multi-subunit complexes exist? What is the molecular basis of the function of their constituent subunits, and how are these subunits organized within a complex? What is the reason for physical connections between certain subunits and not others? In this article, I address these issues through a model of network allostery and its application to the eukaryotic RNA polymerase II Mediator transcription complex. The multiple allosteric networks model (MANM) suggests that protein complexes such as Mediator exist not only as physical but also as functional networks of interconnected proteins through which information is transferred from subunit to subunit by the propagation of an allosteric state known as conformational spread. Additionally, there are multiple distinct sub-networks within the Mediator complex that can be defined by their connections to different subunits; these sub-networks have discrete functions that are activated when specific subunits interact with other activator proteins.

Engineering of chimeric eukaryotic/bacterial Rubisco large subunits in Escherichia coli.

PubMed

Koay, Teng Wei; Wong, Hann Ling; Lim, Boon Hoe

2016-11-26

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a rate-limiting photosynthetic enzyme that catalyzes carbon fixation in the Calvin cycle. Much interest has been devoted to engineering this ubiquitous enzyme with the goal of increasing plant growth. However, experiments that have successfully produced improved Rubisco variants, via directed evolution in Escherichia coli, are limited to bacterial Rubisco because the eukaryotic holoenzyme cannot be produced in E. coli. The present study attempts to determine the specific differences between bacterial and eukaryotic Rubisco large subunit primary structure that are responsible for preventing heterologous eukaryotic holoenzyme formation in E. coli. A series of chimeric Synechococcus Rubiscos were created in which different sections of the large subunit were swapped with those of the homologous Chlamydomonas Rubisco. Chimeric holoenzymes that can form in vivo would indicate that differences within the swapped sections do not disrupt holoenzyme formation. Large subunit residues 1-97, 198-247 and 448-472 were successfully swapped without inhibiting holoenzyme formation. In all ten chimeras, protein expression was observed for the separate subunits at a detectable level. As a first approximation, the regions that can tolerate swapping may be targets for future engineering.

Nuclear 28S rDNA phylogeny supports the basal placement of Noctiluca scintillans (Dinophyceae; Noctilucales) in dinoflagellates.

PubMed

Ki, Jang-Seu

2010-05-01

Noctiluca scintillans (Macartney) Kofoid et Swezy, 1921 is an unarmoured heterotrophic dinoflagellate with a global distribution, and has been considered as one of the ancestral taxa among dinoflagellates. Recently, 18S rDNA, actin, alpha-, beta-tubulin, and Hsp90-based phylogenies have shown the basal position of the noctilucids. However, the relationships of dinoflagellates in the basal lineages are still controversial. Although the nuclear rDNA (e.g. 18S, ITS-5.8S, and 28S) contains much genetic information, DNA sequences of N. scintillans rDNA molecules were insufficiently characterized as yet. Here the author sequenced a long-range nuclear rDNA, spanning from the 18S to the D5 region of the 28S rDNA, of N. scintillans. The present N. scintillans had a nearly identical genotype (>99.0% similarity) compared to other Noctiluca sequences from different geographic origins. Nucleotide divergence in the partial 28S rDNA was significantly high (p<0.05) as compared to the 18S rDNA, demonstrating that the information from 28S rDNA is more variable. The 28S rDNA phylogeny of 17 selected dinoflagellates, two perkinsids, and two apicomplexans as outgroups showed that N. scintillans and Oxyrrhis marina formed a clade that diverged separately from core dinoflagellates. Copyright (c) 2009 Elsevier GmbH. All rights reserved.

Distribution of 45S rDNA in Modern Rose Cultivars (Rosa hybrida), Rosa rugosa, and Their Interspecific Hybrids Revealed by Fluorescence in situ Hybridization.

PubMed

Ding, Xiao-Liu; Xu, Ting-Liang; Wang, Jing; Luo, Le; Yu, Chao; Dong, Gui-Min; Pan, Hui-Tang; Zhang, Qi-Xiang

2016-01-01

To elucidate the evolutionary dynamics of the location and number of rDNA loci in the process of polyploidization in the genus Rosa, we examined 45S rDNA sites in the chromosomes of 6 modern rose cultivars (R. hybrida), 5 R. rugosa cultivars, and 20 hybrid progenies by fluorescence in situ hybridization. Variation in the number of rDNA sites in parents and their interspecific hybrids was detected. As expected, 4 rDNA sites were observed in the genomes of 4 modern rose cultivars, while 3 hybridization sites were observed in the 2 others. Two expected rDNA sites were found in 2 R. rugosa cultivars, while in the other 3 R. rugosa cultivars 4 sites were present. Among the 20 R. hybrida × R. rugosa offspring, 13 carried the expected number of rDNA sites, and 1 had 6 hybridization sites, which exceeded the expected number by far. The other 6 offspring had either 2 or 3 hybridization sites, which was less than expected. Differences in the number of rDNA loci were observed in interspecific offspring, indicating that rDNA loci exhibit instability after distant hybridization events. Abnormal chromosome pairing may be the main factor explaining the variation in rDNA sites during polyploidization. © 2016 S. Karger AG, Basel.

Replication and meiotic transmission of yeast ribosomal RNA genes.

PubMed

Brewer, B J; Zakian, V A; Fangman, W L

1980-11-01

The yeast Saccharomyces cerevisiae has approximately 120 genes for the ribosomal RNAs (rDNA) which are organized in tandem within chromosomal DNA. These multiple-copy genes are homogeneous in sequence but can undergo changes in copy number and topology. To determine if these changes reflect unusual features of rDNA metabolism, we have examined both the replication of rDNA in the mitotic cell cycle and the inheritance of rDNA during meiosis. The results indicate that rDNA behaves identically to chromosomal DNA: each rDNA unit is replicated once during the S phase of each cell cycle and each unit is conserved through meiosis. Therefore, the flexibility in copy number and topology of rDNA does not arise from the selective replication of units in each S phase nor by the selective inheritance of units in meiosis.

Genome re-assignment of Arachis trinitensis (Sect. Arachis, Leguminosae) and its implications for the genetic origin of cultivated peanut

PubMed Central

2010-01-01

The karyotype structure of Arachis trinitensis was studied by conventional Feulgen staining, CMA/DAPI banding and rDNA loci detection by fluorescence in situ hybridization (FISH) in order to establish its genome status and test the hypothesis that this species is a genome donor of cultivated peanut. Conventional staining revealed that the karyotype lacked the small “A chromosomes” characteristic of the A genome. In agreement with this, chromosomal banding showed that none of the chromosomes had the large centromeric bands expected for A chromosomes. FISH revealed one pair each of 5S and 45S rDNA loci, located in different medium-sized metacentric chromosomes. Collectively, these results suggest that A. trinitensis should be removed from the A genome and be considered as a B or non-A genome species. The pattern of heterochromatic bands and rDNA loci of A. trinitensis differ markedly from any of the complements of A. hypogaea, suggesting that the former species is unlikely to be one of the wild diploid progenitors of the latter. PMID:21637581

Fragile sites, dysfunctional telomere and chromosome fusions: What is 5S rDNA role?

PubMed

Barros, Alain Victor; Wolski, Michele Andressa Vier; Nogaroto, Viviane; Almeida, Mara Cristina; Moreira-Filho, Orlando; Vicari, Marcelo Ricardo

2017-04-15

Repetitive DNA regions are known as fragile chromosomal sites which present a high flexibility and low stability. Our focus was characterize fragile sites in 5S rDNA regions. The Ancistrus sp. species shows a diploid number of 50 and an indicative Robertsonian fusion at chromosomal pair 1. Two sequences of 5S rDNA were identified: 5S.1 rDNA and 5S.2 rDNA. The first sequence gathers the necessary structures to gene expression and shows a functional secondary structure prediction. Otherwise, the 5S.2 rDNA sequence does not contain the upstream sequences that are required to expression, furthermore its structure prediction reveals a nonfunctional ribosomal RNA. The chromosomal mapping revealed several 5S.1 and 5S.2 rDNA clusters. In addition, the 5S.2 rDNA clusters were found in acrocentric and metacentric chromosomes proximal regions. The pair 1 5S.2 rDNA cluster is co-located with interstitial telomeric sites (ITS). Our results indicate that its clusters are hotspots to chromosomal breaks. During the meiotic prophase bouquet arrangement, double strand breaks (DSBs) at proximal 5S.2 rDNA of acrocentric chromosomes could lead to homologous and non-homologous repair mechanisms as Robertsonian fusions. Still, ITS sites provides chromosomal instability, resulting in telomeric recombination via TRF2 shelterin protein and a series of breakage-fusion-bridge cycles. Our proposal is that 5S rDNA derived sequences, act as chromosomal fragile sites in association with some chromosomal rearrangements of Loricariidae. Copyright © 2017 Elsevier B.V. All rights reserved.

MUC1 and MUC4: Switching the Emphasis from Large to Small

PubMed Central

Carraway, Kermit L.

2011-01-01

Summation The MUC1 and MUC4 membrane mucins are each composed of a large alpha (α) and a small beta (β) subunit. The α subunits are fully exposed at the cell surface and contain variable numbers of repeated amino acid sequences that are heavily glycosylated. In contrast, the β subunits are much smaller and are anchored within the cell membrane, with their amino-terminal portions exposed at the cell surface and their carboxy-terminal tails facing the cytosol. Studies over the last several years are challenging the long-held belief that α subunits play the predominant role in cancer by conferring cellular properties that allow tumor cells to evade immune recognition and destruction. Indeed, the β subunits of MUC1 and MUC4 have emerged as oncogenes, as they engage signaling pathways responsible for tumor initiation and progression. Thus, a switch in the emphasis from the large α to the small β subunits offers attractive possibilities for successful clinical application. Such a focus shift is further supported by the absence of allelic polymorphism and variable glycosylation in the β subunit as well as by the presence of the β subunit in most MUC1 and MUC4 isoforms expressed by tumors. MUC1α, also known as CA15.3, is a Food and Drug Administration-approved serum biomarker for breast cancer, but its use is no longer recommended by the American Society of Clinical Oncology. However, comparison of β subunit expression in normal and malignant breast tissues may offer a novel approach to the exploitation of membrane mucins as biomarkers, as MUC1β-induced gene signatures with prognostic and predictive values in breast cancer have been reported. Preclinical studies with peptides that interfere with MUC1β oncogenic functions also look promising. PMID:21728842

Quick identification of acetic acid bacteria based on nucleotide sequences of the 16S-23S rDNA internal transcribed spacer region and of the PQQ-dependent alcohol dehydrogenase gene.

PubMed

Trcek, Janja

2005-10-01

Acetic acid bacteria (AAB) are well known for oxidizing different ethanol-containing substrates into various types of vinegar. They are also used for production of some biotechnologically important products, such as sorbose and gluconic acids. However, their presence is not always appreciated since certain species also spoil wine, juice, beer and fruits. To be able to follow AAB in all these processes, the species involved must be identified accurately and quickly. Because of inaccuracy and very time-consuming phenotypic analysis of AAB, the application of molecular methods is necessary. Since the pairwise comparison among the 16S rRNA gene sequences of AAB shows very high similarity (up to 99.9%) other DNA-targets should be used. Our previous studies showed that the restriction analysis of 16S-23S rDNA internal transcribed spacer region is a suitable approach for quick affiliation of an acetic acid bacterium to a distinct group of restriction types and also for quick identification of a potentially novel species of acetic acid bacterium (Trcek & Teuber 2002; Trcek 2002). However, with the exception of two conserved genes, encoding tRNAIle and tRNAAla, the sequences of 16S-23S rDNA are highly divergent among AAB species. For this reason we analyzed in this study a gene encoding PQQ-dependent ADH as a possible DNA-target. First we confirmed the expression of subunit I of PQQ-dependent ADH (AdhA) also in Asaia, the only genus of AAB which exhibits little or no ADH-activity. Further we analyzed the partial sequences of adhA among some representative species of the genera Acetobacter, Gluconobacter and Gluconacetobacter. The conserved and variable regions in these sequences made possible the construction of A. acetispecific oligonucleotide the specificity of which was confirmed in PCR-reaction using 45 well-defined strains of AAB as DNA-templates. The primer was also successfully used in direct identification of A. aceti from home made cider vinegar as well as for revealing the misclassification of strain IFO 3283 into the species A. aceti.

A comparative study of genome organization and inferences for the systematics of two large bushcricket genera of the tribe Barbitistini (Orthoptera: Tettigoniidae: Phaneropterinae)

PubMed Central

2014-01-01

Background Poecilimon and Isophya are the largest genera of the tribe Barbitistini and among the most systematically complicated and evolutionarily intriguing groups of Palearctic tettigoniids. We examined the genomic organization of 79 taxa with a stable chromosome number using classical (C–banding, silver and fluorochrome staining) and molecular (fluorescence in situ hybridization with 18S rDNA and (TTAGG) n telomeric probes) cytogenetic techniques. These tools were employed to establish genetic organization and differences or similarities between genera or species within the same genus and determine if cytogenetic markers can be used for identifying some taxonomic groups of species. Results Differences between the karyotypes of the studied genera include some general changes in the morphology of the X chromosome in Isophya (in contrast to Poecilimon). The number of major rDNA clusters per haploid genome divided Poecilimon into two main almost equal groups (with either one or two clusters), while two rDNA clusters predominated in Isophya. In both genera, rDNA loci were preferentially located in the paracentromeric region of the autosomes and rarely in the sex chromosomes. Our results demonstrate a coincidence between the location of rDNA loci and active NORs and GC-rich heterochromatin regions. The C/DAPI/CMA3 bands observed in most Poecilimon chromosomes suggest the presence of more families of repetitive DNA sequences as compared to the heterochromatin patterns in Isophya. Conclusions The results show both differences and similarities in genome organization among species of the same genus and between genera. Previous views on the systematics and phylogenetic grouping of certain lineages are discussed in light of the present cytogenetic results. In some cases, variation of chromosome markers was observed to correspond with variation in other evolutionary traits, which is related to the processes of ongoing speciation and hybridization in zones of secondary contact. It was concluded that the physical mapping of rDNA sequences and heterochromatin may be used as an additional marker for understanding interspecific relationships in these groups and their routes of speciation. PMID:24625118

Repeated reunions and splits feature the highly dynamic evolution of 5S and 35S ribosomal RNA genes (rDNA) in the Asteraceae family.

PubMed

Garcia, Sònia; Panero, José L; Siroky, Jiri; Kovarik, Ales

2010-08-16

In flowering plants and animals the most common ribosomal RNA genes (rDNA) organisation is that in which 35S (encoding 18S-5.8S-26S rRNA) and 5S genes are physically separated occupying different chromosomal loci. However, recent observations established that both genes have been unified to a single 35S-5S unit in the genus Artemisia (Asteraceae), a genomic arrangement typical of primitive eukaryotes such as yeast, among others. Here we aim to reveal the origin, distribution and mechanisms leading to the linked organisation of rDNA in the Asteraceae by analysing unit structure (PCR, Southern blot, sequencing), gene copy number (quantitative PCR) and chromosomal position (FISH) of 5S and 35S rRNA genes in approximately 200 species representing the family diversity and other closely related groups. Dominant linked rDNA genotype was found within three large groups in subfamily Asteroideae: tribe Anthemideae (93% of the studied cases), tribe Gnaphalieae (100%) and in the "Heliantheae alliance" (23%). The remaining five tribes of the Asteroideae displayed canonical non linked arrangement of rDNA, as did the other groups in the Asteraceae. Nevertheless, low copy linked genes were identified among several species that amplified unlinked units. The conserved position of functional 5S insertions downstream from the 26S gene suggests a unique, perhaps retrotransposon-mediated integration event at the base of subfamily Asteroideae. Further evolution likely involved divergence of 26S-5S intergenic spacers, amplification and homogenisation of units across the chromosomes and concomitant elimination of unlinked arrays. However, the opposite trend, from linked towards unlinked arrangement was also surmised in few species indicating possible reversibility of these processes. Our results indicate that nearly 25% of Asteraceae species may have evolved unusual linked arrangement of rRNA genes. Thus, in plants, fundamental changes in intrinsic structure of rDNA units, their copy number and chromosomal organisation may occur within relatively short evolutionary time. We hypothesize that the 5S gene integration within the 35S unit might have repeatedly occurred during plant evolution, and probably once in Asteraceae.

Repeated reunions and splits feature the highly dynamic evolution of 5S and 35S ribosomal RNA genes (rDNA) in the Asteraceae family

PubMed Central

2010-01-01

Background In flowering plants and animals the most common ribosomal RNA genes (rDNA) organisation is that in which 35S (encoding 18S-5.8S-26S rRNA) and 5S genes are physically separated occupying different chromosomal loci. However, recent observations established that both genes have been unified to a single 35S-5S unit in the genus Artemisia (Asteraceae), a genomic arrangement typical of primitive eukaryotes such as yeast, among others. Here we aim to reveal the origin, distribution and mechanisms leading to the linked organisation of rDNA in the Asteraceae by analysing unit structure (PCR, Southern blot, sequencing), gene copy number (quantitative PCR) and chromosomal position (FISH) of 5S and 35S rRNA genes in ~200 species representing the family diversity and other closely related groups. Results Dominant linked rDNA genotype was found within three large groups in subfamily Asteroideae: tribe Anthemideae (93% of the studied cases), tribe Gnaphalieae (100%) and in the "Heliantheae alliance" (23%). The remaining five tribes of the Asteroideae displayed canonical non linked arrangement of rDNA, as did the other groups in the Asteraceae. Nevertheless, low copy linked genes were identified among several species that amplified unlinked units. The conserved position of functional 5S insertions downstream from the 26S gene suggests a unique, perhaps retrotransposon-mediated integration event at the base of subfamily Asteroideae. Further evolution likely involved divergence of 26S-5S intergenic spacers, amplification and homogenisation of units across the chromosomes and concomitant elimination of unlinked arrays. However, the opposite trend, from linked towards unlinked arrangement was also surmised in few species indicating possible reversibility of these processes. Conclusions Our results indicate that nearly 25% of Asteraceae species may have evolved unusual linked arrangement of rRNA genes. Thus, in plants, fundamental changes in intrinsic structure of rDNA units, their copy number and chromosomal organisation may occur within relatively short evolutionary time. We hypothesize that the 5S gene integration within the 35S unit might have repeatedly occurred during plant evolution, and probably once in Asteraceae. PMID:20712858

Heterochromatic siRNAs and DDM1 Independently Silence Aberrant 5S rDNA Transcripts in Arabidopsis

PubMed Central

Blevins, Todd; Pontes, Olga; Pikaard, Craig S.; Meins, Frederick

2009-01-01

5S ribosomal RNA gene repeats are arranged in heterochromatic arrays (5S rDNA) situated near the centromeres of Arabidopsis chromosomes. The chromatin remodeling factor DDM1 is known to maintain 5S rDNA methylation patterns while silencing transcription through 5S rDNA intergenic spacers (IGS). We mapped small-interfering RNAs (siRNA) to a composite 5S rDNA repeat, revealing a high density of siRNAs matching silenced IGS transcripts. IGS transcript repression requires proteins of the heterochromatic siRNA pathway, including RNA polymerase IV (Pol IV), RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and DICER-LIKE 3 (DCL3). Using molecular and cytogenetic approaches, we show that the DDM1 and siRNA-dependent silencing effects are genetically independent. DDM1 suppresses production of the siRNAs, however, thereby limiting RNA-directed DNA methylation at 5S rDNA repeats. We conclude that DDM1 and siRNA-dependent silencing are overlapping processes that both repress aberrant 5S rDNA transcription and contribute to the heterochromatic state of 5S rDNA arrays. PMID:19529764

Stimulation of ribosomal RNA gene promoter by transcription factor Sp1 involves active DNA demethylation by Gadd45-NER pathway.

PubMed

Rajput, Pallavi; Pandey, Vijaya; Kumar, Vijay

2016-08-01

The well-studied Pol II transcription factor Sp1 has not been investigated for its regulatory role in rDNA transcription. Here, we show that Sp1 bound to specific sites on rDNA and localized into the nucleoli during the G1 phase of cell cycle to activate rDNA transcription. It facilitated the recruitment of Pol I pre-initiation complex and impeded the binding of nucleolar remodeling complex (NoRC) to rDNA resulting in the formation of euchromatin active state. More importantly, Sp1 also orchestrated the site-specific binding of Gadd45a-nucleotide excision repair (NER) complex resulting in active demethylation and transcriptional activation of rDNA. Interestingly, knockdown of Sp1 impaired rDNA transcription due to reduced engagement of the Gadd45a-NER complex and hypermethylation of rDNA. Thus, the present study unveils a novel role of Sp1 in rDNA transcription involving promoter demethylation. Copyright © 2016 Elsevier B.V. All rights reserved.

Reversal of a Neurospora Translocation by Crossing over Involving Displaced Rdna, and Methylation of the Rdna Segments That Result from Recombination

PubMed Central

Perkins, David D.; Metzenberg, Robert L.; Raju, Namboori B.; Selker, Eric U.; Barry, Edward G.

1986-01-01

In translocation OY321 of Neurospora crassa, the nucleolus organizer is divided into two segments, a proximal portion located interstitially in one interchange chromosome, and a distal portion now located terminally on another chromosome, linkage group I. In crosses of Translocation x Translocation, exceptional progeny are recovered nonselectively in which the chromosome sequence has apparently reverted to Normal. Genetic, cytological, and molecular evidence indicates that reversion is the result of meiotic crossing over between homologous displaced rDNA repeats. Marker linkages are wild type in these exceptional progeny. They differ from wild type, however, in retaining an interstitial block of rRNA genes which can be demonstrated cytologically by the presence of a second, small interstitial nucleolus and genetically by linkage of an rDNA restriction site polymorphism to the mating-type locus in linkage group I. The interstitial rDNA is more highly methylated than the terminal rDNA. The mechanism by which methylation enzymes distinguish between interstitial rDNA and terminal rDNA is unknown. Some hypotheses are considered. PMID:2947829

NRPD4, a protein related to the RPB4 subunit of RNA polymerase II, is a component of RNA polymerases IV and V and is required for RNA-directed DNA methylation

PubMed Central

He, Xin-Jian; Hsu, Yi-Feng; Pontes, Olga; Zhu, Jianhua; Lu, Jian; Bressan, Ray A.; Pikaard, Craig; Wang, Co-Shine; Zhu, Jian-Kang

2009-01-01

RNA-directed DNA methylation (RdDM) is an RNAi-based mechanism for establishing transcriptional gene silencing in plants. The plant-specific RNA polymerases IV and V are required for the generation of 24-nucleotide (nt) siRNAs and for guiding sequence-specific DNA methylation by the siRNAs, respectively. However, unlike the extensively studied multisubunit Pol II, our current knowledge about Pol IV and Pol V is restricted to only the two largest subunits NRPD1a/NRPD1 and NRPD1b/NRPE1 and the one second-largest subunit NRPD2a. It is unclear whether other subunits may be required for the functioning of Pol IV and Pol V in RdDM. From a genetic screen for second-site suppressors of the DNA demethylase mutant ros1, we identified a new component (referred to as RDM2) as well as seven known components (NRPD1, NRPE1, NRPD2a, AGO4, HEN1, DRD1, and HDA6) of the RdDM pathway. The differential effects of the mutations on two mechanistically distinct transcriptional silencing reporters suggest that RDM2, NRPD1, NRPE1, NRPD2a, HEN1, and DRD1 function only in the siRNA-dependent pathway of transcriptional silencing, whereas HDA6 and AGO4 have roles in both siRNA-dependent and -independent pathways of transcriptional silencing. In the rdm2 mutants, DNA methylation and siRNA accumulation were reduced substantially at loci previously identified as endogenous targets of Pol IV and Pol V, including 5S rDNA, MEA-ISR, AtSN1, AtGP1, and AtMU1. The amino acid sequence of RDM2 is similar to that of RPB4 subunit of Pol II, but we show evidence that RDM2 has diverged significantly from RPB4 and cannot function in Pol II. An association of RDM2 with both NRPD1 and NRPE1 was observed by coimmunoprecipitation and coimmunolocalization assays. Our results show that RDM2/NRPD4/NRPE4 is a new component of the RdDM pathway in Arabidopsis and that it functions as part of Pol IV and Pol V. PMID:19204117

NRPD4, a Protein Related to the RPB4 Subunit of RNA Polymerase II, is a Component of RNA Polymerases IV and V and is Required for RNA-directed DNA methylation

DOE Office of Scientific and Technical Information (OSTI.GOV)

He, Xin-Jian; Hsu, Yi-Feng; Pontes, Olga

2009-01-01

RNA-directed DNA methylation (RdDM) is an RNAi-based mechanism for establishing transcriptional gene silencing in plants. The plant-specific RNA polymerases IV and V are required for the generation of 24-nucleotide (nt) siRNAs and for guiding sequence-specific DNA methylation by the siRNAs, respectively. However, unlike the extensively studied multisubunit Pol II, our current knowledge about Pol IV and Pol V is restricted to only the two largest subunits NRPD1a/NRPD1 and NRPD1b/NRPE1 and the one second-largest subunit NRPD2a. It is unclear whether other subunits may be required for the functioning of Pol IV and Pol V in RdDM. From a genetic screen formore » second-site suppressors of the DNA demethylase mutant ros1, we identified a new component (referred to as RDM2) as well as seven known components (NRPD1, NRPE1, NRPD2a, AGO4, HEN1, DRD1, and HDA6) of the RdDM pathway. The differential effects of the mutations on two mechanistically distinct transcriptional silencing reporters suggest that RDM2, NRPD1, NRPE1, NRPD2a, HEN1, and DRD1 function only in the siRNA-dependent pathway of transcriptional silencing, whereas HDA6 and AGO4 have roles in both siRNA-dependent and -independent pathways of transcriptional silencing. In the rdm2 mutants, DNA methylation and siRNA accumulation were reduced substantially at loci previously identified as endogenous targets of Pol IV and Pol V, including 5S rDNA, MEA-ISR, AtSN1, AtGP1, and AtMU1. The amino acid sequence of RDM2 is similar to that of RPB4 subunit of Pol II, but we show evidence that RDM2 has diverged significantly from RPB4 and cannot function in Pol II. An association of RDM2 with both NRPD1 and NRPE1 was observed by coimmunoprecipitation and coimmunolocalization assays. Our results show that RDM2/NRPD4/NRPE4 is a new component of the RdDM pathway in Arabidopsis and that it functions as part of Pol IV and Pol V.« less

Mapping of rDNA on the chromosomes of Eleusine species by fluorescence in situ hybridization.

PubMed

Bisht, M S; Mukai, Y

2000-12-01

Mapping of rDNA sites on the chromosomes of four diploid and two tetraploid species of Eleusine has provided valuable information on genome relationship between the species. Presence of 18S-5.8S-26S rDNA on the largest pair of the chromosomes, location of 5S rDNA at four sites on two pairs of chromosomes and presence of 18S-5.8S-26S and 5S rDNA at same location on one pair of chromosomes have clearly differentiated E. multiflora from rest of the species of Eleusine. The two tetraploid species, E. coracana and E. africana have the same number of 18S-5.8S-26S and 5S rDNA sites and located at similar position on the chromosomes. Diploid species, E. indica, E. floccifolia and E. tristachya have the same 18S-5.8S-26S sites and location on the chromosomes which also resembled with the two pairs of 18S-5.8S-26S rDNA locations in tetraploid species, E. coracana and E. africana. The 5S rDNA sites on chromosomes of E. indica and E. floccifolia were also comparable to the 5S rDNA sites of E. africana and E. coracana. The similarity of the rDNA sites and their location on chromosomes in the three diploid and two polyploid species also supports the view that genome donors to tetraploid species may be from these diploid species.

Isolated spinach ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit .sup..epsilon. N-methyltransferase and method of inactivating ribulose-1,5-bisphosphatase carboxylase/oxygenase large subunit .sup..epsilon. N-methyltransferase activity

DOEpatents

Houtz, Robert L.

1999-01-01

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltransferase (protein methylase III or Rubisco LSMT) from a plant which has a des(methyl) lysyl residue in the LS is disclosed. In addition, the full-length cDNA clones for Rubisco LSMT are disclosed. Transgenic plants and methods of producing same which have the Rubisco LSMT gene inserted into the DNA are also provided. Further, methods of inactivating the enzymatic activity of Rubisco LSMT are also disclosed.

Molecular organization and phylogenetic analysis of 5S rDNA in crustaceans of the genus Pollicipes reveal birth-and-death evolution and strong purifying selection.

PubMed

Perina, Alejandra; Seoane, David; González-Tizón, Ana M; Rodríguez-Fariña, Fernanda; Martínez-Lage, Andrés

2011-10-17

The 5S ribosomal DNA (5S rDNA) is organized in tandem arrays with repeat units that consist of a transcribing region (5S) and a variable nontranscribed spacer (NTS), in higher eukaryotes. Until recently the 5S rDNA was thought to be subject to concerted evolution, however, in several taxa, sequence divergence levels between the 5S and the NTS were found higher than expected under this model. So, many studies have shown that birth-and-death processes and selection can drive the evolution of 5S rDNA. In analyses of 5S rDNA evolution is found several 5S rDNA types in the genome, with low levels of nucleotide variation in the 5S and a spacer region highly divergent. Molecular organization and nucleotide sequence of the 5S ribosomal DNA multigene family (5S rDNA) were investigated in three Pollicipes species in an evolutionary context. The nucleotide sequence variation revealed that several 5S rDNA variants occur in Pollicipes genomes. They are clustered in up to seven different types based on differences in their nontranscribed spacers (NTS). Five different units of 5S rDNA were characterized in P. pollicipes and two different units in P. elegans and P. polymerus. Analysis of these sequences showed that identical types were shared among species and that two pseudogenes were present. We predicted the secondary structure and characterized the upstream and downstream conserved elements. Phylogenetic analysis showed an among-species clustering pattern of 5S rDNA types. These results suggest that the evolution of Pollicipes 5S rDNA is driven by birth-and-death processes with strong purifying selection.

Molecular organization and phylogenetic analysis of 5S rDNA in crustaceans of the genus Pollicipes reveal birth-and-death evolution and strong purifying selection

PubMed Central

2011-01-01

Background The 5S ribosomal DNA (5S rDNA) is organized in tandem arrays with repeat units that consist of a transcribing region (5S) and a variable nontranscribed spacer (NTS), in higher eukaryotes. Until recently the 5S rDNA was thought to be subject to concerted evolution, however, in several taxa, sequence divergence levels between the 5S and the NTS were found higher than expected under this model. So, many studies have shown that birth-and-death processes and selection can drive the evolution of 5S rDNA. In analyses of 5S rDNA evolution is found several 5S rDNA types in the genome, with low levels of nucleotide variation in the 5S and a spacer region highly divergent. Molecular organization and nucleotide sequence of the 5S ribosomal DNA multigene family (5S rDNA) were investigated in three Pollicipes species in an evolutionary context. Results The nucleotide sequence variation revealed that several 5S rDNA variants occur in Pollicipes genomes. They are clustered in up to seven different types based on differences in their nontranscribed spacers (NTS). Five different units of 5S rDNA were characterized in P. pollicipes and two different units in P. elegans and P. polymerus. Analysis of these sequences showed that identical types were shared among species and that two pseudogenes were present. We predicted the secondary structure and characterized the upstream and downstream conserved elements. Phylogenetic analysis showed an among-species clustering pattern of 5S rDNA types. Conclusions These results suggest that the evolution of Pollicipes 5S rDNA is driven by birth-and-death processes with strong purifying selection. PMID:22004418

DNA replication stress restricts ribosomal DNA copy number.

PubMed

Salim, Devika; Bradford, William D; Freeland, Amy; Cady, Gillian; Wang, Jianmin; Pruitt, Steven C; Gerton, Jennifer L

2017-09-01

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

Nmd3p Is a Crm1p-Dependent Adapter Protein for Nuclear Export of the Large Ribosomal Subunit

PubMed Central

Ho, Jennifer Hei-Ngam; Kallstrom, George; Johnson, Arlen W.

2000-01-01

In eukaryotic cells, nuclear export of nascent ribosomal subunits through the nuclear pore complex depends on the small GTPase Ran. However, neither the nuclear export signals (NESs) for the ribosomal subunits nor the receptor proteins, which recognize the NESs and mediate export of the subunits, have been identified. We showed previously that Nmd3p is an essential protein from yeast that is required for a late step in biogenesis of the large (60S) ribosomal subunit. Here, we show that Nmd3p shuttles and that deletion of the NES from Nmd3p leads to nuclear accumulation of the mutant protein, inhibition of the 60S subunit biogenesis, and inhibition of the nuclear export of 60S subunits. Moreover, the 60S subunits that accumulate in the nucleus can be coimmunoprecipitated with the NES-deficient Nmd3p. 60S subunit biogenesis and export of truncated Nmd3p were restored by the addition of an exogenous NES. To identify the export receptor for Nmd3p we show that Nmd3p shuttling and 60S export is blocked by the Crm1p-specific inhibitor leptomycin B. These results identify Crm1p as the receptor for Nmd3p export. Thus, export of the 60S subunit is mediated by the adapter protein Nmd3p in a Crm1p-dependent pathway. PMID:11086007

Filarial nematode infection in eclectus parrots (Eclectus roratus) in Taiwan.

PubMed

Huang, Yen-Li; Tsai, Shinn-Shyong; Thongchan, Duangsuda; Khatri-Chhetri, Rupak; Wu, Hung-Yi

2017-04-01

A total of 166 psittacines belonging to 22 species were received by the Animal Hospital of National Pingtung University of Science & Technology (NPUST) from 2013 to 2015. Only eclectus parrots (Eclectus roratus) were identified as hosts for microfilariae. All eclectus parrots were adult birds and had been kept in Taiwan for more than three years. The relevance of filariae to eclectus parrots is evident as indicated by the 35.7% (5/14) infection rate. At necropsy, adult filarial nematodes 57-75 mm in length and 0.4-0.7 mm in width were found in the hepatic veins. The microfilariae were 170-230 μm in length. Histopathological examination confirmed that eggs and larvae were observed in the ovaries and uteri of female filariae. These nematodes were closely related to an unidentified Filaria sp. (KJ612514.1) as indicated by polymerase chain reaction (PCR) analysis and phylogenetic analysis of nucleotide sequences from 18S ribosomal DNA gene (18S rDNA), mitochondrial cytochrome c oxidase subunit 1 (COX1) gene, and internal transcribed spacers 1-5.8S ribosomal DNA gene (ITS 1-5.8S rDNA). However, structurally the filarial nematodes were similar to that of the Pelecitus sp. Eclectus parrot species are important pet birds and are highly traded, resulting in high uncertainty of the origin of the parasite infection. This study is the first of its kind to report the presence and potential impact of filarial nematode infection on eclectus parrots, suggesting parasite inspection prior to the international trade of these pet birds.

Dual Role of a SAS10/C1D Family Protein in Ribosomal RNA Gene Expression and Processing Is Essential for Reproduction in Arabidopsis thaliana

PubMed Central

Chen, Ying-Jiun C.; Wang, Huei-Jing

2016-01-01

In eukaryotic cells, ribosomal RNAs (rRNAs) are transcribed, processed, and assembled with ribosomal proteins in the nucleolus. Regulatory mechanisms of rRNA gene (rDNA) transcription and processing remain elusive in plants, especially their connection to nucleolar organization. We performed an in silico screen for essential genes of unknown function in Arabidopsis thaliana and identified Thallo (THAL) encoding a SAS10/C1D family protein. THAL disruption caused enlarged nucleoli in arrested embryos, aberrant processing of precursor rRNAs at the 5’ External Transcribed Spacer, and repression of the major rDNA variant (VAR1). THAL overexpression lines showed de-repression of VAR1 and overall reversed effects on rRNA processing sites. Strikingly, THAL overexpression also induced formation of multiple nucleoli per nucleus phenotypic of mutants of heterochromatin factors. THAL physically associated with histone chaperone Nucleolin 1 (NUC1), histone-binding NUC2, and histone demethylase Jumonji 14 (JMJ14) in bimolecular fluorescence complementation assay, suggesting that it participates in chromatin regulation. Furthermore, investigation of truncated THAL proteins revealed that the SAS10 C-terminal domain is likely important for its function in chromatin configuration. THAL also interacted with putative Small Subunit processome components, including previously unreported Arabidopsis homologue of yeast M Phase Phosphoprotein 10 (MPP10). Our results uncovering the dual role of THAL in transcription and processing events critical for proper rRNA biogenesis and nucleolar organization during reproduction are the first to define the function of SAS10/C1D family members in plants. PMID:27792779

Molecular systematics of the wood-inhabiting, lichen-forming genus Xylographa (Baeomycetales, Ostropomycetidae) with eight new species

PubMed Central

Spribille, Toby; Resl, Philipp; Ahti, Teuvo; Pérez-Ortega, Sergio; Tønsberg, Tor; Mayrhofer, Helmut; Lumbsch, H. Thorsten

2016-01-01

The ascomycete genus Xylographa includes some of the most abundant species of wood-inhabiting lichenized fungi in boreal and temperate regions. It has never been monographed and little is known of its species diversity and evolutionary relationships. Based on a morphological and secondary metabolite-based assessment of material from North and South America, Europe and Asia, we generated a three-locus phylogeny based on sequences of the internal transcribed spacer, 28S nuclear rDNA and mitochondrial small subunit rDNA. We analyzed the data within the context of putatively related genera in the order Baeomycetales. Xylographa is a strongly supported monophyletic group closely related to Lithographa and Ptychographa, as well as rock-dwelling and lichenicolous species of Rimularia s.lat. The evolution of linearized ascomata in Xylographa appears to have enabled ascomata to grow laterally, and patterns of lateral growth are diagnostic. We recognize twenty species in Xylographa and provide a thorough revision of nomenclature. The following eight species are new: Xylographa bjoerkii T. Sprib., X. constricta T. Sprib., X. erratica T. Sprib., X. lagoi T. Sprib. & Pérez-Ortega, X. schofieldii T. Sprib., X. septentrionalis T. Sprib., X. stenospora T. Sprib. & Resl and X. vermicularis T. Sprib. The combinations Lambiella insularis (Nyl.) T. Sprib. and Xylographa carneopallida (Räsänen) T. Sprib. are newly proposed. Xylographa constricta from southern South America represents the first known case of secondary de-lichenization in the Baeomycetales. Xylographa parallela s.str. is confirmed as bipolar on the basis of sequenced collections from both southern Chile and the northern Hemisphere. PMID:26953522

Phylogeny, phylogeography, and evolution in the Mediterranean region: News from a freshwater mussel (Potomida, Unionida).

PubMed

Froufe, Elsa; Prié, Vincent; Faria, João; Ghamizi, Mohamed; Gonçalves, Duarte V; Gürlek, Mustafa Emre; Karaouzas, Ioannis; Kebapçi, Ümit; Şereflişan, Hülya; Sobral, Carina; Sousa, Ronaldo; Teixeira, Amílcar; Varandas, Simone; Zogaris, Stamatis; Lopes-Lima, Manuel

2016-07-01

The Potomida genus (Bivalvia, Unionida) has a Circum-Mediterranean distribution and like other freshwater mussel species, its populations have suffered dramatic declines. Although this genus is currently considered as monotypic, it has a long history of taxonomic revisions and presently many aspects of its systematics and evolutionary history are unclear. We sampled a total of 323 individuals from 39 different sites across the Potomida genus distribution, and sequenced two mitochondrial (16S rDNA and Cytochrome c Oxidase Subunit I) and one nuclear (28S rDNA) genes to clarify its phylogeny and phylogeographic history. Our results show that the genus includes two well-supported clades, one comprising solely the western Mediterranean species Potomida littoralis, and the other including two eastern Mediterranean species, the Greek endemic P. acarnanica and the Anatolian and Middle Eastern P. semirugata. We suggest that Potomida started radiating during the upper Miocene, and that both vicariance and dispersal events shaped the diversification and distribution of the genus along the Mediterranean region. P. littoralis is further divided in two mitochondrial lineages, one restricted to Europe and the other occurring mostly in North Africa. Moreover, some European basins present both lineages in sympatry. The conservation status of the three recognized species should be reevaluated, particularly P. acarnanica, since it is restricted to two Greek river basins presenting a high risk of extinction. Overall, our results clarify some important gaps in knowledge concerning the phylogeny, phylogeography and evolution of the Potomida genus in the Mediterranean region with important taxonomical, ecological and conservational implications. Copyright © 2016 Elsevier Inc. All rights reserved.

The role of TcdB and TccC subunits in secretion of the Photorhabdus Tcd toxin complex.

PubMed

Yang, Guowei; Waterfield, Nicholas R

2013-01-01

The Toxin Complex (TC) is a large multi-subunit toxin encoded by a range of bacterial pathogens. The best-characterized examples are from the insect pathogens Photorhabdus, Xenorhabdus and Yersinia. They consist of three large protein subunits, designated A, B and C that assemble in a 5∶1∶1 stoichiometry. Oral toxicity to a range of insects means that some have the potential to be developed as pest control technology. The three subunit proteins do not encode any recognisable export sequences and as such little progress has been made in understanding their secretion. We have developed heterologous TC production and secretion models in E. coli and used them to ascribe functions to different domains of the crucial B+C sub-complex. We have determined that the B and C subunits use a secretion mechanism that is either encoded by the proteins themselves or employ an as yet undefined system common to laboratory strains of E. coli. We demonstrate that both the N-terminal domains of the B and C subunits are required for secretion of the whole complex. We propose a model whereby the N-terminus of the C-subunit toxin exports the B+C sub-complex across the inner membrane while that of the B-subunit allows passage across the outer membrane. We also demonstrate that even in the absence of the B-subunit, that the C-subunit can also facilitate secretion of the larger A-subunit. The recognition of this novel export system is likely to be of importance to future protein secretion studies. Finally, the identification of homologues of B and C subunits in diverse bacterial pathogens, including Burkholderia and Pseudomonas, suggests that these toxins are likely to be important in a range of different hosts, including man.

Comparative chromosomal localization of 45S and 5S rDNAs and implications for genome evolution in Cucumis.

PubMed

Zhang, Zhen-Tao; Yang, Shu-Qiong; Li, Zi-Ang; Zhang, Yun-Xia; Wang, Yun-Zhu; Cheng, Chun-Yan; Li, Ji; Chen, Jin-Feng; Lou, Qun-Feng

2016-07-01

Ribosomal DNAs are useful cytogenetic markers for chromosome analysis. Studies investigating site numbers and distributions of rDNAs have provided important information for elucidating genome organization and chromosomal relationships of many species by fluorescence in situ hybridization. But relevant studies are scarce for species of the genus Cucumis, especially in wild species. In the present study, FISH was conducted to investigate the organization of 45S and 5S rDNA among 20 Cucumis accessions, including cultivars and wild accessions. Our results showed that the number of 45S rDNA sites varied from one to five pairs in different accessions, and most of these sites are located at the terminal regions of chromosomes. Interestingly, up to five pairs of 45S rDNA sites were observed in C. sativus var. sativus, the species which has the lowest chromosome number, i.e., 2n = 14. Only one pair of 5S rDNA sites was detected in all accessions, except for C. heptadactylus, C. sp, and C. spp that had two pairs of 5S rDNA sites. The distributions of 5S rDNA sites showed more variation than 45S rDNA sites. The phylogenetic analysis in this study showed that 45S and 5S rDNA have contrasting evolutionary patterns. We find that 5S rDNA has a polyploidization-related tendency towards the terminal location from an interstitial location but maintains a conserved site number, whereas the 45S rDNA showed a trend of increasing site number but a relatively conserved location.

DNA replication initiator Cdc6 also regulates ribosomal DNA transcription initiation.

PubMed

Huang, Shijiao; Xu, Xiaowei; Wang, Guopeng; Lu, Guoliang; Xie, Wenbing; Tao, Wei; Zhang, Hongyin; Jiang, Qing; Zhang, Chuanmao

2016-04-01

RNA-polymerase-I-dependent ribosomal DNA (rDNA) transcription is fundamental to rRNA processing, ribosome assembly and protein synthesis. However, how this process is initiated during the cell cycle is not fully understood. By performing a proteomic analysis of transcription factors that bind RNA polymerase I during rDNA transcription initiation, we identified that the DNA replication initiator Cdc6 interacts with RNA polymerase I and its co-factors, and promotes rDNA transcription in G1 phase in an ATPase-activity-dependent manner. We further showed that Cdc6 is targeted to the nucleolus during late mitosis and G1 phase in a manner that is dependent on B23 (also known as nucleophosmin, NPM1), and preferentially binds to the rDNA promoter through its ATP-binding domain. Overexpression of Cdc6 increases rDNA transcription, whereas knockdown of Cdc6 results in a decreased association of both RNA polymerase I and the RNA polymerase I transcription factor RRN3 with rDNA, and a reduction of rDNA transcription. Furthermore, depletion of Cdc6 impairs the interaction between RRN3 and RNA polymerase I. Taken together, our data demonstrate that Cdc6 also serves as a regulator of rDNA transcription initiation, and indicate a mechanism by which initiation of rDNA transcription and DNA replication can be coordinated in cells. © 2016. Published by The Company of Biologists Ltd.

Three-dimensional architecture of ribosomal DNA within barley nucleoli revealed with electron microscopy.

PubMed

Iwano, Megumi; Che, Fang-Sik; Takayama, Seiji; Fukui, Kiichi; Isogai, Akira

2003-01-01

To elucidate the topological positioning of ribosomal RNA genes (rDNA) and nucleolar structure in three dimensions, we examined the localization of rDNA using in situ hybridization (ISH) analysis by scanning electron microscopy (SEM). The rDNA genes within the three-dimensional architecture of nucleoli were detected on chromatin fibers that connect a thick strand-like structure and a protrusion of rDNA into the inner nuclear hole where the nucleolus is formed. This novel use of ISH together with SEM is useful for the analysis of nucleolar structure in detail. Furthermore, rDNA was detected at the periphery of the fibrillar centers (FCs) of the nucleolus using immuno-gold labeling together with transmission electron microscopy (TEM). In situ hybridization with TEM confirmed that rDNA is naked and thus active in the FCs of nucleoli; ISH with SEM confirmed that rDNA is not covered with ribonucleo proteins at the protruding point and is thus inactive. We also show that the distribution pattern of FCs differs from sample to sample. These results indicate that rDNA is transcribed dynamically in a time- and region-specific manner over the course of the cell cycle.

Crotoxin: Structural Studies, Mechanism of Action and Cloning of its Gene

DTIC Science & Technology

1988-03-01

thirteen amino acids being acidic . Sequencing of the three peptides present in the acidic subunit, two of which are blocked by pyroglutamate ...the sequence determination of both the basic and acidic subunits of crotoxin- The acidic * subunit peptides were d!Tfficult, .sfi~n~e two of-ftflý...fluorescence spectroscopy. Results indicate a large conformational change occurs upon) ccmplex formation between the acidic and basic subunits of all four

Conservation and loss of ribosomal RNA gene sites in diploid and polyploid Fragaria (Rosaceae)

PubMed Central

2011-01-01

Background The genus Fragaria comprises species at ploidy levels ranging from diploid (2n = 2x = 14) to decaploid (2n = 10x = 70). Fluorescence in situ hybridization with 5S and 25S rDNA probes was performed to gather cytogenetic information that illuminates genomic divergence among different taxa at multiple ploidy levels, as well as to explore the evolution of ribosomal RNA genes during polyploidization in Fragaria. Results Root tip cells of diploid taxa were typified by two 5S and six 25S rDNA hybridization signals of varying intensities, providing a baseline for comparisons within the genus. In three exceptional diploid genotypes, F. nilgerrensis (CFRA 1358 and CFRA 1825) and F. vesca 'Yellow Wonder', two 5S but only four 25S rDNA sites were found but with differing site losses. The numbers of 5S and 25S rDNA signals, respectively were three and nine in a triploid F. ×bifera accession, and were four and twelve in three tetraploids, thus occurring in proportional 1.5× and 2× multiples of the typical diploid pattern. In hexaploid F. moschata, a proportional multiple of six 5S rDNA sites was observed, but the number of 25S rDNA sites was one or two less than the proportionate prediction of eighteen. This apparent tendency toward rDNA site loss at higher ploidy was markedly expanded in octoploids, which displayed only two 5S and ten 25S rDNA sites. In the two decaploids examined, the numbers of 5S and 25S rDNA signals, respectively, were four and fifteen in F. virginiana subsp. platypetala, and six and twelve in F. iturupensis. Conclusions Among diploid Fragaria species, a general consistency of rDNA site numbers implies conserved genomic organization, but highly variable 25S signal sizes and intensities and two instances of site loss suggest concurrent high dynamics of rDNA copy numbers among both homologs and non-homologs. General conservation of rDNA site numbers in lower ploidy, but marked site number reductions at higher ploidy levels, suggest complex evolution of rDNA sites during polyploidization and/or independent evolutionary pathways for 6x versus higher ploidy strawberries. Site number comparisons suggest common genomic composition among natural octoploids, and independent origins of the two divergent decaploid accessions. PMID:22074487

Use of molecular hybridization to explore genetic relationships. Progress report, July 1, 1975--March 31, 1976. [Primates, mice, Drosophila

DOE Office of Scientific and Technical Information (OSTI.GOV)

Atwood, K.C.

Progress is reported on the following research projects: distribution of rDNA in lymphocyte chromosomes of the gibbon; site of 55 DNA in chromosomes of the baboon; satellite associations and rDNA; polymorphisms in rDNA of mouse chromosomes; effect of prephotographing on hybridization; histone and immunoglobulin gene mapping; and rDNA magnification in Drosophila. (HLW)

Ribosomal DNA copy number amplification and loss in human cancers is linked to tumor genetic context, nucleolus activity, and proliferation

PubMed Central

2017-01-01

Ribosomal RNAs (rRNAs) are transcribed from two multicopy DNA arrays: the 5S ribosomal DNA (rDNA) array residing in a single human autosome and the 45S rDNA array residing in five human autosomes. The arrays are among the most variable segments of the genome, exhibit concerted copy number variation (cCNV), encode essential components of the ribosome, and modulate global gene expression. Here we combined whole genome data from >700 tumors and paired normal tissues to provide a portrait of rDNA variation in human tissues and cancers of diverse mutational signatures, including stomach and lung adenocarcinomas, ovarian cancers, and others of the TCGA panel. We show that cancers undergo coupled 5S rDNA array expansion and 45S rDNA loss that is accompanied by increased estimates of proliferation rate and nucleolar activity. These somatic changes in rDNA CN occur in a background of over 10-fold naturally occurring rDNA CN variation across individuals and cCNV of 5S-45S arrays in some but not all tissues. Analysis of genetic context revealed associations between cancer rDNA CN amplification or loss and the presence of specific somatic alterations, including somatic SNPs and copy number gain/losses in protein coding genes across the cancer genome. For instance, somatic inactivation of the tumor suppressor gene TP53 emerged with a strong association with coupled 5S expansion / 45S loss in several cancers. Our results uncover frequent and contrasting changes in the 5S and 45S rDNA along rapidly proliferating cell lineages with high nucleolar activity. We suggest that 5S rDNA amplification facilitates increased proliferation, nucleolar activity, and ribosomal synthesis in cancer, whereas 45S rDNA loss emerges as a byproduct of transcription-replication conflict in rapidly replicating tumor cells. The observations raise the prospects of using the rDNA arrays as re-emerging targets for the design of novel strategies in cancer therapy. PMID:28880866

Identifying the bacterial community on the surface of Intralox belting in a meat boning room by culture-dependent and culture-independent 16S rDNA sequence analysis.

PubMed

Brightwell, Gale; Boerema, Jackie; Mills, John; Mowat, Eilidh; Pulford, David

2006-05-25

We examined the bacterial community present on an Intralox conveyor belt system in an operating lamb boning room by sequencing the 16S ribosomal DNA (rDNA) of bacteria extracted in the presence or absence of cultivation. RFLP patterns for 16S rDNA clone library and cultures were generated using HaeIII and MspI restriction endonucleases. 16S rDNA amplicons produced 8 distinct RFLP pattern groups. RFLP groups I-IV were represented in the clone library and RFLP groups I and V-VIII were represented amongst the cultured isolates. Partial DNA sequences from each RFLP group revealed that all group I, II and VIII representatives were Pseudomonas spp., group III were Sphingomonas spp., group IV clones were most similar to an uncultured alpha proteobacterium, group V was similar to a Serratia spp., group VI with an Alcaligenes spp., and group VII with Microbacterium spp. Sphingomonads were numerically dominant in the culture-independent clone library and along with the group IV alpha proteobacterium were not represented amongst the cultured isolates. Serratia, Alcaligenes and Microbacterium spp. were only represented with cultured isolates. Pseudomonads were detected by both culture-dependent (84% of isolates) and culture-independent (12.5% of clones) methods and their presence at high frequency does pose the risk of product spoilage if transferred onto meat stored under aerobic conditions. The detection of sphingomonads in large numbers by the culture-independent method demands further analysis because sphingomonads may represent a new source of meat spoilage that has not been previously recognised in the meat processing environment. The 16S rDNA collections generated by both methods were important at representing the diversity of the bacterial population associated with an Intralox conveyor belt system.

A puzzle assembly strategy for fabrication of large engineered cartilage tissue constructs.

PubMed

Nover, Adam B; Jones, Brian K; Yu, William T; Donovan, Daniel S; Podolnick, Jeremy D; Cook, James L; Ateshian, Gerard A; Hung, Clark T

2016-03-21

Engineering of large articular cartilage tissue constructs remains a challenge as tissue growth is limited by nutrient diffusion. Here, a novel strategy is investigated, generating large constructs through the assembly of individually cultured, interlocking, smaller puzzle-shaped subunits. These constructs can be engineered consistently with more desirable mechanical and biochemical properties than larger constructs (~4-fold greater Young׳s modulus). A failure testing technique was developed to evaluate the physiologic functionality of constructs, which were cultured as individual subunits for 28 days, then assembled and cultured for an additional 21-35 days. Assembled puzzle constructs withstood large deformations (40-50% compressive strain) prior to failure. Their ability to withstand physiologic loads may be enhanced by increases in subunit strength and assembled culture time. A nude mouse model was utilized to show biocompatibility and fusion of assembled puzzle pieces in vivo. Overall, the technique offers a novel, effective approach to scaling up engineered tissues and may be combined with other techniques and/or applied to the engineering of other tissues. Future studies will aim to optimize this system in an effort to engineer and integrate robust subunits to fill large defects. Copyright © 2016 Elsevier Ltd. All rights reserved.

A Puzzle Assembly Strategy for Fabrication of Large Engineered Cartilage Tissue Constructs

PubMed Central

Nover, Adam B.; Jones, Brian K.; Yu, William T.; Donovan, Daniel S.; Podolnick, Jeremy D.; Cook, James L.; Ateshian, Gerard A.; Hung, Clark T.

2016-01-01

Engineering of large articular cartilage tissue constructs remains a challenge as tissue growth is limited by nutrient diffusion. Here, a novel strategy is investigated, generating large constructs through the assembly of individually cultured, interlocking, smaller puzzle-shaped subunits. These constructs can be engineered consistently with more desirable mechanical and biochemical properties than larger constructs (~4-fold greater Young's modulus). A failure testing technique was developed to evaluate the physiologic functionality of constructs, which were cultured as individual subunits for 28 days, then assembled and cultured for an additional 21-35 days. Assembled puzzle constructs withstood large deformations (40-50% compressive strain) prior to failure. Their ability to withstand physiologic loads may be enhanced by increases in subunit strength and assembled culture time. A nude mouse model was utilized to show biocompatibility and fusion of assembled puzzle pieces in vivo. Overall, the technique offers a novel, effective approach to scaling up engineered tissues and may be combined with other techniques and/or applied to the engineering of other tissues. Future studies will aim to optimize this system in an effort to engineer and integrate robust subunits to fill large defects. PMID:26895780

Effects of transgene-encoded high-molecular weight glutenin proteins in wheat flour blends and sponge and dough baking

USDA-ARS?s Scientific Manuscript database

HMW glutenin subunits are the most important determinants of wheat (Triticum aestivum L.) bread-making quality, and subunit composition explains a large percentage of the variability observed between genotypes. Experiments were designed to elevate expression of a key native HMW glutenin subunit (1D...

Contrasting Patterns of rDNA Homogenization within the Zygosaccharomyces rouxii Species Complex

PubMed Central

Chand Dakal, Tikam; Giudici, Paolo; Solieri, Lisa

2016-01-01

Arrays of repetitive ribosomal DNA (rDNA) sequences are generally expected to evolve as a coherent family, where repeats within such a family are more similar to each other than to orthologs in related species. The continuous homogenization of repeats within individual genomes is a recombination process termed concerted evolution. Here, we investigated the extent and the direction of concerted evolution in 43 yeast strains of the Zygosaccharomyces rouxii species complex (Z. rouxii, Z. sapae, Z. mellis), by analyzing two portions of the 35S rDNA cistron, namely the D1/D2 domains at the 5’ end of the 26S rRNA gene and the segment including the internal transcribed spacers (ITS) 1 and 2 (ITS regions). We demonstrate that intra-genomic rDNA sequence variation is unusually frequent in this clade and that rDNA arrays in single genomes consist of an intermixing of Z. rouxii, Z. sapae and Z. mellis-like sequences, putatively evolved by reticulate evolutionary events that involved repeated hybridization between lineages. The levels and distribution of sequence polymorphisms vary across rDNA repeats in different individuals, reflecting four patterns of rDNA evolution: I) rDNA repeats that are homogeneous within a genome but are chimeras derived from two parental lineages via recombination: Z. rouxii in the ITS region and Z. sapae in the D1/D2 region; II) intra-genomic rDNA repeats that retain polymorphisms only in ITS regions; III) rDNA repeats that vary only in their D1/D2 domains; IV) heterogeneous rDNA arrays that have both polymorphic ITS and D1/D2 regions. We argue that an ongoing process of homogenization following allodiplodization or incomplete lineage sorting gave rise to divergent evolutionary trajectories in different strains, depending upon temporal, structural and functional constraints. We discuss the consequences of these findings for Zygosaccharomyces species delineation and, more in general, for yeast barcoding. PMID:27501051

A Portrait of Ribosomal DNA Contacts with Hi-C Reveals 5S and 45S rDNA Anchoring Points in the Folded Human Genome

PubMed Central

Yu, Shoukai; Lemos, Bernardo

2016-01-01

Ribosomal RNAs (rRNAs) account for >60% of all RNAs in eukaryotic cells and are encoded in the ribosomal DNA (rDNA) arrays. The rRNAs are produced from two sets of loci: the 5S rDNA array resides exclusively on human chromosome 1, whereas the 45S rDNA array resides on the short arm of five human acrocentric chromosomes. The 45S rDNA gives origin to the nucleolus, the nuclear organelle that is the site of ribosome biogenesis. Intriguingly, 5S and 45S rDNA arrays exhibit correlated copy number variation in lymphoblastoid cells (LCLs). Here we examined the genomic architecture and repeat content of the 5S and 45S rDNA arrays in multiple human genome assemblies (including PacBio MHAP assembly) and ascertained contacts between the rDNA arrays and the rest of the genome using Hi-C datasets from two human cell lines (erythroleukemia K562 and lymphoblastoid cells). Our analyses revealed that 5S and 45S arrays each have thousands of contacts in the folded genome, with rDNA-associated regions and genes dispersed across all chromosomes. The rDNA contact map displayed conserved and disparate features between two cell lines, and pointed to specific chromosomes, genomic regions, and genes with evidence of spatial proximity to the rDNA arrays; the data also showed a lack of direct physical interaction between the 5S and 45S rDNA arrays. Finally, the analysis identified an intriguing organization in the 5S array with Alu and 5S elements adjacent to one another and organized in opposite orientation along the array. Portraits of genome folding centered on the ribosomal DNA array could help understand the emergence of concerted variation, the control of 5S and 45S expression, as well as provide insights into an organelle that contributes to the spatial localization of human chromosomes during interphase. PMID:27797956

DNA replication stress restricts ribosomal DNA copy number

PubMed Central

Salim, Devika; Bradford, William D.; Freeland, Amy; Cady, Gillian; Wang, Jianmin

2017-01-01

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100–200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how “normal” copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a “normal” rDNA copy number. PMID:28915237

Ribosomal RNA Genes Contribute to the Formation of Pseudogenes and Junk DNA in the Human Genome.

PubMed

Robicheau, Brent M; Susko, Edward; Harrigan, Amye M; Snyder, Marlene

2017-02-01

Approximately 35% of the human genome can be identified as sequence devoid of a selected-effect function, and not derived from transposable elements or repeated sequences. We provide evidence supporting a known origin for a fraction of this sequence. We show that: 1) highly degraded, but near full length, ribosomal DNA (rDNA) units, including both 45S and Intergenic Spacer (IGS), can be found at multiple sites in the human genome on chromosomes without rDNA arrays, 2) that these rDNA sequences have a propensity for being centromere proximal, and 3) that sequence at all human functional rDNA array ends is divergent from canonical rDNA to the point that it is pseudogenic. We also show that small sequence strings of rDNA (from 45S + IGS) can be found distributed throughout the genome and are identifiable as an "rDNA-like signal", representing 0.26% of the q-arm of HSA21 and ∼2% of the total sequence of other regions tested. The size of sequence strings found in the rDNA-like signal intergrade into the size of sequence strings that make up the full-length degrading rDNA units found scattered throughout the genome. We conclude that the displaced and degrading rDNA sequences are likely of a similar origin but represent different stages in their evolution towards random sequence. Collectively, our data suggests that over vast evolutionary time, rDNA arrays contribute to the production of junk DNA. The concept that the production of rDNA pseudogenes is a by-product of concerted evolution represents a previously under-appreciated process; we demonstrate here its importance. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Large conductance Ca(2+)-activated K(+) channel (BKCa) activating properties of a series of novel N-arylbenzamides: Channel subunit dependent effects.

PubMed

Kirby, R W; Martelli, A; Calderone, V; McKay, N G; Lawson, K

2013-07-15

Large conductance calcium activated potassium channels (BKCa) are fundamental in the control of cellular excitability. Thus, compounds that activate BKCa channels could provide potential therapies in the treatment of pathologies of the cardiovascular and central nervous system. A series of novel N-arylbenzamide compounds, and the reference compound NS1619, were evaluated for BKCa channel opener properties in Human Embryonic Kidney (HEK293) cells expressing the human BKCa channel α-subunit alone or α+β1-subunit complex. Channel activity was determined using a non-radioactive Rb(+) efflux assay to construct concentration effect curves for each compound. All N-arylbenzamide compounds and NS1619 evoked significant (p <0.05) concentration related increases in Rb(+) efflux both in cells expressing α-subunit alone or α+β1-subunits. Co-expression of the β1-subunit modified the Rb(+) efflux responses, relative to that obtained in cells expressing the α-subunit alone, for most of the N-arylbenzamide compounds, in contrast to NS1619. The EC40 values of NS1619, BKMe1 and BKOEt1 were not significantly affected by the co-expression of the BKCa channel α+β1-subunits. In contrast, 5 other N-arylbenzamides (BKPr2, BKPr3, BKPr4, BKH1 and BKVV) showed a significant (p <0.05) 2- to 10-fold increase in EC40 values when tested on the BKCa α+β1-subunit expressing cells compared to BKCa α-subunit expressing cells. Further, the Emax values for BKPr4, BKVV and BKH1 were lower in the BKCa channel α+β1-subunit expressing cells. In conclusion, the N-arylbenzamides studied, like NS1619, were able to activate BKCa channels formed of the α-subunit only. The co-expression of the β1-subunit, however, modified the ability of certain compounds to active the channel leading to differentiated pharmacodynamic profiles. Copyright © 2013 Elsevier Ltd. All rights reserved.

Improved purification of brine-shrimp (Artemia saline) (Na+ + K+)-activated adenosine triphosphatase and amino-acid and carbohydrate analyses of the isolated subunits.

PubMed Central

Peterson, G L; Hokin, L E

1980-01-01

Purification of the (Na+ + K+)-activated ATPase has been improved 2-fold the respect to both purity and yield over the previous method [Peterson, Ewing, Hootman & Conte (1978) J. Biol. Chem. 253, 4762-4770] by using Lubrol WX and non-denaturing concentrations of sodium dodecyl sulphate (SDS). The enzyme was purified 200-fold over the homogenate. The preparation had a specific activity of about 600 mumol of Pi/h per mg of protein, and was about 60% pure according to quantification of Coomassie Blue-stained SDS/polyacrylamide gels. The yield of purified enzyme was about 10 mg of protein per 100g of dry brine-shrimp (Artemia salina) cysts. The method is highly suitable for purification either on a small scale (10-25g of dry cysts) or on a large scale (900g of dry cysts) and methods are described for both. The large (Na+ + K+)-activated ATPase subunit (alpha-subunit) was isolated in pure form by SDS-gel filtration on Bio-Gel A 1.5m. The small subunit (beta-subunit) was eluted with other contaminating proteins on the Bio-Gel column, but was isolated in pure form by extraction from SDS/polyacrylamide gels. The amino acid and carbohydrate compositions of both subunits are reported. The alpha-subunit contained 5.2% carbohydrate by weight, and the beta-subunit 9.2%. Sialic acid was absent from both subunits. Images Fig. 3. Fig. 4. PMID:6272692

Utility of 16S rDNA Sequencing for Identification of Rare Pathogenic Bacteria.

PubMed

Loong, Shih Keng; Khor, Chee Sieng; Jafar, Faizatul Lela; AbuBakar, Sazaly

2016-11-01

Phenotypic identification systems are established methods for laboratory identification of bacteria causing human infections. Here, the utility of phenotypic identification systems was compared against 16S rDNA identification method on clinical isolates obtained during a 5-year study period, with special emphasis on isolates that gave unsatisfactory identification. One hundred and eighty-seven clinical bacteria isolates were tested with commercial phenotypic identification systems and 16S rDNA sequencing. Isolate identities determined using phenotypic identification systems and 16S rDNA sequencing were compared for similarity at genus and species level, with 16S rDNA sequencing as the reference method. Phenotypic identification systems identified ~46% (86/187) of the isolates with identity similar to that identified using 16S rDNA sequencing. Approximately 39% (73/187) and ~15% (28/187) of the isolates showed different genus identity and could not be identified using the phenotypic identification systems, respectively. Both methods succeeded in determining the species identities of 55 isolates; however, only ~69% (38/55) of the isolates matched at species level. 16S rDNA sequencing could not determine the species of ~20% (37/187) of the isolates. The 16S rDNA sequencing is a useful method over the phenotypic identification systems for the identification of rare and difficult to identify bacteria species. The 16S rDNA sequencing method, however, does have limitation for species-level identification of some bacteria highlighting the need for better bacterial pathogen identification tools. © 2016 Wiley Periodicals, Inc.

Condensin suppresses recombination and regulates double-strand break processing at the repetitive ribosomal DNA array to ensure proper chromosome segregation during meiosis in budding yeast.

PubMed

Li, Ping; Jin, Hui; Yu, Hong-Guo

2014-10-01

During meiosis, homologues are linked by crossover, which is required for bipolar chromosome orientation before chromosome segregation at anaphase I. The repetitive ribosomal DNA (rDNA) array, however, undergoes little or no meiotic recombination. Hyperrecombination can cause chromosome missegregation and rDNA copy number instability. We report here that condensin, a conserved protein complex required for chromosome organization, regulates double-strand break (DSB) formation and repair at the rDNA gene cluster during meiosis in budding yeast. Condensin is highly enriched at the rDNA region during prophase I, released at the prophase I/metaphase I transition, and reassociates with rDNA before anaphase I onset. We show that condensin plays a dual role in maintaining rDNA stability: it suppresses the formation of Spo11-mediated rDNA breaks, and it promotes DSB processing to ensure proper chromosome segregation. Condensin is unnecessary for the export of rDNA breaks outside the nucleolus but required for timely repair of meiotic DSBs. Our work reveals that condensin coordinates meiotic recombination with chromosome segregation at the repetitive rDNA sequence, thereby maintaining genome integrity. © 2014 Li et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Oligonucleotide fingerprinting of rRNA genes for analysis of fungal community composition.

PubMed

Valinsky, Lea; Della Vedova, Gianluca; Jiang, Tao; Borneman, James

2002-12-01

Thorough assessments of fungal diversity are currently hindered by technological limitations. Here we describe a new method for identifying fungi, oligonucleotide fingerprinting of rRNA genes (OFRG). ORFG sorts arrayed rRNA gene (ribosomal DNA [rDNA]) clones into taxonomic clusters through a series of hybridization experiments, each using a single oligonucleotide probe. A simulated annealing algorithm was used to design an OFRG probe set for fungal rDNA. Analysis of 1,536 fungal rDNA clones derived from soil generated 455 clusters. A pairwise sequence analysis showed that clones with average sequence identities of 99.2% were grouped into the same cluster. To examine the accuracy of the taxonomic identities produced by this OFRG experiment, we determined the nucleotide sequences for 117 clones distributed throughout the tree. For all but two of these clones, the taxonomic identities generated by this OFRG experiment were consistent with those generated by a nucleotide sequence analysis. Eighty-eight percent of the clones were affiliated with Ascomycota, while 12% belonged to BASIDIOMYCOTA: A large fraction of the clones were affiliated with the genera Fusarium (404 clones) and Raciborskiomyces (176 clones). Smaller assemblages of clones had high sequence identities to the Alternaria, Ascobolus, Chaetomium, Cryptococcus, and Rhizoctonia clades.

Structural Variation of Type I-F CRISPR RNA Guided DNA Surveillance.

PubMed

Pausch, Patrick; Müller-Esparza, Hanna; Gleditzsch, Daniel; Altegoer, Florian; Randau, Lennart; Bange, Gert

2017-08-17

CRISPR-Cas systems are prokaryotic immune systems against invading nucleic acids. Type I CRISPR-Cas systems employ highly diverse, multi-subunit surveillance Cascade complexes that facilitate duplex formation between crRNA and complementary target DNA for R-loop formation, retention, and DNA degradation by the subsequently recruited nuclease Cas3. Typically, the large subunit recognizes bona fide targets through the PAM (protospacer adjacent motif), and the small subunit guides the non-target DNA strand. Here, we present the Apo- and target-DNA-bound structures of the I-Fv (type I-F variant) Cascade lacking the small and large subunits. Large and small subunits are functionally replaced by the 5' terminal crRNA cap Cas5fv and the backbone protein Cas7fv, respectively. Cas5fv facilitates PAM recognition from the DNA major groove site, in contrast to all other described type I systems. Comparison of the type I-Fv Cascade with an anti-CRISPR protein-bound I-F Cascade reveals that the type I-Fv structure differs substantially at known anti-CRISPR protein target sites and might therefore be resistant to viral Cascade interception. Copyright © 2017 Elsevier Inc. All rights reserved.

Cytogenetic Analysis of Populus trichocarpa - Ribosomal DNA, Telomere Repeat Sequence, and Marker-selected BACs

Treesearch

M.N. lslam-Faridi; C.D. Nelson; S.P. DiFazio; L.E. Gunter; G.A. Tuskan

2009-01-01

The 185-285 rDNA and 55 rDNA loci in Populus trichocarpa were localized using fluorescent in situ hybridization (FISH). Two 185-285 rDNA sites and one 55 rDNA site were identified and located at the ends of 3 different chromosomes. FISH signals from the Arabidopsis-type telomere repeat sequence were observed at the distal ends of each chromosome. Six BAC clones...

A Tandemly Arranged Pattern of Two 5S rDNA Arrays in Amolops mantzorum (Anura, Ranidae).

PubMed

Liu, Ting; Song, Menghuan; Xia, Yun; Zeng, Xiaomao

2017-01-01

In an attempt to extend the knowledge of the 5S rDNA organization in anurans, the 5S rDNA sequences of Amolops mantzorum were isolated, characterized, and mapped by FISH. Two forms of 5S rDNA, type I (209 bp) and type II (about 870 bp), were found in specimens investigated from various populations. Both of them contained a 118-bp coding sequence, readily differentiated by their non-transcribed spacer (NTS) sizes and compositions. Four probes (the 5S rDNA coding sequences, the type I NTS, the type II NTS, and the entire type II 5S rDNA sequences) were respectively labeled with TAMRA or digoxigenin to hybridize with mitotic chromosomes for samples of all localities. It turned out that all probes showed the same signals that appeared in every centromeric region and in the telomeric regions of chromosome 5, without differences within or between populations. Obviously, both type I and type II of the 5S rDNA arrays arranged in tandem, which was contrasting with other frogs or fishes recorded to date. More interestingly, all the probes detected centromeric regions in all karyotypes, suggesting the presence of a satellite DNA family derived from 5S rDNA. © 2017 S. Karger AG, Basel.

Amino acid-dependent signaling via S6K1 and MYC is essential for regulation of rDNA transcription

PubMed Central

Kang, Jian; Kusnadi, Eric P.; Ogden, Allison J.; Hicks, Rodney J.; Bammert, Lukas; Kutay, Ulrike; Hung, Sandy; Sanij, Elaine; Hannan, Ross D.; Hannan, Katherine M.; Pearson, Richard B.

2016-01-01

Dysregulation of RNA polymerase I (Pol I)-dependent ribosomal DNA (rDNA) transcription is a consistent feature of malignant transformation that can be targeted to treat cancer. Understanding how rDNA transcription is coupled to the availability of growth factors and nutrients will provide insight into how ribosome biogenesis is maintained in a tumour environment characterised by limiting nutrients. We demonstrate that modulation of rDNA transcription initiation, elongation and rRNA processing is an immediate, co-regulated response to altered amino acid abundance, dependent on both mTORC1 activation of S6K1 and MYC activity. Growth factors regulate rDNA transcription initiation while amino acids modulate growth factor-dependent rDNA transcription by primarily regulating S6K1-dependent rDNA transcription elongation and processing. Thus, we show for the first time amino acids regulate rRNA synthesis by a distinct, post-initiation mechanism, providing a novel model for integrated control of ribosome biogenesis that has implications for understanding how this process is dysregulated in cancer. PMID:27385002

Identification of a Recently Active Mammalian SINE Derived from Ribosomal RNA

PubMed Central

Longo, Mark S.; Brown, Judy D.; Zhang, Chu; O’Neill, Michael J.; O’Neill, Rachel J.

2015-01-01

Complex eukaryotic genomes are riddled with repeated sequences whose derivation does not coincide with phylogenetic history and thus is often unknown. Among such sequences, the capacity for transcriptional activity coupled with the adaptive use of reverse transcription can lead to a diverse group of genomic elements across taxa, otherwise known as selfish elements or mobile elements. Short interspersed nuclear elements (SINEs) are nonautonomous mobile elements found in eukaryotic genomes, typically derived from cellular RNAs such as tRNAs, 7SL or 5S rRNA. Here, we identify and characterize a previously unknown SINE derived from the 3′-end of the large ribosomal subunit (LSU or 28S rDNA) and transcribed via RNA polymerase III. This new element, SINE28, is represented in low-copy numbers in the human reference genome assembly, wherein we have identified 27 discrete loci. Phylogenetic analysis indicates these elements have been transpositionally active within primate lineages as recently as 6 MYA while modern humans still carry transcriptionally active copies. Moreover, we have identified SINE28s in all currently available assembled mammalian genome sequences. Phylogenetic comparisons indicate that these elements are frequently rederived from the highly conserved LSU rRNA sequences in a lineage-specific manner. We propose that this element has not been previously recognized as a SINE given its high identity to the canonical LSU, and that SINE28 likely represents one of possibly many unidentified, active transposable elements within mammalian genomes. PMID:25637222

DNA barcoding revealed Nematodospora valgi gen. nov., sp. nov. and Candida cetoniae sp. nov. in the Lodderomyces clade.

PubMed

Gouliamova, Dilnora E; Dimitrov, Roumen A; Smith, Maudy Th; Groenewald, Marizeth; Stoilova-Disheva, Margarita M; Guéorguiev, Borislav V; Boekhout, Teun

2016-02-01

During a yeast biodiversity survey conducted in 2009-2011 in Bulgaria (South Eastern Europe) five strains of a novel ascomycetous yeast species were isolated from the beetle Valgus hemipterus (Cetoniinae) collected from two localities, namely Osogovska Planina Mountain and Nature Park Zlatni Pyasatsi. Phylogenetic analysis using combined sequences of the D1/D2 domains of the large subunit ribosomal DNA (LSU rDNA) and the internal transcribed spacers 1 + 2 regions (ITS1+2) placed the novel species on a separate branch near the basal part of the Lodderomyces clade. The novel species has a unique ascospore morphology distinct from those of the closely related teleomorphic genus Lodderomyces. Based on phylogenetic analysis and morphology of the ascospores we propose Nematodospora valgi gen. nov., sp. nov. to accommodate these isolates (MB811804 D37S(T), MB802458). Two strains of a novel anamorphic yeast species were isolated from the beetles Cetonia aurata and Oxythyrea funesta (Cetoniinae) collected in East Rhodopies and Sofia city, respectively. DNA barcoding analysis placed the new yeast species within the Candida parapsilosis subclade. Here, we present the description of a new yeast species, Candida cetoniae sp. nov. (IMB1R2(T), MB803501) to accommodate these two strains. The ecology and biogeography of the insect-associated yeasts of the Lodderomyces clade is discussed. Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

DNA Barcoding Green Microalgae Isolated from Neotropical Inland Waters

PubMed Central

Hadi, Sámed I. I. A.; Santana, Hugo; Brunale, Patrícia P. M.; Gomes, Taísa G.; Oliveira, Márcia D.; Matthiensen, Alexandre; Oliveira, Marcos E. C.; Silva, Flávia C. P.; Brasil, Bruno S. A. F.

2016-01-01

This study evaluated the feasibility of using the Ribulose Bisphosphate Carboxylase Large subunit gene (rbcL) and the Internal Transcribed Spacers 1 and 2 of the nuclear rDNA (nuITS1 and nuITS2) markers for identifying a very diverse, albeit poorly known group, of green microalgae from neotropical inland waters. Fifty-one freshwater green microalgae strains isolated from Brazil, the largest biodiversity reservoir in the neotropics, were submitted to DNA barcoding. Currently available universal primers for ITS1-5.8S-ITS2 region amplification were sufficient to successfully amplify and sequence 47 (92%) of the samples. On the other hand, new sets of primers had to be designed for rbcL, which allowed 96% of the samples to be sequenced. Thirty-five percent of the strains could be unambiguously identified to the species level based either on nuITS1 or nuITS2 sequences’ using barcode gap calculations. nuITS2 Compensatory Base Change (CBC) and ITS1-5.8S-ITS2 region phylogenetic analysis, together with morphological inspection, confirmed the identification accuracy. In contrast, only 6% of the strains could be assigned to the correct species based solely on rbcL sequences. In conclusion, the data presented here indicates that either nuITS1 or nuITS2 are useful markers for DNA barcoding of freshwater green microalgae, with advantage for nuITS2 due to the larger availability of analytical tools and reference barcodes deposited at databases for this marker. PMID:26900844

Composition of the root mycorrhizal community associated with Coffea arabica in Fifa Mountains (Jazan region, Saudi Arabia).

PubMed

Mahdhi, Mosbah; Tounekti, Taieb; Al-Turki, Turki Ali; Khemira, Habib

2017-08-01

Arbuscular mycorrhizal fungi (AMF) constitute a key functional group of soil biota that can greatly contribute to crop productivity and ecosystem sustainability. They improve nutrient uptake and enhance the ability of plants to cope with abiotic stresses. The presence of AMF in coffee (Coffea arabica L.) plant roots have been reported in several locations but not in Saudi Arabia despite the fact that coffee has been in cultivation here since ancient times. The objective of the present study was to investigate the diversity of AMF communities colonizing the roots of coffee trees growing in two sites of Fifa Mountains (south-west Saudi Arabia): site 1 at 700 m altitude and site 2 at 1400 m. The AMF large subunit rDNA regions (LSU) were subjected to nested PCR, cloning, sequencing, and phylogenetic analysis. Microscopic observations indicated higher mycorrhizal intensity (24.3%) and spore density (256 spores/100 g of soil) in site 2 (higher altitude). Phylogenetic analysis revealed 10 phylotypes, six belonging to the family Glomeraceae, two to Claroideoglomercea, one to Acaulosporaceae and one to Gigasporaceae family. Glomus was the dominant genus at both sites and the genus Gigaspora was detected only at site 2. This is the first study reporting the presence of AMF in coffee roots and the composition of this particular mycorrhizal community in Saudi Arabia. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Botryosphaeriaceae associated with the die-back of ornamental trees in the Western Balkans.

PubMed

Zlatković, Milica; Keča, Nenad; Wingfield, Michael J; Jami, Fahimeh; Slippers, Bernard

2016-04-01

Extensive die-back and mortality of various ornamental trees and shrubs has been observed in parts of the Western Balkans region during the past decade. The disease symptoms have been typical of those caused by pathogens residing in the Botryosphaeriaceae. The aims of this study were to isolate and characterize Botryosphaeriaceae species associated with diseased ornamental trees in Serbia, Montenegro, Bosnia and Herzegovina. Isolates were initially characterized based on the DNA sequence data for the internal transcribed spacer rDNA and six major clades were identified. Representative isolates from each clade were further characterized using DNA sequence data for the translation elongation factor 1-alpha, β-tubulin-2 and large subunit rRNA gene regions, as well as the morphology of the asexual morphs. Ten species of the Botryosphaeriaceae were identified of which eight, i.e., Dothiorella sarmentorum, Neofusicoccum parvum, Botryosphaeria dothidea, Phaeobotryon cupressi, Sphaeropsis visci, Diplodia seriata, D. sapinea and D. mutila were known taxa. The remaining two species could be identified only as Dothiorella spp. Dichomera syn-asexual morphs of D. sapinea, Dothiorella sp. 2 and B. dothidea, as well as unique morphological characters for a number of the known species are described. Based on host plants and geographic distribution, the majority of Botryosphaeriaceae species found represent new records. The results of this study contribute to our knowledge of the distribution, host associations and impacts of these fungi on trees in urban environments.

The Methanol Dehydrogenase Gene, mxaF, as a Functional and Phylogenetic Marker for Proteobacterial Methanotrophs in Natural Environments

PubMed Central

Lau, Evan; Fisher, Meredith C.; Steudler, Paul A.; Cavanaugh, Colleen M.

2013-01-01

The mxaF gene, coding for the large (α) subunit of methanol dehydrogenase, is highly conserved among distantly related methylotrophic species in the Alpha-, Beta- and Gammaproteobacteria. It is ubiquitous in methanotrophs, in contrast to other methanotroph-specific genes such as the pmoA and mmoX genes, which are absent in some methanotrophic proteobacterial genera. This study examined the potential for using the mxaF gene as a functional and phylogenetic marker for methanotrophs. mxaF and 16S rRNA gene phylogenies were constructed based on over 100 database sequences of known proteobacterial methanotrophs and other methylotrophs to assess their evolutionary histories. Topology tests revealed that mxaF and 16S rDNA genes of methanotrophs do not show congruent evolutionary histories, with incongruencies in methanotrophic taxa in the Methylococcaceae, Methylocystaceae, and Beijerinckiacea. However, known methanotrophs generally formed coherent clades based on mxaF gene sequences, allowing for phylogenetic discrimination of major taxa. This feature highlights the mxaF gene’s usefulness as a biomarker in studying the molecular diversity of proteobacterial methanotrophs in nature. To verify this, PCR-directed assays targeting this gene were used to detect novel methanotrophs from diverse environments including soil, peatland, hydrothermal vent mussel tissues, and methanotroph isolates. The placement of the majority of environmental mxaF gene sequences in distinct methanotroph-specific clades (Methylocystaceae and Methylococcaceae) detected in this study supports the use of mxaF as a biomarker for methanotrophic proteobacteria. PMID:23451130

UBF complexes with phosphatidylinositol 4,5-bisphosphate in nucleolar organizer regions regardless of ongoing RNA polymerase I activity

PubMed Central

Sobol, Margarita; Yildirim, Sukriye; Philimonenko, Vlada V; Marášek, Pavel; Castaño, Enrique; Hozák, Pavel

2013-01-01

To maintain growth and division, cells require a large-scale production of rRNAs which occurs in the nucleolus. Recently, we have shown the interaction of nucleolar phosphatidylinositol 4,5-bisphosphate (PIP2) with proteins involved in rRNA transcription and processing, namely RNA polymerase I (Pol I), UBF, and fibrillarin. Here we extend the study by investigating transcription-related localization of PIP2 in regards to transcription and processing complexes of Pol I. To achieve this, we used either physiological inhibition of transcription during mitosis or inhibition by treatment the cells with actinomycin D (AMD) or 5,6-dichloro-1β-d-ribofuranosyl-benzimidazole (DRB). We show that PIP2 is associated with Pol I subunits and UBF in a transcription-independent manner. On the other hand, PIP2/fibrillarin colocalization is dependent on the production of rRNA. These results indicate that PIP2 is required not only during rRNA production and biogenesis, as we have shown before, but also plays a structural role as an anchor for the Pol I pre-initiation complex during the cell cycle. We suggest that throughout mitosis, PIP2 together with UBF is involved in forming and maintaining the core platform of the rDNA helix structure. Thus we introduce PIP2 as a novel component of the NOR complex, which is further engaged in the renewed rRNA synthesis upon exit from mitosis. PMID:24513678

How the structure of the large subunit controls function in an oxygen-tolerant [NiFe]-hydrogenase

PubMed Central

Bowman, Lisa; Flanagan, Lindsey; Fyfe, Paul K.; Parkin, Alison; Hunter, William N.; Sargent, Frank

2014-01-01

Salmonella enterica is an opportunistic pathogen that produces a [NiFe]-hydrogenase under aerobic conditions. In the present study, genetic engineering approaches were used to facilitate isolation of this enzyme, termed Hyd-5. The crystal structure was determined to a resolution of 3.2 Å and the hydro-genase was observed to comprise associated large and small subunits. The structure indicated that His229 from the large subunit was close to the proximal [4Fe–3S] cluster in the small subunit. In addition, His229 was observed to lie close to a buried glutamic acid (Glu73), which is conserved in oxygen-tolerant hydrogenases. His229 and Glu73 of the Hyd-5 large subunit were found to be important in both hydrogen oxidation activity and the oxygen-tolerance mechanism. Substitution of His229 or Glu73 with alanine led to a loss in the ability of Hyd-5 to oxidize hydrogen in air. Furthermore, the H229A variant was found to have lost the overpotential requirement for activity that is always observed with oxygen-tolerant [NiFe]-hydrogenases. It is possible that His229 has a role in stabilizing the super-oxidized form of the proximal cluster in the presence of oxygen, and it is proposed that Glu73could play a supporting role in fine-tuning the chemistry of His229 to enable this function. PMID:24428762

The karyotype of Nothoscordum arenarium Herter (Gilliesioideae, Alliaceae): A populational and cytomolecular analysis

PubMed Central

2009-01-01

The genus Nothoscordum Kunth comprises approximately 20 species native to South America. Karyologically, the genus is remarkable for its large chromosomes and Robertsonian translocations. Variation in chromosome number has been recorded in a few polyploid species and it is unknown among diploids. This study presents the chromosome number and morphology of 53 individuals of seven populations of N. arenarium Herter (2n = 10). In addition, karyotype analyses after C-banding, staining with CMA and DAPI, and in situ hybridization with 5S and 45S rDNA probes were performed in six individuals from one population. All individuals exhibited 2n = 10 (6M + 4A), except for one tetraploid (2n = 20, 12M + 8A) and one triploid (2n = 15, 9M + 6A) plant. C-banding revealed the presence of CMA+ /DAPI - heterochromatin in the short arm and in the proximal region of the long arm of all acrocentric chromosomes. The 45S rDNA sites co-localized with the CMA + regions of the acrocentrics short arms, while the 5S rDNA probe only hybridized with the subterminal region of a pair of metacentric chromosomes. A change in the pattern of CMA bands and rDNA sites was observed in only one individual bearing a reciprocal translocation involving the long arm of a metacentric and the long arm of an acrocentric chromosome. These data suggest that, despite isolated cases of polyploidy and translocation, the karyotype of N. arenarium is very stable and the karyotypic instability described for other species may be associated with their polyploid condition. PMID:21637654

Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit N-methyltransferase

DOEpatents

Houtz, Robert L.

1998-01-01

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .epsilon.N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the .epsilon.-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed.

Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase

DOEpatents

Houtz, Robert L.

1999-01-01

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the .epsilon.-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed.

Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit N-methyltransferase

DOEpatents

Houtz, R.L.

1998-03-03

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) {epsilon}N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the {epsilon}-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed. 5 figs.

Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit epsilon N-methyltransferase

DOEpatents

Houtz, R.L.

1999-02-02

The gene sequence for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS){sup {epsilon}}N-methyltransferase (protein methylase III or Rubisco LSMT) is disclosed. This enzyme catalyzes methylation of the {epsilon}-amine of lysine-14 in the large subunit of Rubisco. In addition, a full-length cDNA clone for Rubisco LSMT is disclosed. Transgenic plants and methods of producing same which (1) have the Rubisco LSMT gene inserted into the DNA, and (2) have the Rubisco LSMT gene product or the action of the gene product deleted from the DNA are also provided. Further, methods of using the gene to selectively deliver desired agents to a plant are also disclosed. 8 figs.

Myc-induced anchorage of the rDNA IGS region to nucleolar matrix modulates growth-stimulated changes in higher-order rDNA architecture

PubMed Central

Shiue, Chiou-Nan; Nematollahi-Mahani, Amir; Wright, Anthony P.H.

2014-01-01

Chromatin domain organization and the compartmentalized distribution of chromosomal regions are essential for packaging of deoxyribonucleic acid (DNA) in the eukaryotic nucleus as well as regulated gene expression. Nucleoli are the most prominent morphological structures of cell nuclei and nucleolar organization is coupled to cell growth. It has been shown that nuclear scaffold/matrix attachment regions often define the base of looped chromosomal domains in vivo and that they are thereby critical for correct chromosome architecture and gene expression. Here, we show regulated organization of mammalian ribosomal ribonucleic acid genes into distinct chromatin loops by tethering to nucleolar matrix via the non-transcribed inter-genic spacer region of the ribosomal DNA (rDNA). The rDNA gene loop structures are induced specifically upon growth stimulation and are dependent on the activity of the c-Myc protein. Matrix-attached rDNA genes are hypomethylated at the promoter and are thus available for transcriptional activation. rDNA genes silenced by methylation are not recruited to the matrix. c-Myc, which has been shown to induce rDNA transcription directly, is physically associated with rDNA gene looping structures and the intergenic spacer sequence in growing cells. Such a role of Myc proteins in gene activation has not been reported previously. PMID:24609384

Myc-induced anchorage of the rDNA IGS region to nucleolar matrix modulates growth-stimulated changes in higher-order rDNA architecture.

PubMed

Shiue, Chiou-Nan; Nematollahi-Mahani, Amir; Wright, Anthony P H

2014-05-01

Chromatin domain organization and the compartmentalized distribution of chromosomal regions are essential for packaging of deoxyribonucleic acid (DNA) in the eukaryotic nucleus as well as regulated gene expression. Nucleoli are the most prominent morphological structures of cell nuclei and nucleolar organization is coupled to cell growth. It has been shown that nuclear scaffold/matrix attachment regions often define the base of looped chromosomal domains in vivo and that they are thereby critical for correct chromosome architecture and gene expression. Here, we show regulated organization of mammalian ribosomal ribonucleic acid genes into distinct chromatin loops by tethering to nucleolar matrix via the non-transcribed inter-genic spacer region of the ribosomal DNA (rDNA). The rDNA gene loop structures are induced specifically upon growth stimulation and are dependent on the activity of the c-Myc protein. Matrix-attached rDNA genes are hypomethylated at the promoter and are thus available for transcriptional activation. rDNA genes silenced by methylation are not recruited to the matrix. c-Myc, which has been shown to induce rDNA transcription directly, is physically associated with rDNA gene looping structures and the intergenic spacer sequence in growing cells. Such a role of Myc proteins in gene activation has not been reported previously. © 2014 The Author(s). Published by Oxford University Press [on behalf of Nucleic Acids Research].

Noncoding transcripts in sense and antisense orientation regulate the epigenetic state of ribosomal RNA genes.

PubMed

Bierhoff, H; Schmitz, K; Maass, F; Ye, J; Grummt, I

2010-01-01

Alternative transcription of the same gene in sense and antisense orientation regulates expression of protein-coding genes. Here we show that noncoding RNA (ncRNA) in sense and antisense orientation also controls transcription of rRNA genes (rDNA). rDNA exists in two types of chromatin--a euchromatic conformation that is permissive to transcription and a heterochromatic conformation that is transcriptionally silent. Silencing of rDNA is mediated by NoRC, a chromatin-remodeling complex that triggers heterochromatin formation. NoRC function requires RNA that is complementary to the rDNA promoter (pRNA). pRNA forms a DNA:RNA triplex with a regulatory element in the rDNA promoter, and this triplex structure is recognized by DNMT3b. The results imply that triplex-mediated targeting of DNMT3b to specific sequences may be a common pathway in epigenetic regulation. We also show that rDNA is transcribed in antisense orientation. The level of antisense RNA (asRNA) is down-regulated in cancer cells and up-regulated in senescent cells. Ectopic asRNA triggers trimethylation of histone H4 at lysine 20 (H4K20me3), suggesting that antisense transcripts guide the histone methyltransferase Suv4-20 to rDNA. The results reveal that noncoding RNAs in sense and antisense orientation are important determinants of the epigenetic state of rDNA.

A Portrait of Ribosomal DNA Contacts with Hi-C Reveals 5S and 45S rDNA Anchoring Points in the Folded Human Genome.

PubMed

Yu, Shoukai; Lemos, Bernardo

2016-12-31

Ribosomal RNAs (rRNAs) account for >60% of all RNAs in eukaryotic cells and are encoded in the ribosomal DNA (rDNA) arrays. The rRNAs are produced from two sets of loci: the 5S rDNA array resides exclusively on human chromosome 1, whereas the 45S rDNA array resides on the short arm of five human acrocentric chromosomes. The 45S rDNA gives origin to the nucleolus, the nuclear organelle that is the site of ribosome biogenesis. Intriguingly, 5S and 45S rDNA arrays exhibit correlated copy number variation in lymphoblastoid cells (LCLs). Here we examined the genomic architecture and repeat content of the 5S and 45S rDNA arrays in multiple human genome assemblies (including PacBio MHAP assembly) and ascertained contacts between the rDNA arrays and the rest of the genome using Hi-C datasets from two human cell lines (erythroleukemia K562 and lymphoblastoid cells). Our analyses revealed that 5S and 45S arrays each have thousands of contacts in the folded genome, with rDNA-associated regions and genes dispersed across all chromosomes. The rDNA contact map displayed conserved and disparate features between two cell lines, and pointed to specific chromosomes, genomic regions, and genes with evidence of spatial proximity to the rDNA arrays; the data also showed a lack of direct physical interaction between the 5S and 45S rDNA arrays. Finally, the analysis identified an intriguing organization in the 5S array with Alu and 5S elements adjacent to one another and organized in opposite orientation along the array. Portraits of genome folding centered on the ribosomal DNA array could help understand the emergence of concerted variation, the control of 5S and 45S expression, as well as provide insights into an organelle that contributes to the spatial localization of human chromosomes during interphase. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Evolutional dynamics of 45S and 5S ribosomal DNA in ancient allohexaploid Atropa belladonna.

PubMed

Volkov, Roman A; Panchuk, Irina I; Borisjuk, Nikolai V; Hosiawa-Baranska, Marta; Maluszynska, Jolanta; Hemleben, Vera

2017-01-23

Polyploid hybrids represent a rich natural resource to study molecular evolution of plant genes and genomes. Here, we applied a combination of karyological and molecular methods to investigate chromosomal structure, molecular organization and evolution of ribosomal DNA (rDNA) in nightshade, Atropa belladonna (fam. Solanaceae), one of the oldest known allohexaploids among flowering plants. Because of their abundance and specific molecular organization (evolutionarily conserved coding regions linked to variable intergenic spacers, IGS), 45S and 5S rDNA are widely used in plant taxonomic and evolutionary studies. Molecular cloning and nucleotide sequencing of A. belladonna 45S rDNA repeats revealed a general structure characteristic of other Solanaceae species, and a very high sequence similarity of two length variants, with the only difference in number of short IGS subrepeats. These results combined with the detection of three pairs of 45S rDNA loci on separate chromosomes, presumably inherited from both tetraploid and diploid ancestor species, example intensive sequence homogenization that led to substitution/elimination of rDNA repeats of one parent. Chromosome silver-staining revealed that only four out of six 45S rDNA sites are frequently transcriptionally active, demonstrating nucleolar dominance. For 5S rDNA, three size variants of repeats were detected, with the major class represented by repeats containing all functional IGS elements required for transcription, the intermediate size repeats containing partially deleted IGS sequences, and the short 5S repeats containing severe defects both in the IGS and coding sequences. While shorter variants demonstrate increased rate of based substitution, probably in their transition into pseudogenes, the functional 5S rDNA variants are nearly identical at the sequence level, pointing to their origin from a single parental species. Localization of the 5S rDNA genes on two chromosome pairs further supports uniparental inheritance from the tetraploid progenitor. The obtained molecular, cytogenetic and phylogenetic data demonstrate complex evolutionary dynamics of rDNA loci in allohexaploid species of Atropa belladonna. The high level of sequence unification revealed in 45S and 5S rDNA loci of this ancient hybrid species have been seemingly achieved by different molecular mechanisms.

Identification of a Kinase in Wheat Germ that Phosphorylates the Large Subunit of Initiation Factor 4F 1

PubMed Central

Humphreys, Jean; Browning, Karen S.; Ravel, Joanne M.

1988-01-01

A kinase has been isolated from wheat (Triticum aestivum) germ that phosphorylates the 220 kilodaltons (kD) subunit of wheat germ initiation factor (eIF) 4F, the 80 kD subunit of eIF-4B (an isozyme form of eIF-4F) and eIF-4G (the functional equivalent to mammalian eIF-4B). The kinase elutes from Sephacryl S-200 slightly in front of ovalbumin. The kinase phosphorylates casein and histone IIA to a small extent, but does not phosphorylate phosvitin. Of the wheat germ initiation factors, elongation factors, and small and large ribosomal subunits, only eIF-4F, eIF-4B, and eIF-4G are phosphorylated to a significant extent. The kinase phosphorylates eIF-4F to the extent of two phosphates per mole of the 220 kD subunit and phosphorylates eIF-4B to the extent of one phosphate per mole of the 80 kD subunit. The 26 kD subunit of eIF-4F and the 28 kD subunit of eIF-4B are not phosphorylated by the kinase. The kinase phosphorylates the 59 kD component of eIF-4G to the extent of 0.25 phosphate per mole of eIF-4G. Phosphorylation of eIF-4F and eIF-4B does not affect their ability to support the binding of mRNA to small ribosomal subunits in vitro. Images Fig. 2 Fig. 3 PMID:16666331

Multiple effects of S13 in modulating the strength of intersubunit interactions in the ribosome during translation.

PubMed

Cukras, Anthony R; Green, Rachel

2005-05-27

The ribosomal protein S13 is found in the head region of the small subunit, where it interacts with the central protuberance of the large ribosomal subunit and with the P site-bound tRNA through its extended C terminus. The bridging interactions between the large and small subunits are dynamic, and are thought to be critical in orchestrating the molecular motions of the translation cycle. S13 provides a direct link between the tRNA-binding site and the movements in the head of the small subunit seen during translocation, thereby providing a possible pathway of signal transduction. We have created and characterized an rpsM(S13)-deficient strain of Escherichia coli and have found significant defects in subunit association, initiation and translocation through in vitro assays of S13-deficient ribosomes. Targeted mutagenesis of specific bridge and tRNA contact elements in S13 provides evidence that these two interaction domains play critical roles in maintaining the fidelity of translation. This ribosomal protein thus appears to play a non-essential, yet important role by modulating subunit interactions in multiple steps of the translation cycle.

BEND3 represses rDNA transcription by stabilizing a NoRC component via USP21 deubiquitinase

PubMed Central

Khan, Abid; Giri, Sumanprava; Wang, Yating; Chakraborty, Arindam; Ghosh, Archit K.; Anantharaman, Aparna; Aggarwal, Vasudha; Sathyan, Kizhakke M.; Ha, Taekjip; Prasanth, Kannanganattu V.; Prasanth, Supriya G.

2015-01-01

Ribosome biogenesis dictates the translational capacity of cells. Several mechanisms establish and maintain transcriptional output from eukaryotic ribosomal DNA (rDNA) loci. rDNA silencing is one such mechanism that ensures the inactivity and hence the maintenance of a silenced state of a subset of rRNA gene copies. Whereas oncogenic agents stimulate rRNA gene transcription, tumor suppressors decrease rRNA gene transcription. We demonstrate in mammalian cells that BANP, E5R, and Nac1 (BEN) domain 3 (BEND3), a quadruple BEN domain-containing protein, localizes in nucleoli and binds to ribosomal RNA gene promoters to help repress rRNA genes. Loss of BEND3 increases histone H3K4 trimethylation and, correspondingly, decreases rDNA promoter DNA methylation, consistent with a role for BEND3 in rDNA silencing. BEND3 associates with the nucleolar-remodeling complex (NoRC), and SUMOylated BEND3 stabilizes NoRC component TTF-1–interacting protein 5 via association with ubiquitin specific protease 21 (USP21) debiquitinase. Our results provide mechanistic insights into how the novel rDNA transcription repressor BEND3 acts together with NoRC to actively coordinate the establishment of rDNA silencing. PMID:26100909

BEND3 represses rDNA transcription by stabilizing a NoRC component via USP21 deubiquitinase.

PubMed

Khan, Abid; Giri, Sumanprava; Wang, Yating; Chakraborty, Arindam; Ghosh, Archit K; Anantharaman, Aparna; Aggarwal, Vasudha; Sathyan, Kizhakke M; Ha, Taekjip; Prasanth, Kannanganattu V; Prasanth, Supriya G

2015-07-07

Ribosome biogenesis dictates the translational capacity of cells. Several mechanisms establish and maintain transcriptional output from eukaryotic ribosomal DNA (rDNA) loci. rDNA silencing is one such mechanism that ensures the inactivity and hence the maintenance of a silenced state of a subset of rRNA gene copies. Whereas oncogenic agents stimulate rRNA gene transcription, tumor suppressors decrease rRNA gene transcription. We demonstrate in mammalian cells that BANP, E5R, and Nac1 (BEN) domain 3 (BEND3), a quadruple BEN domain-containing protein, localizes in nucleoli and binds to ribosomal RNA gene promoters to help repress rRNA genes. Loss of BEND3 increases histone H3K4 trimethylation and, correspondingly, decreases rDNA promoter DNA methylation, consistent with a role for BEND3 in rDNA silencing. BEND3 associates with the nucleolar-remodeling complex (NoRC), and SUMOylated BEND3 stabilizes NoRC component TTF-1-interacting protein 5 via association with ubiquitin specific protease 21 (USP21) debiquitinase. Our results provide mechanistic insights into how the novel rDNA transcription repressor BEND3 acts together with NoRC to actively coordinate the establishment of rDNA silencing.

Homogeneity of the 16S rDNA sequence among geographically disparate isolates of Taylorella equigenitalis

PubMed Central

Matsuda, M; Tazumi, A; Kagawa, S; Sekizuka, T; Murayama, O; Moore, JE; Millar, BC

2006-01-01

Background At present, six accessible sequences of 16S rDNA from Taylorella equigenitalis (T. equigenitalis) are available, whose sequence differences occur at a few nucleotide positions. Thus it is important to determine these sequences from additional strains in other countries, if possible, in order to clarify any anomalies regarding 16S rDNA sequence heterogeneity. Here, we clone and sequence the approximate full-length 16S rDNA from additional strains of T. equigenitalis isolated in Japan, Australia and France and compare these sequences to the existing published sequences. Results Clarification of any anomalies regarding 16S rDNA sequence heterogeneity of T. equigenitalis was carried out. When cloning, sequencing and comparison of the approximate full-length 16S rDNA from 17 strains of T. equigenitalis isolated in Japan, Australia and France, nucleotide sequence differences were demonstrated at the six loci in the 1,469 nucleotide sequence. Moreover, 12 polymorphic sites occurred among 23 sequences of the 16S rDNA, including the six reference sequences. Conclusion High sequence similarity (99.5% or more) was observed throughout, except from nucleotide positions 138 to 501 where substitutions and deletions were noted. PMID:16398935

FISH-aimed karyotyping and characterization of Renner complexes in permanent heterozygote Rhoeo spathacea.

PubMed

Golczyk, Hieronim; Hasterok, Robert; Joachimiak, Andrzej J

2005-02-01

Fluorescence in situ hybridization (FISH) using 25S rDNA, 5S rDNA, and telomere sequences as probes was carried out in the complex permanent heterozygote Rhoeo spathacea. Telomere sites were exclusively terminal. All 10 25S rDNA loci were located distally and appeared transcriptionally active after silver staining. Six distal and 2 interstitial 5S rDNA sites were detected; 2 of the distal sites strictly colocalized with 25S rDNA loci. The 2 intercalary 5S rDNA loci occurred in short arms of 2 chromosomes that conjoined at meiosis. Chromosomes differed as to the amount of AT-rich centric heterochromatin, suggesting involvement of pericentromeric regions in translocations. The possibility of Robertsonian-like rearrangements was discussed. Double target FISH with ribosomal probes along with DAPI fluorescence gave the basis for full chromosome identification in mitosis. The 2 Renner complexes are structurally balanced, both having 5 25S and 4 5S rDNA sites. Centromere clustering, telomere association, a high number of NOR sites, and a strong tendency for formation of joint nucleoli contribute to the preservation of highly polarized Rabl arrangement at interphase. These findings were discussed in relation to meiotic catenation in Rhoeo.

Deletion of Marek’s disease virus large subunit of ribonucleotide reductase (RR) impairs virus growth in vitro and in vivo

USDA-ARS?s Scientific Manuscript database

Marek’s disease virus (MDV), a highly cell-associated lymphotropic alphaherpesvirus, is the causative agent of a neoplastic disease in domestic chickens, called Marek’s disease (MD). In the unique long region of the MDV genome, open reading frames UL39 and UL40 encode the large and small subunits o...

Marek’s Disease Virus Encoded Ribonucleotide Reductase Large Subunit is not Essential for In Vitro Replication

USDA-ARS?s Scientific Manuscript database

Marek’s disease virus (MDV) infected cells express a viral ribonucleotide reductase (RR) that is distinguishable from that present in uninfected cells by monoclonal antibody T81. Open reading frames UL39 and UL40 of the MDV genome encode the large (RR1) and small (RR2) subunits of RR enzyme, respe...

SSU rDNA divergence in planktonic foraminifera: molecular taxonomy and biogeographic implications.

PubMed

André, Aurore; Quillévéré, Frédéric; Morard, Raphaël; Ujiié, Yurika; Escarguel, Gilles; de Vargas, Colomban; de Garidel-Thoron, Thibault; Douady, Christophe J

2014-01-01

The use of planktonic foraminifera in paleoceanography requires taxonomic consistency and precise assessment of the species biogeography. Yet, ribosomal small subunit (SSUr) DNA analyses have revealed that most of the modern morpho-species of planktonic foraminifera are composed of a complex of several distinct genetic types that may correspond to cryptic or pseudo-cryptic species. These genetic types are usually delimitated using partial sequences located at the 3'end of the SSUrDNA, but typically based on empirical delimitation. Here, we first use patristic genetic distances calculated within and among genetic types of the most common morpho-species to show that intra-type and inter-type genetic distances within morpho-species may significantly overlap, suggesting that genetic types have been sometimes inconsistently defined. We further apply two quantitative and independent methods, ABGD (Automatic Barcode Gap Detection) and GMYC (General Mixed Yule Coalescent) to a dataset of published and newly obtained partial SSU rDNA for a more objective assessment of the species status of these genetic types. Results of these complementary approaches are highly congruent and lead to a molecular taxonomy that ranks 49 genetic types of planktonic foraminifera as genuine (pseudo)cryptic species. Our results advocate for a standardized sequencing procedure allowing homogenous delimitations of (pseudo)cryptic species. On the ground of this revised taxonomic framework, we finally provide an integrative taxonomy synthesizing geographic, ecological and morphological differentiations that can occur among the genuine (pseudo)cryptic species. Due to molecular, environmental or morphological data scarcities, many aspects of our proposed integrative taxonomy are not yet fully resolved. On the other hand, our study opens up the potential for a correct interpretation of environmental sequence datasets.

SSU rDNA Divergence in Planktonic Foraminifera: Molecular Taxonomy and Biogeographic Implications

PubMed Central

André, Aurore; Quillévéré, Frédéric; Morard, Raphaël; Ujiié, Yurika; Escarguel, Gilles; de Vargas, Colomban; de Garidel-Thoron, Thibault; Douady, Christophe J.

2014-01-01

The use of planktonic foraminifera in paleoceanography requires taxonomic consistency and precise assessment of the species biogeography. Yet, ribosomal small subunit (SSUr) DNA analyses have revealed that most of the modern morpho-species of planktonic foraminifera are composed of a complex of several distinct genetic types that may correspond to cryptic or pseudo-cryptic species. These genetic types are usually delimitated using partial sequences located at the 3′end of the SSUrDNA, but typically based on empirical delimitation. Here, we first use patristic genetic distances calculated within and among genetic types of the most common morpho-species to show that intra-type and inter-type genetic distances within morpho-species may significantly overlap, suggesting that genetic types have been sometimes inconsistently defined. We further apply two quantitative and independent methods, ABGD (Automatic Barcode Gap Detection) and GMYC (General Mixed Yule Coalescent) to a dataset of published and newly obtained partial SSU rDNA for a more objective assessment of the species status of these genetic types. Results of these complementary approaches are highly congruent and lead to a molecular taxonomy that ranks 49 genetic types of planktonic foraminifera as genuine (pseudo)cryptic species. Our results advocate for a standardized sequencing procedure allowing homogenous delimitations of (pseudo)cryptic species. On the ground of this revised taxonomic framework, we finally provide an integrative taxonomy synthesizing geographic, ecological and morphological differentiations that can occur among the genuine (pseudo)cryptic species. Due to molecular, environmental or morphological data scarcities, many aspects of our proposed integrative taxonomy are not yet fully resolved. On the other hand, our study opens up the potential for a correct interpretation of environmental sequence datasets. PMID:25119900

Cryptic infection of a broad taxonomic and geographic diversity of tadpoles by Perkinsea protists.

PubMed

Chambouvet, Aurélie; Gower, David J; Jirků, Miloslav; Yabsley, Michael J; Davis, Andrew K; Leonard, Guy; Maguire, Finlay; Doherty-Bone, Thomas M; Bittencourt-Silva, Gabriela Bueno; Wilkinson, Mark; Richards, Thomas A

2015-08-25

The decline of amphibian populations, particularly frogs, is often cited as an example in support of the claim that Earth is undergoing its sixth mass extinction event. Amphibians seem to be particularly sensitive to emerging diseases (e.g., fungal and viral pathogens), yet the diversity and geographic distribution of infectious agents are only starting to be investigated. Recent work has linked a previously undescribed protist with mass-mortality events in the United States, in which infected frog tadpoles have an abnormally enlarged yellowish liver filled with protist cells of a presumed parasite. Phylogenetic analyses revealed that this infectious agent was affiliated with the Perkinsea: a parasitic group within the alveolates exemplified by Perkinsus sp., a "marine" protist responsible for mass-mortality events in commercial shellfish populations. Using small subunit (SSU) ribosomal DNA (rDNA) sequencing, we developed a targeted PCR protocol for preferentially sampling a clade of the Perkinsea. We tested this protocol on freshwater environmental DNA, revealing a wide diversity of Perkinsea lineages in these environments. Then, we used the same protocol to test for Perkinsea-like lineages in livers of 182 tadpoles from multiple families of frogs. We identified a distinct Perkinsea clade, encompassing a low level of SSU rDNA variation different from the lineage previously associated with tadpole mass-mortality events. Members of this clade were present in 38 tadpoles sampled from 14 distinct genera/phylogroups, from five countries across three continents. These data provide, to our knowledge, the first evidence that Perkinsea-like protists infect tadpoles across a wide taxonomic range of frogs in tropical and temperate environments, including oceanic islands.

Molecular Detection, Isolation, and Physiological Characterization of Functionally Dominant Phenol-Degrading Bacteria in Activated Sludge

PubMed Central

Watanabe, Kazuya; Teramoto, Maki; Futamata, Hiroyuki; Harayama, Shigeaki

1998-01-01

DNA was isolated from phenol-digesting activated sludge, and partial fragments of the 16S ribosomal DNA (rDNA) and the gene encoding the largest subunit of multicomponent phenol hydroxylase (LmPH) were amplified by PCR. An analysis of the amplified fragments by temperature gradient gel electrophoresis (TGGE) demonstrated that two major 16S rDNA bands (bands R2 and R3) and two major LmPH gene bands (bands P2 and P3) appeared after the activated sludge became acclimated to phenol. The nucleotide sequences of these major bands were determined. In parallel, bacteria were isolated from the activated sludge by direct plating or by plating after enrichment either in batch cultures or in a chemostat culture. The bacteria isolated were classified into 27 distinct groups by a repetitive extragenic palindromic sequence PCR analysis. The partial nucleotide sequences of 16S rDNAs and LmPH genes of members of these 27 groups were then determined. A comparison of these nucleotide sequences with the sequences of the major TGGE bands indicated that the major bacterial populations, R2 and R3, possessed major LmPH genes P2 and P3, respectively. The dominant populations could be isolated either by direct plating or by chemostat culture enrichment but not by batch culture enrichment. One of the dominant strains (R3) which contained a novel type of LmPH (P3), was closely related to Valivorax paradoxus, and the result of a kinetic analysis of its phenol-oxygenating activity suggested that this strain was the principal phenol digester in the activated sludge. PMID:9797297

Development of a rapid assay to detect the dinoflagellate Amyloodinium ocellatum using loop-mediated isothermal amplification (LAMP).

PubMed

Picón-Camacho, Sara M; Thompson, William P; Blaylock, Reginald B; Lotz, Jeffrey M

2013-09-23

Amyloodinium ocellatum is a highly pathogenic dinoflagellate parasite with global distribution that causes high mortalities in the culture of tropical and sub-tropical marine and estuarine fishes. Diagnosis typically occurs through gross examination following the onset of morbidity, at which point treatment is of limited benefit. In the present study, a new molecular diagnostic tool for the rapid detection of A. ocellatum (AO) was developed using the loop-mediated isothermal amplification method (LAMP). The AO-LAMP assay designed is highly specific using a set of four primers - two outer and two inner primers targeting six different regions on the 5' end of the Small Subunit rDNA region (SSU rDNA) of A. ocellatum. The AO-LAMP assay, optimized for 25-30 min at 62°C, amplified the DNA from A. ocellatum extracted from both water and gill tissue samples and did not amplify DNA from four closely related dinoflagellate sp ecies. The detection limit of the AO-LAMP assay was 10 fg, exceptionally higher than the conventional PCR (1 pg). In addition, the standardized AO-LAMP assay was capable of detecting single tomonts and trophonts; the assay was not affected by the presence of possible inhibitory substances present in environmental water samples or gill samples. The AO-LAMP assay developed in the present study provides a novel useful tool for the simple, rapid and sensitive detection of A. ocellatum in water and gill tissue samples, which could assist in the early detection and improved control of A. ocellatum infections in aquaculture systems. Copyright © 2013 Elsevier B.V. All rights reserved.

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers.

PubMed

Koester, Meike; Gergs, René

2017-10-05

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (δ 15 N and δ 13 C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Molecular Analysis of Ammonia-Oxidizing Bacteria of the β Subdivision of the Class Proteobacteria in Compost and Composted Materials

PubMed Central

Kowalchuk, George A.; Naoumenko, Zinaida S.; Derikx, Piet J. L.; Felske, Andreas; Stephen, John R.; Arkhipchenko, Irina A.

1999-01-01

Although the practice of composting animal wastes for use as biofertilizers has increased in recent years, little is known about the microorganisms responsible for the nitrogen transformations which occur in compost and during the composting process. Ammonia is the principle available nitrogenous compound in composting material, and the conversion of this compound to nitrite in the environment by chemolithotrophic ammonia-oxidizing bacteria is an essential step in nitrogen cycling. Therefore, the distribution of ammonia-oxidizing members of the β subdivision of the class Proteobacteria in a variety of composting materials was assessed by amplifying 16S ribosomal DNA (rDNA) and 16S rRNA by PCR and reverse transcriptase PCR (RT-PCR), respectively. The PCR and RT-PCR products were separated by denaturing gradient gel electrophoresis (DGGE) and were identified by hybridization with a hierarchical set of oligonucleotide probes designed to detect ammonia oxidizer-like sequence clusters in the genera Nitrosospira and Nitrosomonas. Ammonia oxidizer-like 16S rDNA was detected in almost all of the materials tested, including industrial and experimental composts, manure, and commercial biofertilizers. A comparison of the DGGE and hybridization results after specific PCR and RT-PCR suggested that not all of the different ammonia oxidizer groups detected in compost are equally active. amoA, the gene encoding the active-site-containing subunit of ammonia monooxygenase, was also targeted by PCR, and template concentrations were estimated by competitive PCR. Detection of ammonia-oxidizing bacteria in the composts tested suggested that such materials may not be biologically inert with respect to nitrification and that the fate of nitrogen during composting and compost storage may be affected by the presence of these organisms. PMID:9925559

In vivo effects on intron retention and exon skipping by the U2AF large subunit and SF1/BBP in the nematode Caenorhabditis elegans

PubMed Central

Ma, Long; Tan, Zhiping; Teng, Yanling; Hoersch, Sebastian; Horvitz, H. Robert

2011-01-01

The in vivo analysis of the roles of splicing factors in regulating alternative splicing in animals remains a challenge. Using a microarray-based screen, we identified a Caenorhabditis elegans gene, tos-1, that exhibited three of the four major types of alternative splicing: intron retention, exon skipping, and, in the presence of U2AF large subunit mutations, the use of alternative 3′ splice sites. Mutations in the splicing factors U2AF large subunit and SF1/BBP altered the splicing of tos-1. 3′ splice sites of the retained intron or before the skipped exon regulate the splicing pattern of tos-1. Our study provides in vivo evidence that intron retention and exon skipping can be regulated largely by the identities of 3′ splice sites. PMID:22033331

Ultrastructure and molecular phylogenetic position of a novel phagotrophic stramenopile from low oxygen environments: Rictus lutensis gen. et sp. nov. (Bicosoecida, incertae sedis).

PubMed

Yubuki, Naoji; Leander, Brian S; Silberman, Jeffrey D

2010-04-01

A novel free free-living phagotrophic flagellate, Rictus lutensis gen. et sp. nov., with two heterodynamic flagella, a permanent cytostome and a cytopharynx was isolated from muddy, low oxygen coastal sediments in Cape Cod, MA, USA. We cultivated and characterized this flagellate with transmission electron microscopy, scanning electron microscopy and molecular phylogenetic analyses inferred from small subunit (SSU) rDNA sequences. These data demonstrated that this organism has the key ultrastructural characters of the Bicosoecida, including similar transitional zones and a similar overall flagellar apparatus consisting of an x fiber and an L-shape microtubular root 2 involved in food capture. Although the molecular phylogenetic analyses were concordant with the ultrastructural data in placing R. lutensis with the bicosoecid clade, the internal position of this relatively divergent sequence within the clade was not resolved. Therefore, we interpret R. lutensis gen. et sp. nov. as a novel bicosoecid incertae sedis. Copyright 2009 Elsevier GmbH. All rights reserved.

Taxonomic revision of Aegista subchinensis (Möllendorff, 1884) (Stylommatophora, Bradybaenidae) and a description of a new species of Aegista from eastern Taiwan based on multilocus phylogeny and comparative morphology

PubMed Central

Huang, Chih-Wei; Lee, Yen-Chen; Lin, Si-Min; Wu, Wen-Lung

2014-01-01

Abstract Aegista subchinensis (Möllendorff, 1884) is a widely distributed land snail species with morphological variation and endemic to Taiwan. Three genetic markers (partial sequence of the mitochondrial cytochrome c oxidase subunit I [COI], the 16S rDNA and the nuclear internal transcribed spacer 2 [ITS2]) were analysed to infer phylogenetic relationships and genetic divergence of closely related species of the genus Aegista, Aegista vermis (Reeve, 1852) and Aegista oculus (Pfeiffer, 1850). A new species from Aegista subchinensis has been recognized on the basis of phylogenetic and morphological evidences. The nominal new species, Aegista diversifamilia sp. n. is distinguished from Aegista subchinensis (Möllendorff, 1884) by its larger shell size, aperture and apex angle; wider umbilicus and flatter shell shape. The northernmost distribution of Aegista diversifamilia sp. n. is limited by the Lanyang River, which is presumed to mark the geographic barrier between Aegista diversifamilia sp. n. and Aegista subchinensis. PMID:25349506

Morphological and Molecular Characterization of Paramecium (Viridoparamecium nov. subgen.) chlorelligerum Kahl, 1935 (Ciliophora)

PubMed Central

KREUTZ, MARTIN; STOECK, THORSTEN; FOISSNER, WILHELM

2013-01-01

We redescribe Paramecium chlorelligerum, a forgotten species, which Kahl (1935) briefly but precisely described in the addendum to his ciliate monographs as a Paramecium with symbiotic green algae. The redescription is based on classical morphological methods and the analysis of the small subunit (SSU) rDNA. Morphologically, P. chlorelligerum differs from P. (Chloroparamecium) bursaria, the second green species in the genus, by having a special swimming shape, the length of the caudal cilia, the size of the micronucleus, the size of the symbiotic algae, the contractile vacuoles (with collecting vesicles vs. collecting canals), and the number of excretory pores /contractile vacuole (1 vs. 2--3). The molecular investigations show that P. chlorelligerum forms a distinct branch distant from the P. (Chloroparamecium) bursaria clade. Thus, we classify P. chlorelligerum in a new subgenus: Paramecium (Viridoparamecium) chlorelligerum. The symbiotic alga belongs to the little-known genus Meyerella, as yet recorded only from the plankton of a North American lake. PMID:22827482

Morphology, morphogenesis, and phylogeny of an Anteholosticha intermedia (Ciliophora, Urostylida) population from the United States.

PubMed

Chen, Lingyun; Wu, Weining; El-Serehy, Hamed A; Hu, Xiaozhong; Clamp, John C

2018-04-30

A distinct population of Anteholosticha intermedia was isolated from soil in the Great Smoky Mountains of North Carolina, USA, and its morphology, morphogenesis and molecular phylogeny investigated by microscopic observations of live and protargol-prepared specimens and analyses of the sequence of small subunit (SSU) rDNA. Our population closely resembles the populations from Austria and Korea. Members of the genus Anteholosticha have been regarded as ontogenetically diverse, which is confirmed by the present work. The most noteworthy ontogenetic feature of the American population of A. intermedia is that the oral primordium in the proter appears apokinetally at the posterior end of the undulating membranes anlage at the beginning of division and then dedifferentiates midway through morphogenesis. Molecular phylogenetic analyses demonstrate, with high support, that the American population of A. intermedia is clearly distinct from congeners and branches as part of a sister lineage to the Bakuella-Urostyla clade that belongs to the major clade comprising the order Urostylida. Copyright © 2018 Elsevier GmbH. All rights reserved.

Genotyping of Giardia lamblia isolates from humans in China and Korea using ribosomal DNA Sequences.

PubMed

Yong, T S; Park, S J; Hwang, U W; Yang, H W; Lee, K W; Min, D Y; Rim, H J; Wang, Y; Zheng, F

2000-08-01

Genetic characterization of a total of 15 Giardia lamblia isolates, 8 from Anhui Province, China (all from purified cysts) and 7 from Seoul, Korea (2 from axenic cultures and 5 from purified cysts), was performed by polymerase chain reaction amplification and sequencing of a 295-bp region near the 5' end of the small subunit ribosomal DNA (eukaryotic 16S rDNA). Phylogenetic analyses were subsequently conducted using sequence data obtained in this study, as well as sequences published from other Giardia isolates. The maximum parsimony method revealed that G. lamblia isolates from humans in China and Korea are divided into 2 major lineages, assemblages A and B. All 7 Korean isolates were grouped into assemblage A, whereas 4 Chinese isolates were grouped into assemblage A and 4 into assemblage B. Two Giardia microti isolates and 2 dog-derived Giardia isolates also grouped into assemblage B, whereas Giardia ardeae and Giardia muris were unique.

Reengineering ribosome export.

PubMed

Lo, Kai-Yin; Johnson, Arlen W

2009-03-01

Large cargoes require multiple receptors for efficient transport through the nuclear pore complex. The 60S ribosomal subunit is one of the bulkiest transport cargoes, and in yeast three different receptors, Crm1, Mex67/Mtr2, and Arx1, collaborate in its export. However, only Crm1, recruited by the adapter Nmd3, appears to be conserved for 60S export in higher eukaryotes. We asked if export of the large subunit requires specific receptors. We made protein fusions between mutant Nmd3 and various export receptors. Surprisingly, fusions of Mex67, the tRNA exportin Los1, Mtr2, Cse1, or Msn5 to Nmd3, lacking its Crm1-dependent nuclear export signal (NES), all functioned in export. Furthermore, these chimeric proteins supported 60S export even in the presence of the Crm1 inhibitor leptomycin B, indicating that export was now independent of Crm1. These results suggest that there is not a requirement for a specific export receptor for the large subunit, as recruitment of any receptor will suffice. Finally we show that the addition of an NES directly to the 60S ribosomal subunit protein Rpl3 promotes export. These results imply remarkable flexibility in the export pathway for the 60S subunit and help explain how different export receptors could have evolved in different eukaryotic lineages.

Reengineering Ribosome Export

PubMed Central

Lo, Kai-Yin

2009-01-01

Large cargoes require multiple receptors for efficient transport through the nuclear pore complex. The 60S ribosomal subunit is one of the bulkiest transport cargoes, and in yeast three different receptors, Crm1, Mex67/Mtr2, and Arx1, collaborate in its export. However, only Crm1, recruited by the adapter Nmd3, appears to be conserved for 60S export in higher eukaryotes. We asked if export of the large subunit requires specific receptors. We made protein fusions between mutant Nmd3 and various export receptors. Surprisingly, fusions of Mex67, the tRNA exportin Los1, Mtr2, Cse1, or Msn5 to Nmd3, lacking its Crm1-dependent nuclear export signal (NES), all functioned in export. Furthermore, these chimeric proteins supported 60S export even in the presence of the Crm1 inhibitor leptomycin B, indicating that export was now independent of Crm1. These results suggest that there is not a requirement for a specific export receptor for the large subunit, as recruitment of any receptor will suffice. Finally we show that the addition of an NES directly to the 60S ribosomal subunit protein Rpl3 promotes export. These results imply remarkable flexibility in the export pathway for the 60S subunit and help explain how different export receptors could have evolved in different eukaryotic lineages. PMID:19144820

Beta3 subunits promote expression and nicotine-induced up-regulation of human nicotinic alpha6* nicotinic acetylcholine receptors expressed in transfected cell lines.

PubMed

Tumkosit, Prem; Kuryatov, Alexander; Luo, Jie; Lindstrom, Jon

2006-10-01

Nicotinic acetylcholine receptors (AChRs) containing alpha6 subunits are typically found at aminergic nerve endings where they play important roles in nicotine addiction and Parkinson's disease. alpha6* AChRs usually contain beta3 subunits. beta3 subunits are presumed to assemble only in the accessory subunit position within AChRs where they do not participate in forming acetylcholine binding sites. Assembly of subunits in the accessory position may be a critical final step in assembly of mature AChRs. Human alpha6 AChRs subtypes were permanently transfected into human tsA201 human embryonic kidney (HEK) cell lines. alpha6beta2beta3 and alpha6beta4beta3 cell lines were found to express much larger amounts of AChRs and were more sensitive to nicotine-induced increase in the amount of AChRs than were alpha6beta2 or alpha6beta4 cell lines. The increased sensitivity to nicotine-induced up-regulation was due not to a beta3-induced increase in affinity for nicotine but probably to a direct effect on assembly of AChR subunits. HEK cells express only a small amount of mature alpha6beta2 AChRs, but many of these subunits are on the cell surface. This contrasts with Xenopus laevis oocytes, which express a large amount of incorrectly assembled alpha6beta2 subunits that bind cholinergic ligands but form large amorphous intracellular aggregates. Monoclonal antibodies (mAbs) were made to the alpha6 and beta3 subunits to aid in the characterization of these AChRs. The alpha6 mAbs bind to epitopes C-terminal of the extracellular domain. These data demonstrate that both cell type and the accessory subunit beta3 can play important roles in alpha6* AChR expression, stability, and up-regulation by nicotine.

Variation in Ribosomal DNA among Isolates of the Mycorrhizal Fungus Cenococcum Geophilum FR.

NASA Astrophysics Data System (ADS)

Lobuglio, Katherine Frances

1990-01-01

Cenococcum geophilum Fr., a cosmopolitan mycorrhizal fungus, is well-known for its extremely wide host and habitat range. The ecological diversity of C. geophilum sharply contrasts its present taxonomic status as a monotypic form -genus. Restriction fragment length polymorphisms (RFLPs) in nuclear ribosomal DNA (rDNA) was used to assess the degree of genetic variation among 72 isolates of C. geophilum. The probe used in this study was the rDNA repeat cloned from C. geophilum isolate A145 (pCG15). Length of the rDNA repeat was approximately 9 kb. The rDNA clone was mapped for 5 restriction endonucleases. Hybridization with cloned Saccharomyces cerevisiae rDNA (pSR118, and pSR125 containing the 18S, and 5.8-25S rRNA genes respectively), and alignment of restriction endonuclease sites conserved in the rDNA genes of other fungi, were used to position the corresponding rDNAs of C. geophilum. Southern hybridizations with EcoRI, HindIII, XhoI, and PstI digested DNAs indicated extensive variation among the C. geophilum isolates, greater than has been previously reported to occur within a fungal species. Most of the rDNA polymorphisms occurred in the IGS region. Restriction endonuclease site and length polymorphisms were also observed in the 5.8S-26S genic regions. Sixteen size categories of length mutations, 6 restriction endonuclease site additions, and 4 restriction endonuclease site deletions were determined using isolate A145 as a reference. The rDNA repeat length among the isolates varied from approximately 8.5 to 10.2 kb. RFLPs were also observed in the mitochondrial (mt) 24S rRNA gene and flanking regions of HindIII digested DNAs of C. geophilum isolates representing both geographically distinct and similar origins. Among the C. geophilum isolates analyzed there were fewer RFLPs in mt-DNA than in nuclear rDNA. EcoRI rDNA phenotypes between C. geophilum and Elaphomyces anthracinus, its proposed teleomorph or sexual state, did not correspond. In addition, the four Elaphomyces species examined had smaller rDNA repeat sizes (approximately 7.3 to 8.0 kb) than that observed among C. geophilum isolates. UPGMA cluster analysis grouped C. geophilum isolates, on the basis of shared nuclear rDNA phenotypes, into a broad range of clusters ranging from 100% to 44% similarity. Limited correlation was observed among nuclear rDNA phenotypes and culture morphology, mycorrhizal characteristics, or geographic origins of the isolates. The amount of genetic variation demonstrated in this study indicates that C. geophilum is either an extremely heterogenous species or it represents more than one species and possibly more than one genus.

Evolution in the block: common elements of 5S rDNA organization and evolutionary patterns in distant fish genera.

PubMed

Campo, Daniel; García-Vázquez, Eva

2012-01-01

The 5S rDNA is organized in the genome as tandemly repeated copies of a structural unit composed of a coding sequence plus a nontranscribed spacer (NTS). The coding region is highly conserved in the evolution, whereas the NTS vary in both length and sequence. It has been proposed that 5S rRNA genes are members of a gene family that have arisen through concerted evolution. In this study, we describe the molecular organization and evolution of the 5S rDNA in the genera Lepidorhombus and Scophthalmus (Scophthalmidae) and compared it with already known 5S rDNA of the very different genera Merluccius (Merluccidae) and Salmo (Salmoninae), to identify common structural elements or patterns for understanding 5S rDNA evolution in fish. High intra- and interspecific diversity within the 5S rDNA family in all the genera can be explained by a combination of duplications, deletions, and transposition events. Sequence blocks with high similarity in all the 5S rDNA members across species were identified for the four studied genera, with evidences of intense gene conversion within noncoding regions. We propose a model to explain the evolution of the 5S rDNA, in which the evolutionary units are blocks of nucleotides rather than the entire sequences or single nucleotides. This model implies a "two-speed" evolution: slow within blocks (homogenized by recombination) and fast within the gene family (diversified by duplications and deletions).

Marek’s disease virus encoded ribonucleotide reductase large subunit is essential for in vivo replication and plays a critical role in viral pathogenesis.

USDA-ARS?s Scientific Manuscript database

Marek’s disease virus encodes a ribonucleotide reductase (RR) that consists of two subunits namely RR1 and RR2, both of which associate to form an active holoenzyme and both subunits are necessary for enzyme activity. It is an essential enzyme for the conversion of ribonucleotides to deoxyribonucleo...

Bird ticks in Hungary reflect western, southern, eastern flyway connections and two genetic lineages of Ixodes frontalis and Haemaphysalis concinna.

PubMed

Hornok, S; Flaisz, B; Takács, N; Kontschán, J; Csörgő, T; Csipak, Á; Jaksa, B R; Kováts, D

2016-02-24

Birds play an important role in short- and long-distance transportation of ticks and tick-borne pathogens. The aim of the present study was to provide comprehensive information on the species and genetic diversity of ixodid ticks transported by migratory and non-migratory bird species in Central Europe, and to evaluate relevant data in a geographical, as well as in an ecological context. During a three year period (2012-2014), altogether 3339 ixodid ticks were collected from 1167 passerine birds (representatives of 47 species) at ringing stations in Hungary. These ticks were identified, and the tick-infestations of bird species were compared according to various traits. In addition, PCR and sequencing of part of the cytochrome oxidase subunit-I (COI) and 16S rDNA genes were performed from representatives of five tick species. The most abundant tick species found were Ixodes ricinus and Haemaphysalis concinna (with 2296 and 989 immature stages, respectively). In addition, 48 I. frontalis (all stages), three Hyalomma rufipes nymphs, one I. lividus and two I. festai females were collected. The majority of I. ricinus and I. frontalis specimens occurred on ground-feeding bird species, as contrasted to Ha. concinna. Hy. rufipes showed the highest degree of sequence identity to an Ethiopian hybrid of the same tick species. Based on both COI and 16S rDNA gene analyses, two genetic lineages of I. frontalis were recognized (with only 91.4 % identity in their partial COI gene). These were highly similar to South-Western European isolates of the same tick species. Phylogenetic analysis of Ha. concinna specimens collected from birds in Hungary also revealed two genetic lineages, one of which showed high (≥99 %) degree of 16S rDNA sequence identity to conspecific East Asian isolates. Two genetic lineages of I. frontalis and Ha. concinna are transported by birds in Central Europe, which reflect a high degree of sequence identity to South-Western European and East Asian isolates of the same tick species, respectively. In addition, I. festai was collected for the first time in Hungary. These findings highlight the importance of western and eastern migratory connections by birds (in addition to the southern direction), which are also relevant to the epidemiology of tick-borne diseases.

Description of two new gill myxozoans from smallmouth (Micropterus dolomieu) and largemouth bass (Micropterus salmoides)

USGS Publications Warehouse

Walsh, Heather L.; Iwanowicz, Luke R.; Glenney, Gavin W.; Iwanowicz, Deborah D.; Blazer, Vicki

2012-01-01

Two previously undescribed species of myxozoan parasites were observed in the gills of bass inhabiting the Potomac and James River basins. They are described using morphological characteristics and small-subunit (SSU) rDNA gene sequences. Both were taxonomically identified as new species of Myxobolus; Myxobolus branchiarum n. sp. was found exclusively in smallmouth bass, and Myxobolus micropterii n. sp. was found in largemouth and smallmouth bass. Small, spherical, white plasmodia of M. branchiarum from smallmouth bass were observed grossly in the gills; these plasmodia had an average length of 320.3 µm and width of 246.1 µm. The development of the plasmodia is intralamellar in the secondary lamellae of the gills. Mature spores were pyriform in shape with a length of 12.8 ± 1.4 (8.1–15.1) µm and width of 6.9 ± 1.1 (4.0–9.0) µm. Analysis of SSU rDNA identified M. branchiarum in a sister-group to 3 species of Henneguya, although morphologically caudal appendages were absent. Myxobolus micropterii observed in the gills of largemouth and smallmouth bass had larger, ovoid, cream-colored plasmodia with an average length of 568.1 µm and width of 148.1 µm. The cysts developed at the distal end of the gill filament within the primary lamellae. The mature spores were ovoid in shape with a length of 10.8 ± 0.7 (9.2–12.2) µm and width of 10.6 ± 0.6 (9.0–11.8) µm. SSU rDNA analysis placed M. micropterii in a sister group with Henneguya lobosa and Myxobolus oliveirai. The highest prevalence of M. branchiarum was observed in the gills of bass collected from the Cowpasture River (50.9%). Prevalence was 44.6% in bass from the Potomac River and only 4.3% in bass collected from the Shenandoah River. A seasonal study of M. branchiarum, which included both infected and uninfected smallmouth bass, determined that a significantly higher intensity was observed in the spring than in the summer (P < 0.001) or fall (P  =  0.004). In an analysis excluding uninfected bass, a higher intensity was observed in the spring than in the summer (P  =  0.001) or fall (P  =  0.008). Prevalence and seasonal differences were not determined for M. micropterii.

Description of two new gill myxozoans from smallmouth (Micropterus dolomieu) and largemouth (Micropterus salmoides) bass.

PubMed

Walsh, Heather L; Iwanowicz, Luke R; Glenney, Gavin W; Iwanowicz, Deborah D; Blazer, Vicki S

2012-04-01

Two previously undescribed species of myxozoan parasites were observed in the gills of bass inhabiting the Potomac and James River basins. They are described using morphological characteristics and small-subunit (SSU) rDNA gene sequences. Both were taxonomically identified as new species of Myxobolus; Myxobolus branchiarum n. sp. was found exclusively in smallmouth bass, and Myxobolus micropterii n. sp. was found in largemouth and smallmouth bass. Small, spherical, white plasmodia of M. branchiarum from smallmouth bass were observed grossly in the gills; these plasmodia had an average length of 320.3 µm and width of 246.1 µm. The development of the plasmodia is intralamellar in the secondary lamellae of the gills. Mature spores were pyriform in shape with a length of 12.8 ± 1.4 (8.1-15.1) µm and width of 6.9 ± 1.1 (4.0-9.0) µm. Analysis of SSU rDNA identified M. branchiarum in a sister-group to 3 species of Henneguya , although morphologically caudal appendages were absent. Myxobolus micropterii observed in the gills of largemouth and smallmouth bass had larger, ovoid, cream-colored plasmodia with an average length of 568.1 µm and width of 148.1 µm. The cysts developed at the distal end of the gill filament within the primary lamellae. The mature spores were ovoid in shape with a length of 10.8 ± 0.7 (9.2-12.2) µm and width of 10.6 ± 0.6 (9.0-11.8) µm. SSU rDNA analysis placed M. micropterii in a sister group with Henneguya lobosa and Myxobolus oliveirai . The highest prevalence of M. branchiarum was observed in the gills of bass collected from the Cowpasture River (50.9%). Prevalence was 44.6% in bass from the Potomac River and only 4.3% in bass collected from the Shenandoah River. A seasonal study of M. branchiarum , which included both infected and uninfected smallmouth bass, determined that a significantly higher intensity was observed in the spring than in the summer (P < 0.001) or fall (P  =  0.004). In an analysis excluding uninfected bass, a higher intensity was observed in the spring than in the summer (P  =  0.001) or fall (P  =  0.008). Prevalence and seasonal differences were not determined for M. micropterii .

Biology of Symbioses between Marine Invertebrates and Intracellular Bacteria

DTIC Science & Technology

1990-01-30

bisphosphate carboxylase We designed from published sequence information oligonucleotide primers which are complementary to conserved regions on RubisCO ...large and small subunit genes. These primers were used successfully to amplify using polymerase chain reaction (PCR) specific regions of RubisCO ...for the large subunit of ribulose bisphosphate carboxylase/oxygenase ( RubisCO ) to symbiont DNA shows that the symbionts from both deep-sea and shallow

Pharmacological consequences of the coexpression of BK channel α and auxiliary β subunits

PubMed Central

Torres, Yolima P.; Granados, Sara T.; Latorre, Ramón

2014-01-01

Coded by a single gene (Slo1, KCM) and activated by depolarizing potentials and by a rise in intracellular Ca2+ concentration, the large conductance voltage- and Ca2+-activated K+ channel (BK) is unique among the superfamily of K+ channels. BK channels are tetramers characterized by a pore-forming α subunit containing seven transmembrane segments (instead of the six found in voltage-dependent K+ channels) and a large C terminus composed of two regulators of K+ conductance domains (RCK domains), where the Ca2+-binding sites reside. BK channels can be associated with accessory β subunits and, although different BK modulatory mechanisms have been described, greater interest has recently been placed on the role that the β subunits may play in the modulation of BK channel gating due to its physiological importance. Four β subunits have currently been identified (i.e., β1, β2, β3, and β4) and despite the fact that they all share the same topology, it has been shown that every β subunit has a specific tissue distribution and that they modify channel kinetics as well as their pharmacological properties and the apparent Ca2+ sensitivity of the α subunit in different ways. Additionally, different studies have shown that natural, endogenous, and synthetic compounds can modulate BK channels through β subunits. Considering the importance of these channels in different pathological conditions, such as hypertension and neurological disorders, this review focuses on the mechanisms by which these compounds modulate the biophysical properties of BK channels through the regulation of β subunits, as well as their potential therapeutic uses for diseases such as those mentioned above. PMID:25346693

Pharmacological consequences of the coexpression of BK channel α and auxiliary β subunits.

PubMed

Torres, Yolima P; Granados, Sara T; Latorre, Ramón

2014-01-01

Coded by a single gene (Slo1, KCM) and activated by depolarizing potentials and by a rise in intracellular Ca(2+) concentration, the large conductance voltage- and Ca(2+)-activated K(+) channel (BK) is unique among the superfamily of K(+) channels. BK channels are tetramers characterized by a pore-forming α subunit containing seven transmembrane segments (instead of the six found in voltage-dependent K(+) channels) and a large C terminus composed of two regulators of K(+) conductance domains (RCK domains), where the Ca(2+)-binding sites reside. BK channels can be associated with accessory β subunits and, although different BK modulatory mechanisms have been described, greater interest has recently been placed on the role that the β subunits may play in the modulation of BK channel gating due to its physiological importance. Four β subunits have currently been identified (i.e., β1, β2, β3, and β4) and despite the fact that they all share the same topology, it has been shown that every β subunit has a specific tissue distribution and that they modify channel kinetics as well as their pharmacological properties and the apparent Ca(2+) sensitivity of the α subunit in different ways. Additionally, different studies have shown that natural, endogenous, and synthetic compounds can modulate BK channels through β subunits. Considering the importance of these channels in different pathological conditions, such as hypertension and neurological disorders, this review focuses on the mechanisms by which these compounds modulate the biophysical properties of BK channels through the regulation of β subunits, as well as their potential therapeutic uses for diseases such as those mentioned above.

Development and validation of an rDNA operon based primer walking strategy applicable to de novo bacterial genome finishing

PubMed Central

Eastman, Alexander W.; Yuan, Ze-Chun

2015-01-01

Advances in sequencing technology have drastically increased the depth and feasibility of bacterial genome sequencing. However, little information is available that details the specific techniques and procedures employed during genome sequencing despite the large numbers of published genomes. Shotgun approaches employed by second-generation sequencing platforms has necessitated the development of robust bioinformatics tools for in silico assembly, and complete assembly is limited by the presence of repetitive DNA sequences and multi-copy operons. Typically, re-sequencing with multiple platforms and laborious, targeted Sanger sequencing are employed to finish a draft bacterial genome. Here we describe a novel strategy based on the identification and targeted sequencing of repetitive rDNA operons to expedite bacterial genome assembly and finishing. Our strategy was validated by finishing the genome of Paenibacillus polymyxa strain CR1, a bacterium with potential in sustainable agriculture and bio-based processes. An analysis of the 38 contigs contained in the P. polymyxa strain CR1 draft genome revealed 12 repetitive rDNA operons with varied intragenic and flanking regions of variable length, unanimously located at contig boundaries and within contig gaps. These highly similar but not identical rDNA operons were experimentally verified and sequenced simultaneously with multiple, specially designed primer sets. This approach also identified and corrected significant sequence rearrangement generated during the initial in silico assembly of sequencing reads. Our approach reduces the required effort associated with blind primer walking for contig assembly, increasing both the speed and feasibility of genome finishing. Our study further reinforces the notion that repetitive DNA elements are major limiting factors for genome finishing. Moreover, we provided a step-by-step workflow for genome finishing, which may guide future bacterial genome finishing projects. PMID:25653642

Ribosomal Internal Transcribed Spacer of Prototheca wickerhamii Has Characteristic Structure Useful for Identification and Genotyping

PubMed Central

Hirose, Noriyuki; Nishimura, Kazuko; Inoue-Sakamoto, Maki; Masuda, Michiaki

2013-01-01

Prototheca species are achlorophyllous algae ubiquitous in nature and known to cause localized and systemic infection both in humans and animals. Although identification of the Prototheca species in clinical specimens is a challenge, there are an increasing number of cases in which molecular techniques have successfully been used for diagnosis of protothecosis. In this study, we characterized nuclear ribosomal DNA (rDNA) of a strain of Prototheca (FL11-0001) isolated from a dermatitis patient in Japan for its species identification. When nuclear rDNA of FL11-0001 and that of various other Prototheca strains were compared by polymerase chain reaction (PCR), the results indicated that the sizes of ribosomal internal transcribed spacer (ITS) were different in a species-dependent manner, suggesting that the variation might be useful for differentiation of Prototheca spp. Especially, ITS of P. wickerhamii, the most common cause of human protothecosis, was distinctively larger than that of other Prototheca spp. FL11-0001, whose ITS was comparably large, could easily be identified as P. wickerhamii. The usefulness of the PCR analysis of ITS was also demonstrated by the discovery that one of the clinical isolates that had previously been designated as P. wickerhamii was likely a novel species. Furthermore, our data demonstrated that nucleotide sequences of P. wickerhamii ITS are heterogenous between different rDNA copies in each strain and also polymorphic between strains. Phylogenetic analysis suggested that the ITS sequences could be classified to four clades, based on which P. wickerhamii strains might be grouped into at least two genotypes. Comprehensive characterization of Prototheca rDNA may provide valuable insights into diagnosis and epidemiology of protothecosis, as well as evolution and taxonomy of Prototheca and related organisms. PMID:24312279

Ribosomal internal transcribed spacer of Prototheca wickerhamii has characteristic structure useful for identification and genotyping.

PubMed

Hirose, Noriyuki; Nishimura, Kazuko; Inoue-Sakamoto, Maki; Masuda, Michiaki

2013-01-01

Prototheca species are achlorophyllous algae ubiquitous in nature and known to cause localized and systemic infection both in humans and animals. Although identification of the Prototheca species in clinical specimens is a challenge, there are an increasing number of cases in which molecular techniques have successfully been used for diagnosis of protothecosis. In this study, we characterized nuclear ribosomal DNA (rDNA) of a strain of Prototheca (FL11-0001) isolated from a dermatitis patient in Japan for its species identification. When nuclear rDNA of FL11-0001 and that of various other Prototheca strains were compared by polymerase chain reaction (PCR), the results indicated that the sizes of ribosomal internal transcribed spacer (ITS) were different in a species-dependent manner, suggesting that the variation might be useful for differentiation of Prototheca spp. Especially, ITS of P. wickerhamii, the most common cause of human protothecosis, was distinctively larger than that of other Prototheca spp. FL11-0001, whose ITS was comparably large, could easily be identified as P. wickerhamii. The usefulness of the PCR analysis of ITS was also demonstrated by the discovery that one of the clinical isolates that had previously been designated as P. wickerhamii was likely a novel species. Furthermore, our data demonstrated that nucleotide sequences of P. wickerhamii ITS are heterogenous between different rDNA copies in each strain and also polymorphic between strains. Phylogenetic analysis suggested that the ITS sequences could be classified to four clades, based on which P. wickerhamii strains might be grouped into at least two genotypes. Comprehensive characterization of Prototheca rDNA may provide valuable insights into diagnosis and epidemiology of protothecosis, as well as evolution and taxonomy of Prototheca and related organisms.

ZZ/ZW sex chromosome system in the endangered fish Lignobrycon myersi Miranda-Ribeiro, 1956 (Teleostei, Characiformes, Triportheidae).

PubMed

Rodrigues, Alexandre Dos Santos; Medrado, Aline Souza; Diniz, Débora; Oliveira, Claudio; Affonso, Paulo Roberto Antunes de Mello

2016-01-01

Lignobrycon myersi is an endemic fish species from a few coastal rivers in northeastern Brazil. Based on molecular evidence, Lignobrycon myersi and genera Triportheus Cope, 1872, Agoniates Müller & Troschel, 1845, Clupeacharax Pearson, 1924 and Engraulisoma Castro, 1981 were placed in the family Triportheidae. In the present work, we report the first cytogenetic data for Lignobrycon myersi to test the hypothesis that Lignobrycon and Triportheus are closely related. Studied specimens presented 2n=52 with 28 metacentric (m), 18 submetacentric (sm) and six subtelocentric (st) chromosomes for males and 27 m, 19 sm and 6 st for females, characterizing a ZZ/ZW sex chromosome system. The Z chromosome corresponds to the largest chromosome in karyotype while the W is about 50% smaller than the Z and largely heterochromatic. Terminal nucleolus organizer regions, GC-rich sites and 18S rDNA signals were detected on pair 14. However, additional 18S rDNA sites were observed in the W chromosome. The 5S rDNA was mainly detected on long arms of pair 7. The apparent synapomorphic chromosomal traits of Triportheus and Lignobrycon myersi reinforce their close phylogenetic relationship, suggesting that the ZZ/ZW chromosome system in both genera has arisen before cladogenic events.

Asymmetric Epigenetic Modification and Elimination of rDNA Sequences by Polyploidization in Wheat[W

PubMed Central

Guo, Xiang

2014-01-01

rRNA genes consist of long tandem repeats clustered on chromosomes, and their products are important functional components of the ribosome. In common wheat (Triticum aestivum), rDNA loci from the A and D genomes were largely lost during the evolutionary process. This biased DNA elimination may be related to asymmetric transcription and epigenetic modifications caused by the polyploid formation. Here, we observed both sets of parental nucleolus organizing regions (NORs) were expressed after hybridization, but asymmetric silencing of one parental NOR was immediately induced by chromosome doubling, and reversing the ploidy status could not reactivate silenced NORs. Furthermore, increased CHG and CHH DNA methylation on promoters was accompanied by asymmetric silencing of NORs. Enrichment of H3K27me3 and H3K9me2 modifications was also observed to be a direct response to increased DNA methylation and transcriptional inactivation of NOR loci. Both A and D genome NOR loci with these modifications started to disappear in the S4 generation and were completely eliminated by the S7 generation in synthetic tetraploid wheat. Our results indicated that asymmetric epigenetic modification and elimination of rDNA sequences between different donor genomes may lead to stable allopolyploid wheat with increased differentiation and diversity. PMID:25415973

Asymmetric epigenetic modification and elimination of rDNA sequences by polyploidization in wheat.

PubMed

Guo, Xiang; Han, Fangpu

2014-11-01

rRNA genes consist of long tandem repeats clustered on chromosomes, and their products are important functional components of the ribosome. In common wheat (Triticum aestivum), rDNA loci from the A and D genomes were largely lost during the evolutionary process. This biased DNA elimination may be related to asymmetric transcription and epigenetic modifications caused by the polyploid formation. Here, we observed both sets of parental nucleolus organizing regions (NORs) were expressed after hybridization, but asymmetric silencing of one parental NOR was immediately induced by chromosome doubling, and reversing the ploidy status could not reactivate silenced NORs. Furthermore, increased CHG and CHH DNA methylation on promoters was accompanied by asymmetric silencing of NORs. Enrichment of H3K27me3 and H3K9me2 modifications was also observed to be a direct response to increased DNA methylation and transcriptional inactivation of NOR loci. Both A and D genome NOR loci with these modifications started to disappear in the S4 generation and were completely eliminated by the S7 generation in synthetic tetraploid wheat. Our results indicated that asymmetric epigenetic modification and elimination of rDNA sequences between different donor genomes may lead to stable allopolyploid wheat with increased differentiation and diversity. © 2014 American Society of Plant Biologists. All rights reserved.

Detection and Phylogenetic Analysis of Wolbachia in the Asiatic Rice Leafroller, Cnaphalocrocis medinalis, in Chinese Populations

PubMed Central

Chai, Huan-Na; Du, Yu-Zhou; Qiu, Bao-Li; Zhai, Bao-Ping

2011-01-01

Wolbachia are a group of intracellular inherited endosymbiontic bacteria infecting a wide range of insects. In this study the infection status of Wolbachia (Rickettsiales: Rickettsiaceae) was measured in the Asiatic rice leafroller, Cnaphalocrocis medinalis (Guenée) (Lepidoptera: Pyralidae), from twenty locations in China by sequencing wsp, ftsZ and 16S rDNA genes. The results showed high infection rates of Wolbachia in C. medinalis populations. Wolbachia was detected in all geographically separate populations; the average infection rate was ∼ 62.5%, and the highest rates were 90% in Wenzhou and Yangzhou populations. The Wolbachia detected in different C. medinalis populations were 100% identical to each other when wsp, ftsZ, and 16S rDNA sequences were compared, with all sequences belonging to the Wolbachia B supergroup. Based on wsp, ftsZ and 16S rDNA sequences of Wolbachia, three phylogenetic trees of similar pattern emerged. This analysis indicated the possibility of inter-species and intra-species horizontal transmission of Wolbachia in different arthropods in related geographical regions. The migration route of C. medinalis in mainland China was also discussed since large differentiation had been found between the wsp sequences of Chinese and Thai populations. PMID:22233324

Rare but active taxa contribute to community dynamics of benthic biofilms in glacier-fed streams.

PubMed

Wilhelm, Linda; Besemer, Katharina; Fasching, Christina; Urich, Tim; Singer, Gabriel A; Quince, Christopher; Battin, Tom J

2014-08-01

Glaciers harbour diverse microorganisms, which upon ice melt can be released downstream. In glacier-fed streams microorganisms can attach to stones or sediments to form benthic biofilms. We used 454-pyrosequencing to explore the bulk (16S rDNA) and putatively active (16S rRNA) microbial communities of stone and sediment biofilms across 26 glacier-fed streams. We found differences in community composition between bulk and active communities among streams and a stronger congruence between biofilm types. Relative abundances of rRNA and rDNA were positively correlated across different taxa and taxonomic levels, but at lower taxonomic levels, the higher abundance in either the active or the bulk communities became more apparent. Here, environmental variables played a minor role in structuring active communities. However, we found a large number of rare taxa with higher relative abundances in rRNA compared with rDNA. This suggests that rare taxa contribute disproportionately to microbial community dynamics in glacier-fed streams. Our findings propose that high community turnover, where taxa repeatedly enter and leave the 'seed bank', contributes to the maintenance of microbial biodiversity in harsh ecosystems with continuous environmental perturbations, such as glacier-fed streams. © 2014 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

Concerted copy number variation balances ribosomal DNA dosage in human and mouse genomes

PubMed Central

Gibbons, John G.; Branco, Alan T.; Godinho, Susana A.; Yu, Shoukai; Lemos, Bernardo

2015-01-01

Tandemly repeated ribosomal DNA (rDNA) arrays are among the most evolutionary dynamic loci of eukaryotic genomes. The loci code for essential cellular components, yet exhibit extensive copy number (CN) variation within and between species. CN might be partly determined by the requirement of dosage balance between the 5S and 45S rDNA arrays. The arrays are nonhomologous, physically unlinked in mammals, and encode functionally interdependent RNA components of the ribosome. Here we show that the 5S and 45S rDNA arrays exhibit concerted CN variation (cCNV). Despite 5S and 45S rDNA elements residing on different chromosomes and lacking sequence similarity, cCNV between these loci is strong, evolutionarily conserved in humans and mice, and manifested across individual genotypes in natural populations and pedigrees. Finally, we observe that bisphenol A induces rapid and parallel modulation of 5S and 45S rDNA CN. Our observations reveal a novel mode of genome variation, indicate that natural selection contributed to the evolution and conservation of cCNV, and support the hypothesis that 5S CN is partly determined by the requirement of dosage balance with the 45S rDNA array. We suggest that human disease variation might be traced to disrupted rDNA dosage balance in the genome. PMID:25583482

Molecular organization and chromosomal localization of 5S rDNA in Amazonian Engystomops (Anura, Leiuperidae)

PubMed Central

2012-01-01

Background For anurans, knowledge of 5S rDNA is scarce. For Engystomops species, chromosomal homeologies are difficult to recognize due to the high level of inter- and intraspecific cytogenetic variation. In an attempt to better compare the karyotypes of the Amazonian species Engystomops freibergi and Engystomops petersi, and to extend the knowledge of 5S rDNA organization in anurans, the 5S rDNA sequences of Amazonian Engystomops species were isolated, characterized, and mapped. Results Two types of 5S rDNA, which were readily differentiated by their NTS (non-transcribed spacer) sizes and compositions, were isolated from specimens of E. freibergi from Brazil and E. petersi from two Ecuadorian localities (Puyo and Yasuní). In the E. freibergi karyotypes, the entire type I 5S rDNA repeating unit hybridized to the pericentromeric region of 3p, whereas the entire type II 5S rDNA repeating unit mapped to the distal region of 6q, suggesting a differential localization of these sequences. The type I NTS probe clearly detected the 3p pericentromeric region in the karyotypes of E. freibergi and E. petersi from Puyo and the 5p pericentromeric region in the karyotype of E. petersi from Yasuní, but no distal or interstitial signals were observed. Interestingly, this probe also detected many centromeric regions in the three karyotypes, suggesting the presence of a satellite DNA family derived from 5S rDNA. The type II NTS probe detected only distal 6q regions in the three karyotypes, corroborating the differential distribution of the two types of 5S rDNA. Conclusions Because the 5S rDNA types found in Engystomops are related to those of Physalaemus with respect to their nucleotide sequences and chromosomal locations, their origin likely preceded the evolutionary divergence of these genera. In addition, our data indicated homeology between Chromosome 5 in E. petersi from Yasuní and Chromosomes 3 in E. freibergi and E. petersi from Puyo. In addition, the chromosomal location of the type II 5S rDNA corroborates the hypothesis that the Chromosomes 6 of E. petersi and E. freibergi are homeologous despite the great differences observed between the karyotypes of the Yasuní specimens and the others. PMID:22433220

Molecular organization and chromosomal localization of 5S rDNA in Amazonian Engystomops (Anura, Leiuperidae).

PubMed

Rodrigues, Débora Silva; Rivera, Miryan; Lourenço, Luciana Bolsoni

2012-03-20

For anurans, knowledge of 5S rDNA is scarce. For Engystomops species, chromosomal homeologies are difficult to recognize due to the high level of inter- and intraspecific cytogenetic variation. In an attempt to better compare the karyotypes of the Amazonian species Engystomops freibergi and Engystomops petersi, and to extend the knowledge of 5S rDNA organization in anurans, the 5S rDNA sequences of Amazonian Engystomops species were isolated, characterized, and mapped. Two types of 5S rDNA, which were readily differentiated by their NTS (non-transcribed spacer) sizes and compositions, were isolated from specimens of E. freibergi from Brazil and E. petersi from two Ecuadorian localities (Puyo and Yasuní). In the E. freibergi karyotypes, the entire type I 5S rDNA repeating unit hybridized to the pericentromeric region of 3p, whereas the entire type II 5S rDNA repeating unit mapped to the distal region of 6q, suggesting a differential localization of these sequences. The type I NTS probe clearly detected the 3p pericentromeric region in the karyotypes of E. freibergi and E. petersi from Puyo and the 5p pericentromeric region in the karyotype of E. petersi from Yasuní, but no distal or interstitial signals were observed. Interestingly, this probe also detected many centromeric regions in the three karyotypes, suggesting the presence of a satellite DNA family derived from 5S rDNA. The type II NTS probe detected only distal 6q regions in the three karyotypes, corroborating the differential distribution of the two types of 5S rDNA. Because the 5S rDNA types found in Engystomops are related to those of Physalaemus with respect to their nucleotide sequences and chromosomal locations, their origin likely preceded the evolutionary divergence of these genera. In addition, our data indicated homeology between Chromosome 5 in E. petersi from Yasuní and Chromosomes 3 in E. freibergi and E. petersi from Puyo. In addition, the chromosomal location of the type II 5S rDNA corroborates the hypothesis that the Chromosomes 6 of E. petersi and E. freibergi are homeologous despite the great differences observed between the karyotypes of the Yasuní specimens and the others.

Amino acid sequence of the human fibronectin receptor

PubMed Central

1987-01-01

The amino acid sequence deduced from cDNA of the human placental fibronectin receptor is reported. The receptor is composed of two subunits: an alpha subunit of 1,008 amino acids which is processed into two polypeptides disulfide bonded to one another, and a beta subunit of 778 amino acids. Each subunit has near its COOH terminus a hydrophobic segment. This and other sequence features suggest a structure for the receptor in which the hydrophobic segments serve as transmembrane domains anchoring each subunit to the membrane and dividing each into a large ectodomain and a short cytoplasmic domain. The alpha subunit ectodomain has five sequence elements homologous to consensus Ca2+- binding sites of several calcium-binding proteins, and the beta subunit contains a fourfold repeat strikingly rich in cysteine. The alpha subunit sequence is 46% homologous to the alpha subunit of the vitronectin receptor. The beta subunit is 44% homologous to the human platelet adhesion receptor subunit IIIa and 47% homologous to a leukocyte adhesion receptor beta subunit. The high degree of homology (85%) of the beta subunit with one of the polypeptides of a chicken adhesion receptor complex referred to as integrin complex strongly suggests that the latter polypeptide is the chicken homologue of the fibronectin receptor beta subunit. These receptor subunit homologies define a superfamily of adhesion receptors. The availability of the entire protein sequence for the fibronectin receptor will facilitate studies on the functions of these receptors. PMID:2958481

African swine fever virus encodes two genes which share significant homology with the two largest subunits of DNA-dependent RNA polymerases.

PubMed Central

Yáñez, R J; Boursnell, M; Nogal, M L; Yuste, L; Viñuela, E

1993-01-01

A random sequencing strategy applied to two large SalI restriction fragments (SB and SD) of the African swine fever virus (ASFV) genome revealed that they might encode proteins similar to the two largest RNA polymerase subunits of eukaryotes, poxviruses and Escherichia coli. After further mapping by dot-blot hybridization, two large open reading frames (ORFs) were completely sequenced. The first ORF (NP1450L) encodes a protein of 1450 amino acids with extensive similarity to the largest subunit of RNA polymerases. The second one (EP1242L) codes for a protein of 1242 amino acids similar to the second largest RNA polymerase subunit. Proteins NP1450L and EP1242L are more similar to the corresponding subunits of eukaryotic RNA polymerase II than to those of vaccinia virus, the prototype poxvirus, which shares many functional characteristics with ASFV. ORFs NP1450L and EP1242L are mainly expressed late in ASFV infection, after the onset of DNA replication. Images PMID:8506138

Decreased surface expression of the δ subunit of the GABAA receptor contributes to reduced tonic inhibition in dentate granule cells in a mouse model of fragile X syndrome.

PubMed

Zhang, Nianhui; Peng, Zechun; Tong, Xiaoping; Lindemeyer, A Kerstin; Cetina, Yliana; Huang, Christine S; Olsen, Richard W; Otis, Thomas S; Houser, Carolyn R

2017-11-01

While numerous changes in the GABA system have been identified in models of Fragile X Syndrome (FXS), alterations in subunits of the GABA A receptors (GABA A Rs) that mediate tonic inhibition are particularly intriguing. Considering the key role of tonic inhibition in controlling neuronal excitability, reduced tonic inhibition could contribute to FXS-associated disorders such as hyperactivity, hypersensitivity, and increased seizure susceptibility. The current study has focused on the expression and function of the δ subunit of the GABA A R, a major subunit involved in tonic inhibition, in granule cells of the dentate gyrus in the Fmr1 knockout (KO) mouse model of FXS. Electrophysiological studies of dentate granule cells revealed a marked, nearly four-fold, decrease in tonic inhibition in the Fmr1 KO mice, as well as reduced effects of two δ subunit-preferring pharmacological agents, THIP and DS2, supporting the suggestion that δ subunit-containing GABA A Rs are compromised in the Fmr1 KO mice. Immunohistochemistry demonstrated a small but statistically significant decrease in δ subunit labeling in the molecular layer of the dentate gyrus in Fmr1 KO mice compared to wildtype (WT) littermates. The discrepancy between the large deficits in GABA-mediated tonic inhibition in granule cells in the Fmr1 KO mice and only modest reductions in immunolabeling of the δ subunit led to studies of surface expression of the δ subunit. Cross-linking experiments followed by Western blot analysis demonstrated a small, non-significant decrease in total δ subunit protein in the hippocampus of Fmr1 KO mice, but a four-fold decrease in surface expression of the δ subunit in these mice. No significant changes were observed in total or surface expression of the α4 subunit protein, a major partner of the δ subunit in the forebrain. Postembedding immunogold labeling for the δ subunit demonstrated a large, three-fold, decrease in the number of symmetric synapses with immunolabeling at perisynaptic locations in Fmr1 KO mice. While α4 immunogold particles were also reduced at perisynaptic locations in the Fmr1 KO mice, the labeling was increased at synaptic sites. Together these findings suggest that, in the dentate gyrus, altered surface expression of the δ subunit, rather than a decrease in δ subunit expression alone, could be limiting δ subunit-mediated tonic inhibition in this model of FXS. Finding ways to increase surface expression of the δ subunit of the GABA A R could be a novel approach to treatment of hyperexcitability-related alterations in FXS. Copyright © 2017 Elsevier Inc. All rights reserved.

Reduced rDNA Copy Number Does Not Affect “Competitive” Chromosome Pairing in XYY Males of Drosophila melanogaster

PubMed Central

Maggert, Keith A.

2014-01-01

The ribosomal DNA (rDNA) arrays are causal agents in X-Y chromosome pairing in meiosis I of Drosophila males. Despite broad variation in X-linked and Y-linked rDNA copy number, polymorphisms in regulatory/spacer sequences between rRNA genes, and variance in copy number of interrupting R1 and R2 retrotransposable elements, there is little evidence that different rDNA arrays affect pairing efficacy. I investigated whether induced rDNA copy number polymorphisms affect chromosome pairing in a “competitive” situation in which complex pairing configurations were possible using males with XYY constitution. Using a common normal X chromosome, one of two different full-length Y chromosomes, and a third chromosome from a series of otherwise-isogenic rDNA deletions, I detected no differences in X-Y or Y-Y pairing or chromosome segregation frequencies that could not be attributed to random variation alone. This work was performed in the context of an undergraduate teaching program at Texas A&M University, and I discuss the pedagogical utility of this and other such experiments. PMID:24449686

Ribulose-1,5-bis-phosphate carboxylase/oxygenase accumulation factor1 is required for holoenzyme assembly in maize.

PubMed

Feiz, Leila; Williams-Carrier, Rosalind; Wostrikoff, Katia; Belcher, Susan; Barkan, Alice; Stern, David B

2012-08-01

Most life is ultimately sustained by photosynthesis and its rate-limiting carbon fixing enzyme, ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco). Although the structurally comparable cyanobacterial Rubisco is amenable to in vitro assembly, the higher plant enzyme has been refractory to such manipulation due to poor understanding of its assembly pathway. Here, we report the identification of a chloroplast protein required for Rubisco accumulation in maize (Zea mays), RUBISCO ACCUMULATION FACTOR1 (RAF1), which lacks any characterized functional domains. Maize lines lacking RAF1 due to Mutator transposon insertions are Rubisco deficient and seedling lethal. Analysis of transcripts and proteins showed that Rubisco large subunit synthesis in raf1 plants is not compromised; however, newly synthesized Rubisco large subunit appears in a high molecular weight form whose accumulation requires a specific chaperonin 60 isoform. Gel filtration analysis and blue native gels showed that endogenous and recombinant RAF1 are trimeric; however, following in vivo cross-linking, RAF1 copurifies with Rubisco large subunit, suggesting that they interact weakly or transiently. RAF1 is predominantly expressed in bundle sheath chloroplasts, consistent with a Rubisco accumulation function. Our results support the hypothesis that RAF1 acts during Rubisco assembly by releasing and/or sequestering the large subunit from chaperonins early in the assembly process.

PCR amplification and sequences of cDNA clones for the small and large subunits of ADP-glucose pyrophosphorylase from barley tissues.

PubMed

Villand, P; Aalen, R; Olsen, O A; Lüthi, E; Lönneborg, A; Kleczkowski, L A

1992-06-01

Several cDNAs encoding the small and large subunit of ADP-glucose pyrophosphorylase (AGP) were isolated from total RNA of the starchy endosperm, roots and leaves of barley by polymerase chain reaction (PCR). Sets of degenerate oligonucleotide primers, based on previously published conserved amino acid sequences of plant AGP, were used for synthesis and amplification of the cDNAs. For either the endosperm, roots and leaves, the restriction analysis of PCR products (ca. 550 nucleotides each) has revealed heterogeneity, suggesting presence of three transcripts for AGP in the endosperm and roots, and up to two AGP transcripts in the leaf tissue. Based on the derived amino acid sequences, two clones from the endosperm, beps and bepl, were identified as coding for the small and large subunit of AGP, respectively, while a leaf transcript (blpl) encoded the putative large subunit of AGP. There was about 50% identity between the endosperm clones, and both of them were about 60% identical to the leaf cDNA. Northern blot analysis has indicated that beps and bepl are expressed in both the endosperm and roots, while blpl is detectable only in leaves. Application of the PCR technique in studies on gene structure and gene expression of plant AGP is discussed.

Correlation of 16S Ribosomal DNA Signature Sequences with Temperature-Dependent Growth Rates of Mesophilic and Psychrotolerant Strains of the Bacillus cereus Group

PubMed Central

Prüß, Birgit M.; Francis, Kevin P.; von Stetten, Felix; Scherer, Siegfried

1999-01-01

Sequences of the 16S ribosomal DNA (rDNA) from psychrotolerant and mesophilic strains of the Bacillus cereus group revealed signatures which were specific for these two thermal groups of bacteria. Further analysis of the genomic DNA from a wide range of food and soil isolates showed that B. cereus group strains have between 6 and 10 copies of 16S rDNA. Moreover, a number of these environmental strains have both rDNA operons with psychrotolerant signatures and rDNA operons with mesophilic signatures. The ability of these isolates to grow at low temperatures correlates with the prevalence of rDNA operons with psychrotolerant signatures, indicating specific nucleotides within the 16S rRNA to play a role in psychrotolerance. PMID:10198030

Contrasting patterns of evolution of 45S and 5S rDNA families uncover new aspects in the genome constitution of the agronomically important grass Thinopyrum intermedium (Triticeae).

PubMed

Mahelka, Václav; Kopecky, David; Baum, Bernard R

2013-09-01

We employed sequencing of clones and in situ hybridization (genomic and fluorescent in situ hybridization [GISH and rDNA-FISH]) to characterize both the sequence variation and genomic organization of 45S (herein ITS1-5.8S-ITS2 region) and 5S (5S gene + nontranscribed spacer) ribosomal DNA (rDNA) families in the allohexaploid grass Thinopyrum intermedium. Both rDNA families are organized within several rDNA loci within all three subgenomes of the allohexaploid species. Both families have undergone different patterns of evolution. The 45S rDNA family has evolved in a concerted manner: internal transcribed spacer (ITS) sequences residing within the arrays of two subgenomes out of three got homogenized toward one major ribotype, whereas the third subgenome contained a minor proportion of distinct unhomogenized copies. Homogenization mechanisms such as unequal crossover and/or gene conversion were coupled with the loss of certain 45S rDNA loci. Unlike in the 45S family, the data suggest that neither interlocus homogenization among homeologous chromosomes nor locus loss occurred in 5S rDNA. Consistently with other Triticeae, the 5S rDNA family in intermediate wheatgrass comprised two distinct array types-the long- and short-spacer unit classes. Within the long and short units, we distinguished five and three different types, respectively, likely representing homeologous unit classes donated by putative parental species. Although the major ITS ribotype corresponds in our phylogenetic analysis to the E-genome species, the minor ribotype corresponds to Dasypyrum. 5S sequences suggested the contributions from Pseudoroegneria, Dasypyrum, and Aegilops. The contribution from Aegilops to the intermediate wheatgrass' genome is a new finding with implications in wheat improvement. We discuss rDNA evolution and potential origin of intermediate wheatgrass.

Organization and variation analysis of 5S rDNA in different ploidy-level hybrids of red crucian carp × topmouth culter.

PubMed

He, Weiguo; Qin, Qinbo; Liu, Shaojun; Li, Tangluo; Wang, Jing; Xiao, Jun; Xie, Lihua; Zhang, Chun; Liu, Yun

2012-01-01

Through distant crossing, diploid, triploid and tetraploid hybrids of red crucian carp (Carassius auratus red var., RCC♀, Cyprininae, 2n = 100) × topmouth culter (Erythroculter ilishaeformis Bleeker, TC♂, Cultrinae, 2n = 48) were successfully produced. Diploid hybrids possessed 74 chromosomes with one set from RCC and one set from TC; triploid hybrids harbored 124 chromosomes with two sets from RCC and one set from TC; tetraploid hybrids had 148 chromosomes with two sets from RCC and two sets from TC. The 5S rDNA of the three different ploidy-level hybrids and their parents were sequenced and analyzed. There were three monomeric 5S rDNA classes (designated class I: 203 bp; class II: 340 bp; and class III: 477 bp) in RCC and two monomeric 5S rDNA classes (designated class IV: 188 bp, and class V: 286 bp) in TC. In the hybrid offspring, diploid hybrids inherited three 5S rDNA classes from their female parent (RCC) and only class IV from their male parent (TC). Triploid hybrids inherited class II and class III from their female parent (RCC) and class IV from their male parent (TC). Tetraploid hybrids gained class II and class III from their female parent (RCC), and generated a new 5S rDNA sequence (designated class I-N). The specific paternal 5S rDNA sequence of class V was not found in the hybrid offspring. Sequence analysis of 5S rDNA revealed the influence of hybridization and polyploidization on the organization and variation of 5S rDNA in fish. This is the first report on the coexistence in vertebrates of viable diploid, triploid and tetraploid hybrids produced by crossing parents with different chromosome numbers, and these new hybrids are novel specimens for studying the genomic variation in the first generation of interspecific hybrids, which has significance for evolution and fish genetics.

Analyses of the Large Subunit Histidine-Rich Motif Expose an Alternative Proton Transfer Pathway in [NiFe] Hydrogenases

PubMed Central

Szőri-Dorogházi, Emma; Maróti, Gergely; Szőri, Milán; Nyilasi, Andrea; Rákhely, Gábor; Kovács, Kornél L.

2012-01-01

A highly conserved histidine-rich region with unknown function was recognized in the large subunit of [NiFe] hydrogenases. The HxHxxHxxHxH sequence occurs in most membrane-bound hydrogenases, but only two of these histidines are present in the cytoplasmic ones. Site-directed mutagenesis of the His-rich region of the T. roseopersicina membrane-attached Hyn hydrogenase disclosed that the enzyme activity was significantly affected only by the replacement of the His104 residue. Computational analysis of the hydrogen bond network in the large subunits indicated that the second histidine of this motif might be a component of a proton transfer pathway including Arg487, Asp103, His104 and Glu436. Substitutions of the conserved amino acids of the presumed transfer route impaired the activity of the Hyn hydrogenase. Western hybridization was applied to demonstrate that the cellular level of the mutant hydrogenases was similar to that of the wild type. Mostly based on theoretical modeling, few proton transfer pathways have already been suggested for [NiFe] hydrogenases. Our results propose an alternative route for proton transfer between the [NiFe] active center and the surface of the protein. A novel feature of this model is that this proton pathway is located on the opposite side of the large subunit relative to the position of the small subunit. This is the first study presenting a systematic analysis of an in silico predicted proton translocation pathway in [NiFe] hydrogenases by site-directed mutagenesis. PMID:22511957

[18S-25S rDNA variation in tissue culture of some Gentiana L. species].

PubMed

Mel'nyk, V M; Andrieiev, I O; Spiridonova, K V; Strashniuk, N M; Kunakh, V A

2007-01-01

18S-25S rDNA of intact plants and tissue cultures of G. acaulis, G. punctata and G. lutea have been investigated by using blot-hybridization. The decrease of rDNA amount was found in the callus cultures as compared with the plants. In contrast to other species, G. lutea showed intragenome heterogeneity of rRNA genes as well as qualitative rDNA changes in tissue culture, in particular appearance of altered repeats. The relationship between the peculiarities of rRNA gene structure and their rearrangements in in vitro culture was suggested.

Tandem repeats of the 5' non-transcribed spacer of Tetrahymena rDNA function as high copy number autonomous replicons in the macronucleus but do not prevent rRNA gene dosage regulation.

PubMed Central

Pan, W J; Blackburn, E H

1995-01-01

The rRNA genes in the somatic macronucleus of Tetrahymena thermophila are normally on 21 kb linear palindromic molecules (rDNA). We examined the effect on rRNA gene dosage of transforming T.thermophila macronuclei with plasmid constructs containing a pair of tandemly repeated rDNA replication origin regions unlinked to the rRNA gene. A significant proportion of the plasmid sequences were maintained as high copy circular molecules, eventually consisting solely of tandem arrays of origin regions. As reported previously for cells transformed by a construct in which the same tandem rDNA origins were linked to the rRNA gene [Yu, G.-L. and Blackburn, E. H. (1990) Mol. Cell. Biol., 10, 2070-2080], origin sequences recombined to form linear molecules bearing several tandem repeats of the origin region, as well as rRNA genes. The total number of rDNA origin sequences eventually exceeded rRNA gene copies by approximately 20- to 40-fold and the number of circular replicons carrying only rDNA origin sequences exceeded rRNA gene copies by 2- to 3-fold. However, the rRNA gene dosage was unchanged. Hence, simply monitoring the total number of rDNA origin regions is not sufficient to regulate rRNA gene copy number. Images PMID:7784211

Evidence of birth-and-death evolution of 5S rRNA gene in Channa species (Teleostei, Perciformes).

PubMed

Barman, Anindya Sundar; Singh, Mamta; Singh, Rajeev Kumar; Lal, Kuldeep Kumar

2016-12-01

In higher eukaryotes, minor rDNA family codes for 5S rRNA that is arranged in tandem arrays and comprises of a highly conserved 120 bp long coding sequence with a variable non-transcribed spacer (NTS). Initially the 5S rDNA repeats are considered to be evolved by the process of concerted evolution. But some recent reports, including teleost fishes suggested that evolution of 5S rDNA repeat does not fit into the concerted evolution model and evolution of 5S rDNA family may be explained by a birth-and-death evolution model. In order to study the mode of evolution of 5S rDNA repeats in Perciformes fish species, nucleotide sequence and molecular organization of five species of genus Channa were analyzed in the present study. Molecular analyses revealed several variants of 5S rDNA repeats (four types of NTS) and networks created by a neighbor net algorithm for each type of sequences (I, II, III and IV) did not show a clear clustering in species specific manner. The stable secondary structure is predicted and upstream and downstream conserved regulatory elements were characterized. Sequence analyses also shown the presence of two putative pseudogenes in Channa marulius. Present study supported that 5S rDNA repeats in genus Channa were evolved under the process of birth-and-death.

Role of ART-27, a Novel Androgen Receptor Coactivator, in Normal Prostate and Prostate Cancer

DTIC Science & Technology

2005-04-01

associates with pro- teins that include RBP5, a subunit shared by RNA polymerases I, II , and Ill, an RBP5 binding protein called unconventional prefoldin ...of a large multiprotein complex that contains RNA polymerase II subunit 5, a subunit shared by all three RNA polymerases; unconventional prefoldin ...dithiothreitol; GRIP, glucocorticoid re- ceptor Interacting p rotein; HA, hemagglutinin; MMTV, mouse mamm ary tumor virus ; PAIS, partial AIS; SDS

Stalled RNAP-II molecules bound to non-coding rDNA spacers are required for normal nucleolus architecture.

PubMed

Freire-Picos, M A; Landeira-Ameijeiras, V; Mayán, María D

2013-07-01

The correct distribution of nuclear domains is critical for the maintenance of normal cellular processes such as transcription and replication, which are regulated depending on their location and surroundings. The most well-characterized nuclear domain, the nucleolus, is essential for cell survival and metabolism. Alterations in nucleolar structure affect nuclear dynamics; however, how the nucleolus and the rest of the nuclear domains are interconnected is largely unknown. In this report, we demonstrate that RNAP-II is vital for the maintenance of the typical crescent-shaped structure of the nucleolar rDNA repeats and rRNA transcription. When stalled RNAP-II molecules are not bound to the chromatin, the nucleolus loses its typical crescent-shaped structure. However, the RNAP-II interaction with Seh1p, or cryptic transcription by RNAP-II, is not critical for morphological changes. Copyright © 2013 John Wiley & Sons, Ltd.

Systematic analysis and evolution of 5S ribosomal DNA in metazoans.

PubMed

Vierna, J; Wehner, S; Höner zu Siederdissen, C; Martínez-Lage, A; Marz, M

2013-11-01

Several studies on 5S ribosomal DNA (5S rDNA) have been focused on a subset of the following features in mostly one organism: number of copies, pseudogenes, secondary structure, promoter and terminator characteristics, genomic arrangements, types of non-transcribed spacers and evolution. In this work, we systematically analyzed 5S rDNA sequence diversity in available metazoan genomes, and showed organism-specific and evolutionary-conserved features. Putatively functional sequences (12,766) from 97 organisms allowed us to identify general features of this multigene family in animals. Interestingly, we show that each mammal species has a highly conserved (housekeeping) 5S rRNA type and many variable ones. The genomic organization of 5S rDNA is still under debate. Here, we report the occurrence of several paralog 5S rRNA sequences in 58 of the examined species, and a flexible genome organization of 5S rDNA in animals. We found heterogeneous 5S rDNA clusters in several species, supporting the hypothesis of an exchange of 5S rDNA from one locus to another. A rather high degree of variation of upstream, internal and downstream putative regulatory regions appears to characterize metazoan 5S rDNA. We systematically studied the internal promoters and described three different types of termination signals, as well as variable distances between the coding region and the typical termination signal. Finally, we present a statistical method for detection of linkage among noncoding RNA (ncRNA) gene families. This method showed no evolutionary-conserved linkage among 5S rDNAs and any other ncRNA genes within Metazoa, even though we found 5S rDNA to be linked to various ncRNAs in several clades.

Systematic analysis and evolution of 5S ribosomal DNA in metazoans

PubMed Central

Vierna, J; Wehner, S; Höner zu Siederdissen, C; Martínez-Lage, A; Marz, M

2013-01-01

Several studies on 5S ribosomal DNA (5S rDNA) have been focused on a subset of the following features in mostly one organism: number of copies, pseudogenes, secondary structure, promoter and terminator characteristics, genomic arrangements, types of non-transcribed spacers and evolution. In this work, we systematically analyzed 5S rDNA sequence diversity in available metazoan genomes, and showed organism-specific and evolutionary-conserved features. Putatively functional sequences (12 766) from 97 organisms allowed us to identify general features of this multigene family in animals. Interestingly, we show that each mammal species has a highly conserved (housekeeping) 5S rRNA type and many variable ones. The genomic organization of 5S rDNA is still under debate. Here, we report the occurrence of several paralog 5S rRNA sequences in 58 of the examined species, and a flexible genome organization of 5S rDNA in animals. We found heterogeneous 5S rDNA clusters in several species, supporting the hypothesis of an exchange of 5S rDNA from one locus to another. A rather high degree of variation of upstream, internal and downstream putative regulatory regions appears to characterize metazoan 5S rDNA. We systematically studied the internal promoters and described three different types of termination signals, as well as variable distances between the coding region and the typical termination signal. Finally, we present a statistical method for detection of linkage among noncoding RNA (ncRNA) gene families. This method showed no evolutionary-conserved linkage among 5S rDNAs and any other ncRNA genes within Metazoa, even though we found 5S rDNA to be linked to various ncRNAs in several clades. PMID:23838690

Comparative analysis of bacteria associated with different mosses by 16S rRNA and 16S rDNA sequencing.

PubMed

Tian, Yang; Li, Yan Hong

2017-01-01

To understand the differences of the bacteria associated with different mosses, a phylogenetic study of bacterial communities in three mosses was carried out based on 16S rDNA and 16S rRNA sequencing. The mosses used were Hygroamblystegium noterophilum, Entodon compressus and Grimmia montana, representing hygrophyte, shady plant and xerophyte, respectively. In total, the operational taxonomic units (OTUs), richness and diversity were different regardless of the moss species and the library level. All the examined 1183 clones were assigned to 248 OTUs, 56 genera were assigned in rDNA libraries and 23 genera were determined at the rRNA level. Proteobacteria and Bacteroidetes were considered as the most dominant phyla in all the libraries, whereas abundant Actinobacteria and Acidobacteria were detected in the rDNA library of Entodon compressus and approximately 24.7% clones were assigned to Candidate division TM7 in Grimmia montana at rRNA level. The heatmap showed the bacterial profiles derived from rRNA and rDNA were partly overlapping. However, the principle component analysis of all the profiles derived from rDNA showed sharper differences between the different mosses than that of rRNA-based profiles. This suggests that the metabolically active bacterial compositions in different mosses were more phylogenetically similar and the differences of the bacteria associated with different mosses were mainly detected at the rDNA level. Obtained results clearly demonstrate that combination of 16S rDNA and 16S rRNA sequencing is preferred approach to have a good understanding on the constitution of the microbial communities in mosses. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[Comparative analysis between diatom nitric acid digestion method and plankton 16S rDNA PCR method].

PubMed

Han, Jun-ge; Wang, Cheng-bao; Li, Xing-biao; Fan, Yan-yan; Feng, Xiang-ping

2013-10-01

To compare and explore the application value of diatom nitric acid digestion method and plankton 16S rDNA PCR method for drowning identification. Forty drowning cases from 2010 to 2011 were collected from Department of Forensic Medicine of Wenzhou Medical University. Samples including lung, kidney, liver and field water from each case were tested with diatom nitric acid digestion method and plankton 16S rDNA PCR method, respectively. The Diatom nitric acid digestion method and plankton 16S rDNA PCR method required 20 g and 2 g of each organ, and 15 mL and 1.5 mL of field water, respectively. The inspection time and detection rate were compared between the two methods. Diatom nitric acid digestion method mainly detected two species of diatoms, Centriae and Pennatae, while plankton 16S rDNA PCR method amplified a length of 162 bp band. The average inspection time of each case of the Diatom nitric acid digestion method was (95.30 +/- 2.78) min less than (325.33 +/- 14.18) min of plankton 16S rDNA PCR method (P < 0.05). The detection rates of two methods for field water and lung were both 100%. For liver and kidney, the detection rate of plankton 16S rDNA PCR method was both 80%, higher than 40% and 30% of diatom nitric acid digestion method (P < 0.05), respectively. The laboratory testing method needs to be appropriately selected according to the specific circumstances in the forensic appraisal of drowning. Compared with diatom nitric acid digestion method, plankton 16S rDNA PCR method has practice values with such advantages as less quantity of samples, huge information and high specificity.

A redescription of Myxobolus inornatus from young-of-the-year smallmouth bass (Micropterus dolomieu).

PubMed

Walsh, H L; Blazer, V S; Iwanowicz, L R; Smith, G

2012-12-01

During investigations of young-of-the year smallmouth bass ( Micropterus dolomieu ) mortalities in the Susquehanna River, Pennsylvania, U.S.A. and affected tributaries, raised areas were noted in the muscle in the vicinity of the caudal peduncle. The raised areas were caused by plasmodia of a myxozoan parasite. Spores found within plasmodia were similar to those of Myxobolus inornatus previously described from the caudal peduncle of fingerling largemouth bass ( Micropterus salmoides ) in Montana. Here, M. inornatus is redescribed based on histologic comparisons and spore measurements. The addition of spore photographs, line drawings, a voucher specimen, and partial small-subunit ribosomal (rSSU) DNA gene sequence are new in this study. This is also the first description of M. inornatus from smallmouth bass. The plasmodia of M. inornatus were grossly observed at the base of the caudal and dorsal fins and were 280.3 ± 33.5 (range 77.1-920.3) μm long and 320.6 ± 41.0 (range 74.85-898.4) μm wide. In some instances, plasmodia of M. inornatus were large enough to rupture the epidermis or were associated with misaligned vertebrae. The slightly pyriform spores were 11.3 ± 0.2 (range 8.6-17.4) μm in length and 8.6 ± 0.2 (range 7.1-13.7) μm wide with an iodinophilous vacuole and a sutural ridge with 8 to 10 sutural folds. The SSU rDNA gene sequence places M. inornatus in a sister group with Myxobolus osburni .

FISH-Flow: a quantitative molecular approach for describing mixed clade communities of Symbiodinium

NASA Astrophysics Data System (ADS)

McIlroy, S. E.; Smith, G. J.; Geller, J. B.

2014-03-01

Our understanding of reef corals and their fate in a changing climate is limited by our ability to monitor the diversity and abundance of the dinoflagellate endosymbionts that sustain them. This study combined two well-known methods in tandem: fluorescent in situ hybridization (FISH) for genotype-specific labeling of Symbiodinium and flow cytometry to quantify the abundance of each symbiont clade in a sample. This technique (FISH-Flow) was developed with cultured Symbiodinium representing four distinct clades (based on large subunit rDNA) and was used to distinguish and quantify these types with high efficiency and few false positives. This technique was also applied to freshly isolated symbionts of Orbicella faveolata and Orbicella annularis. Isolates from acutely bleached coral tissues had significantly lower labeling efficiency; however, isolates from healthy tissue had efficiencies comparable to cultured Symbiodinium trials. RNA degradation in bleaching samples may have interfered with labeling of cells. Nevertheless, we were able to determine that, with and without thermal stress, experimental columns of the coral O. annularis hosted a majority of clade B and B/C symbionts on the top and side of the coral column, respectively. We demonstrated that, for cultured Symbiodinium and Symbiodinium freshly isolated from healthy host tissues, the relative ratio of clades could be accurately determined for clades present at as low as 7 % relative abundance. While this method does not improve upon PCR-based techniques in identifying clades at background levels, FISH-Flow provides a high precision, flexible system for targeting, quantifying and isolating Symbiodinium genotypes of interest.

Phylogenetic Diversity of Sponge-Associated Fungi from the Caribbean and the Pacific of Panama and Their In Vitro Effect on Angiotensin and Endothelin Receptors.

PubMed

Bolaños, Jessica; De León, Luis Fernando; Ochoa, Edgardo; Darias, José; Raja, Huzefa A; Shearer, Carol A; Miller, Andrew N; Vanderheyden, Patrick; Porras-Alfaro, Andrea; Caballero-George, Catherina

2015-10-01

Fungi occupy an important ecological niche in the marine environment, and marine fungi possess an immense biotechnological potential. This study documents the fungal diversity associated with 39 species of sponges and determines their potential to produce secondary metabolites capable of interacting with mammalian G-protein-coupled receptors involved in blood pressure regulation. Total genomic DNA was extracted from 563 representative fungal strains obtained from marine sponges collected by SCUBA from the Caribbean and the Pacific regions of Panama. A total of 194 operational taxonomic units were found with 58% represented by singletons based on the internal transcribed spacer (ITS) and partial large subunit (LSU) rDNA regions. Marine sponges were highly dominated by Ascomycota fungi (95.6%) and represented by two major classes, Sordariomycetes and Dothideomycetes. Rarefaction curves showed no saturation, indicating that further efforts are needed to reveal the entire diversity at this site. Several unique clades were found during phylogenetic analysis with the highest diversity of unique clades in the order Pleosporales. From the 65 cultures tested to determine their in vitro effect on angiotensin and endothelin receptors, the extracts of Fusarium sp. and Phoma sp. blocked the activation of these receptors by more than 50% of the control and seven others inhibited between 30 and 45%. Our results indicate that marine sponges from Panama are a "hot spot" of fungal diversity as well as a rich resource for capturing, cataloguing, and assessing the pharmacological potential of substances present in previously undiscovered fungi associated with marine sponges.

Genotype and Phenotype of Echinococcus granulosus Derived from Wild Sheep (Ovis orientalis) in Iran.

PubMed

Eslami, Ali; Meshgi, Behnam; Jalousian, Fatemeh; Rahmani, Shima; Salari, Mohammad Ali

2016-02-01

The aim of the present study is to determine the characteristics of genotype and phenotype of Echinococcus granulosus derived from wild sheep and to compare them with the strains of E. granulosus sensu stricto (sheep-dog) and E. granulosus camel strain (camel-dog) in Iran. In Khojir National Park, near Tehran, Iran, a fertile hydatid cyst was recently found in the liver of a dead wild sheep (Ovis orientalis). The number of protoscolices (n=6,000) proved enough for an experimental infection in a dog. The characteristics of large and small hooks of metacestode were statistically determined as the sensu stricto strain but not the camel strain (P=0.5). To determine E. granulosus genotype, 20 adult worms of this type were collected from the infected dog. The second internal transcribed spacer (ITS2) of the nuclear ribosomal DNA (rDNA) and cytochrome c oxidase 1 subunit (COX1) of the mitochondrial DNA were amplified from individual adult worm by PCR. Subsequently, the PCR product was sequenced by Sanger method. The lengths of ITS2 and COX1 sequences were 378 and 857 bp, respectively, for all the sequenced samples. The amplified DNA sequences from both ribosomal and mitochondrial genes were highly similar (99% and 98%, respectively) to that of the ovine strain in the GenBank database. The results of the present study indicate that the morpho-molecular features and characteristics of E. granulosus in the Iranian wild sheep are the same as those of the sheep-dog E. granulosus sensu stricto strain.

Immunohistochemical analyses of alpha1 and alpha3 Na+/K+-ATPase subunit expression in medulloblastomas.

PubMed

Suñol, Mariona; Cusi, Victoria; Cruz, Ofelia; Kiss, Robert; Lefranc, Florence

2011-03-01

The levels of expression of the α1 and α3 subunits of the Na(+)/K(+)-ATPase (the NaK sodium pump) in medulloblastomas are unclear. This study investigated the expression of the NaK subunits using immunohistochemical methods in 29 medulloblastomas including 23 classic, three large-cell/anaplastic and three nodular/desmoplastic medulloblastomas, as well as in three atypical teratoid/rhabdoid tumors (AT/RTs). There was overexpression of the α1 or α3 NaK subunits in more than half of the medulloblastomas and atypical AT/RTs, with about one-third of these tumours displaying overexpression of both subunits. These preliminary data suggest that targeting these subunits in AT/RTs and medulloblastomas that overexpress these proteins may lead to therapeutic benefit. These findings warrant confirmation in larger numbers of patients than those used in this study. Moreover, it should be determined whether inhibition of the α1/α3 NaK subunits can be integrated into the risk stratification schemes already in use for medulloblastoma patients.

DiBAC4(3) hits a “sweet spot” for the activation of arterial large-conductance Ca2+-activated potassium channels independently of the β1-subunit

PubMed Central

Scornik, Fabiana S.; Bucciero, Ronald S.; Wu, Yuesheng; Selga, Elisabet; Bosch Calero, Cristina; Brugada, Ramon

2013-01-01

The voltage-sensitive dye bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC4(3)] has been reported as a novel large-conductance Ca2+-activated K+ (BK) channel activator with selectivity for its β1- or β4-subunits. In arterial smooth muscle, BK channels are formed by a pore-forming α-subunit and a smooth muscle-abundant regulatory β1-subunit. This tissue specificity has driven extensive pharmacological research aimed at regulating arterial tone. Using animals with a disruption of the gene for the β1-subunit, we explored the effects of DiBAC4(3) in native channels from arterial smooth muscle. We tested the hypothesis that, in native BK channels, activation by DiBAC4(3) relies mostly on its α-subunit. We studied BK channels from wild-type and transgenic β1-knockout mice in excised patches. BK channels from brain arteries, with or without the β1-subunit, were similarly activated by DiBAC4(3). In addition, we found that saturating concentrations of DiBAC4(3) (∼30 μM) promote an unprecedented persistent activation of the channel that negatively shifts its voltage dependence by as much as −300 mV. This “sweet spot” for persistent activation is independent of Ca2+ and/or the β1–4-subunits and is fully achieved when DiBAC4(3) is applied to the intracellular side of the channel. Arterial BK channel response to DiBAC4(3) varies across species and/or vascular beds. DiBAC4(3) unique effects can reveal details of BK channel gating mechanisms and help in the rational design of BK channel activators. PMID:23542916

Inter-subunit electrostatic interactions in ferritin molecule: comparison with inter-molecular interactions in crystals

NASA Astrophysics Data System (ADS)

Takahashi, Takuya; Hogyoku, Michiru; Nagayama, Kuniaki

1996-10-01

We evaluated the contribution of electrostatic interactions to the stability of macromolecular assembly in a horse L ferritin molecule composed of 24 subunits and the three-dimensional crystal of the ferritin molecules with numerical calculation of Poisson-Boltzmann equation based on dielectric model. The calculation showed that the electrostatic energy both favors the assembly of the 24 subunits and the crystalline assembly of the ferritin molecules (i.e., 24-mers). Short-range interactions less than 5 Å such as salt bridges and hydrogen bonds were important for both the subunit assembly and the crystalline assembly. To elucidate the strong stabilization by electrostatic interactions in both the ferritin 24-mer and its crystal, we analyzed the contribution of individual atoms. It revealed that the stabilization was arising from buried salt bridges or hydrogen bonds, which yielded more than 5 kcal/mol in some interactions. These large electrostatic stabilization and also the unexpected small ionic strength dependence was different from those of bovine pancreatic trypsin inhibitor (BPTI) orthorhombic and pig-insulin cubic crystals previously calculated. We also evaluated changes of the accessible surface area (ASA) and hydration free energy in accordance with the process of the subunit assembly. The change of hydration free energy, which was very large (i.e. ˜ + 100 kcal/mol/subunit) and unfavorable for the assembly, was proportional to the electrostatic hydration energy (i.e. Born energy change in hydration process). Hydrophobic groups were likely to appear more frequently than hydrophilic groups at the subunit interfaces. These results suggest that the molecular structure of the ferritin 24-mer and the crystal structure of the 24-mers were both stabilized by local electrostatic interactions, in particular. We view protein crystals as an extension of the protein oligomer to an infinite number of subunits association.

Subunit mass fingerprinting of mitochondrial complex I.

PubMed

Morgner, Nina; Zickermann, Volker; Kerscher, Stefan; Wittig, Ilka; Abdrakhmanova, Albina; Barth, Hans-Dieter; Brutschy, Bernhard; Brandt, Ulrich

2008-10-01

We have employed laser induced liquid bead ion desorption (LILBID) mass spectrometry to determine the total mass and to study the subunit composition of respiratory chain complex I from Yarrowia lipolytica. Using 5-10 pmol of purified complex I, we could assign all 40 known subunits of this membrane bound multiprotein complex to peaks in LILBID subunit fingerprint spectra by comparing predicted protein masses to observed ion masses. Notably, even the highly hydrophobic subunits encoded by the mitochondrial genome were easily detectable. Moreover, the LILBID approach allowed us to spot and correct several errors in the genome-derived protein sequences of complex I subunits. Typically, the masses of the individual subunits as determined by LILBID mass spectrometry were within 100 Da of the predicted values. For the first time, we demonstrate that LILBID spectrometry can be successfully applied to a complex I band eluted from a blue-native polyacrylamide gel, making small amounts of large multiprotein complexes accessible for subunit mass fingerprint analysis even if they are membrane bound. Thus, the LILBID subunit mass fingerprint method will be of great value for efficient proteomic analysis of complex I and its assembly intermediates, as well as of other water soluble and membrane bound multiprotein complexes.

Increasing the starch content and grain weight of common wheat by overexpression of the cytosolic AGPase large subunit gene.

PubMed

Kang, Guozhang; Liu, Guoqin; Peng, Xiaoqi; Wei, Liting; Wang, Chenyang; Zhu, YunJi; Ma, Ying; Jiang, Yumei; Guo, Tiancai

2013-12-01

ADP-glucose pyrophosphorylase (AGPase) catalyzes the first committed step of starch synthesis. AGPase is a heterotetramer composed of two large subunits and two small subunits, has cytosolic and plastidial isoforms, and is detected mainly in the cytosol of endosperm in cereal crops. To investigate the effects of AGPase cytosolic large subunit gene (LSU I) on starch biosynthesis in higher plant, in this study, a TaLSU I gene from wheat was overexpressed under the control of an endosperm-specific promoter in a wheat cultivar (Yumai 34). PCR, Southern blot, and real-time RT-PCR analyses indicated that the transgene was integrated into the genome of transgenic plants and was overexpressed in their progeny. The overexpression of the TaLSU I gene remarkably enhanced AGPase activity, endosperm starch weight, grain number per spike, and single grain weight. Therefore, we conclude that overexpression of the TaLSU I gene enhances the starch biosynthesis in endosperm of wheat grains, having potential applications in wheat breeding to develop a high-yield wheat cultivar with high starch weight and kernel weight. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Cytochrome c oxidase subunit 1 barcode data of fish of the Nayband National Park in the Persian Gulf and analysis using meta-data flag several cryptic species.

PubMed

Asgharian, Hosseinali; Sahafi, Homayoun Hosseinzadeh; Ardalan, Aria Ashja; Shekarriz, Shahrokh; Elahi, Elahe

2011-05-01

We provide cytochrome c oxidase subunit 1 (COI) barcode sequences of fishes of the Nayband National Park, Persian Gulf, Iran. Industrial activities, ecological considerations and goals of The Fish Barcode of Life campaign make it crucial that fish species residing in the park be identified. To the best of our knowledge, this is the first report of barcoding data on fishes of the Persian Gulf. We examined 187 individuals representing 76 species, 56 genera and 32 families. The data flagged potentially cryptic species of Gerres filamentosus and Plectorhinchus schotaf. 16S rDNA data on these species are provided. Exclusion of these two potential cryptic species resulted in a mean COI intraspecific distance of 0.18%, and a mean inter- to intraspecific divergence ratio of 66.7. There was no overlap between maximum Kimura 2-parameter distances among conspecifics (1.66%) and minimum distance among congeneric species (6.19%). Barcodes shared among species were not observed. Neighbour-joining analysis showed that most species formed cohesive sequence units with little variation. Finally, the comparison of 16 selected species from this study with meta-data of conspecifics from Australia, India, China and South Africa revealed high interregion divergences and potential existence of six cryptic species. Pairwise interregional comparisons were more informative than global divergence assessments with regard to detection of cryptic variation. Our analysis exemplifies optimal use of the expanding barcode data now becoming available. © 2011 Blackwell Publishing Ltd.

Topography of succinate dehydrogenase in the mitochondrial inner membrane. A study using limited proteolysis and immunoblotting.

PubMed Central

Clarkson, G H; Neagle, J; Lindsay, J G

1991-01-01

The arrangement of the large (70,000-Mr) and small (30,000-Mr) subunits of succinate dehydrogenase in the mitochondrial inner membrane was investigated by immunoblot analysis of bovine heart mitochondria (right-side-out, outer membrane disrupted) or submitochondrial particles (inside-out) that had been subjected to surface-specific proteolysis. Both subunits were resistant to proteinase treatment provided that the integrity of the inner membrane was preserved, suggesting that neither subunit is exposed at the cytoplasmic surface of the membrane. The bulk of the small subunit appears to protrude into the matrix compartment, since the 30,000-Mr polypeptide is degraded extensively during limited proteolysis of submitochondrial particles without the appearance of an immunologically reactive membrane-associated fragment: moreover, a soluble 27,000-Mr peptide derived from this subunit is observed transiently on incubation with trypsin. Similar data obtained from the large subunit suggest that this polypeptide interacts with the matrix side of the inner membrane via two distinct domains; these are detected as stable membrane-associated fragments of 32,000 Mr and 27,000 Mr after treatment of submitochondrial particles with papain or proteinase K, although the 27,000-Mr fragment can be degraded further to low-Mr peptides with trypsin or alpha-chymotrypsin. A stable 32,000-34,000-Mr fragment is generated by a variety of specific and non-specific proteinases, indicating that it may be embedded largely within the lipid bilayer, or is inaccessible to proteolytic attack owing to its proximity to the surface of the intact membrane, possibly interacting with the hydrophobic membrane anchoring polypeptides of the succinate: ubiquinone reductase complex. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:1996968

Physical Localization and DNA Methylation of 45S rRNA Gene Loci in Jatropha curcas L.

PubMed Central

Gong, Zhiyun; Xue, Chao; Zhang, Mingliang; Guo, Rui; Zhou, Yong; Shi, Guoxin

2013-01-01

In eukaryotes, 45S rRNA genes are arranged in tandem arrays of repeat units, and not all copies are transcribed during mitosis. DNA methylation is considered to be an epigenetic marker for rDNA activation. Here, we established a clear and accurate karyogram for Jatropha curcas L. The chromosomal formula was found to be 2n = 2x = 22 = 12m+10sm. We found that the 45S rDNA loci were located at the termini of chromosomes 7 and 9 in J. curcas. The distribution of 45S rDNA has no significant difference in J. curcas from different sources. Based on the hybridization signal patterns, there were two forms of rDNA - dispersed and condensed. The dispersed type of signals appeared during interphase and prophase, while the condensed types appeared during different stages of mitosis. DNA methylation analysis showed that when 45S rDNA stronger signals were dispersed and connected to the nucleolus, DNA methylation levels were lower at interphase and prophase. However, when the 45S rDNA loci were condensed, especially during metaphase, they showed different forms of DNA methylation. PMID:24386362

Evolutionary insight on localization of 18S, 28S rDNA genes on homologous chromosomes in Primates genomes

PubMed Central

Mazzoleni, Sofia; Rovatsos, Michail; Schillaci, Odessa; Dumas, Francesca

2018-01-01

Abstract We explored the topology of 18S and 28S rDNA units by fluorescence in situ hybridization (FISH) in the karyotypes of thirteen species representatives from major groups of Primates and Tupaia minor (Günther, 1876) (Scandentia), in order to expand our knowledge of Primate genome reshuffling and to identify the possible dispersion mechanisms of rDNA sequences. We documented that rDNA probe signals were identified on one to six pairs of chromosomes, both acrocentric and metacentric ones. In addition, we examined the potential homology of chromosomes bearing rDNA genes across different species and in a wide phylogenetic perspective, based on the DAPI-inverted pattern and their synteny to human. Our analysis revealed an extensive variability in the topology of the rDNA signals across studied species. In some cases, closely related species show signals on homologous chromosomes, thus representing synapomorphies, while in other cases, signal was detected on distinct chromosomes, leading to species specific patterns. These results led us to support the hypothesis that different mechanisms are responsible for the distribution of the ribosomal DNA cluster in Primates. PMID:29416829

Sharp switches between regular and swinger mitochondrial replication: 16S rDNA systematically exchanging nucleotides A<->T+C<->G in the mitogenome of Kamimuria wangi.

PubMed

Seligmann, Hervé

2016-07-01

Swinger DNAs are sequences whose homology with known sequences is detected only by assuming systematic exchanges between nucleotides. Nine symmetric (X<->Y, i.e. A<->C) and fourteen asymmetric (X->Y->Z, i.e. A->C->G) exchanges exist. All swinger DNA previously detected in GenBank follow the A<->T+C<->G exchange, while mitochondrial swinger RNAs distribute among different swinger types. Here different alignment criteria detect 87 additional swinger mitochondrial DNAs (86 from insects), including the first swinger gene embedded within a complete genome, corresponding to the mitochondrial 16S rDNA of the stonefly Kamimuria wangi. Other Kamimuria mt genome regions are "regular", stressing unanswered questions on (a) swinger polymerization regulation; (b) swinger 16S rDNA functions; and (c) specificity to rDNA, in particular 16S rDNA. Sharp switches between regular and swinger replication, together with previous observations on swinger transcription, suggest that swinger replication might be due to a switch in polymerization mode of regular polymerases and the possibility of swinger-encoded information, predicted in primordial genes such as rDNA.

Conformational Sampling and Nucleotide-Dependent Transitions of the GroEL Subunit Probed by Unbiased Molecular Dynamics Simulations

PubMed Central

Skjaerven, Lars; Grant, Barry; Muga, Arturo; Teigen, Knut; McCammon, J. Andrew; Reuter, Nathalie; Martinez, Aurora

2011-01-01

GroEL is an ATP dependent molecular chaperone that promotes the folding of a large number of substrate proteins in E. coli. Large-scale conformational transitions occurring during the reaction cycle have been characterized from extensive crystallographic studies. However, the link between the observed conformations and the mechanisms involved in the allosteric response to ATP and the nucleotide-driven reaction cycle are not completely established. Here we describe extensive (in total long) unbiased molecular dynamics (MD) simulations that probe the response of GroEL subunits to ATP binding. We observe nucleotide dependent conformational transitions, and show with multiple 100 ns long simulations that the ligand-induced shift in the conformational populations are intrinsically coded in the structure-dynamics relationship of the protein subunit. Thus, these simulations reveal a stabilization of the equatorial domain upon nucleotide binding and a concomitant “opening” of the subunit, which reaches a conformation close to that observed in the crystal structure of the subunits within the ADP-bound oligomer. Moreover, we identify changes in a set of unique intrasubunit interactions potentially important for the conformational transition. PMID:21423709

Subunit interface dynamics in hexadecameric rubisco.

PubMed

van Lun, Michiel; van der Spoel, David; Andersson, Inger

2011-09-02

Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) plays an important role in the global carbon cycle as a hub for biomass. Rubisco catalyzes not only the carboxylation of RuBP with carbon dioxide but also a competing oxygenation reaction of RuBP with a negative impact on photosynthetic yield. The functional active site is built from two large (L) subunits that form a dimer. The octameric core of four L(2) dimers is held at each end by a cluster of four small (S) subunits, forming a hexadecamer. Each large subunit contacts more than one S subunit. These interactions exploit the dynamic flexibility of Rubisco, which we address in this study. Here, we describe seven different types of interfaces of hexadecameric Rubisco. We have analyzed these interfaces with respect to the size of the interface area and the number of polar interactions, including salt bridges and hydrogen bonds in a variety of Rubisco enzymes from different organisms and different kingdoms of life, including the Rubisco-like proteins. We have also performed molecular dynamics simulations of Rubisco from Chlamydomonas reinhardtii and mutants thereof. From our computational analyses, we propose structural checkpoints of the S subunit to ensure the functionality and/or assembly of the Rubisco holoenzyme. These checkpoints appear to fine-tune the dynamics of the enzyme in a way that could influence enzyme performance. Copyright © 2011 Elsevier Ltd. All rights reserved.

Cytochrome b in human complex II (succinate-ubiquinone oxidoreductase): cDNA cloning of the components in liver mitochondria and chromosome assignment of the genes for the large (SDHC) and small (SDHD) subunits to 1q21 and 11q23.

PubMed

Hirawake, H; Taniwaki, M; Tamura, A; Kojima, S; Kita, K

1997-01-01

Complex II (succinate-ubiquinone oxidoreductase) is an important enzyme complex in both the tricarboxylic acid cycle and the aerobic respiratory chains of mitochondria in eukaryotic cells and prokaryotic organisms. In this study, the amino acid sequences of the large (cybL) and small (cybS) subunits of cytochrome b in human liver complex II were deduced from cDNAs isolated by homology probing with mixed primers for the polymerase chain reaction. The mature cybL and cybS contain 140 and 103 amino acids, respectively, and show little similarity to the amino acid sequences of the subunits from other species in contrast to the highly conserved features of the flavoprotein (Fp) subunit and iron-sulfur protein (Ip) subunit. From hydrophobicity analysis, both cybL and cybS appear to have three transmembrane segments, indicating their role as membrane-anchors for the enzyme complex. Histidine residues, which are possible heme axial ligands in cytochrome b of complex II, were found in the second transmembrane segment of each subunit. The genes for cybL (SDHC) and cybS (SDHD) were mapped to chromosome 1q21 and 11q23, respectively by fluorescent in situ hybridization (FISH).

A protein with an inactive pterin-4a-carbinolamine dehydratase domain is required for Rubisco biogenesis in plants.

PubMed

Feiz, Leila; Williams-Carrier, Rosalind; Belcher, Susan; Montano, Monica; Barkan, Alice; Stern, David B

2014-12-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) plays a critical role in sustaining life by catalysis of carbon fixation in the Calvin-Benson pathway. Incomplete knowledge of the assembly pathway of chloroplast Rubisco has hampered efforts to fully delineate the enzyme's properties, or seek improved catalytic characteristics via directed evolution. Here we report that a Mu transposon insertion in the Zea mays (maize) gene encoding a chloroplast dimerization co-factor of hepatocyte nuclear factor 1 (DCoH)/pterin-4α-carbinolamine dehydratases (PCD)-like protein is the causative mutation in a seedling-lethal, Rubisco-deficient mutant named Rubisco accumulation factor 2 (raf2-1). In raf2 mutants newly synthesized Rubisco large subunit accumulates in a high-molecular weight complex, the formation of which requires a specific chaperonin 60-kDa isoform. Analogous observations had been made previously with maize mutants lacking the Rubisco biogenesis proteins RAF1 and BSD2. Chemical cross-linking of maize leaves followed by immunoprecipitation with antibodies to RAF2, RAF1 or BSD2 demonstrated co-immunoprecipitation of each with Rubisco small subunit, and to a lesser extent, co-immunoprecipitation with Rubisco large subunit. We propose that RAF2, RAF1 and BSD2 form transient complexes with the Rubisco small subunit, which in turn assembles with the large subunit as it is released from chaperonins. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

Photoinduced reduction of the medial FeS center in the hydrogenase small subunit HupS from Nostoc punctiforme.

PubMed

Raleiras, Patrícia; Hammarström, Leif; Lindblad, Peter; Styring, Stenbjörn; Magnuson, Ann

2015-07-01

The small subunit from the NiFe uptake hydrogenase, HupSL, in the cyanobacterium Nostoc punctiforme ATCC 29133, has been isolated in the absence of the large subunit (P. Raleiras, P. Kellers, P. Lindblad, S. Styring, A. Magnuson, J. Biol. Chem. 288 (2013) 18,345-18,352). Here, we have used flash photolysis to reduce the iron-sulfur clusters in the isolated small subunit, HupS. We used ascorbate as electron donor to the photogenerated excited state of Ru(II)-trisbipyridine (Ru(bpy)3), to generate Ru(I)(bpy)3 as reducing agent. Our results show that the isolated small subunit can be reduced by the Ru(I)(bpy)3 generated through flash photolysis. Copyright © 2015 Elsevier Inc. All rights reserved.

Ribulose-1,5-Bis-Phosphate Carboxylase/Oxygenase Accumulation Factor1 Is Required for Holoenzyme Assembly in Maize[C][W

PubMed Central

Feiz, Leila; Williams-Carrier, Rosalind; Wostrikoff, Katia; Belcher, Susan; Barkan, Alice; Stern, David B.

2012-01-01

Most life is ultimately sustained by photosynthesis and its rate-limiting carbon fixing enzyme, ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco). Although the structurally comparable cyanobacterial Rubisco is amenable to in vitro assembly, the higher plant enzyme has been refractory to such manipulation due to poor understanding of its assembly pathway. Here, we report the identification of a chloroplast protein required for Rubisco accumulation in maize (Zea mays), RUBISCO ACCUMULATION FACTOR1 (RAF1), which lacks any characterized functional domains. Maize lines lacking RAF1 due to Mutator transposon insertions are Rubisco deficient and seedling lethal. Analysis of transcripts and proteins showed that Rubisco large subunit synthesis in raf1 plants is not compromised; however, newly synthesized Rubisco large subunit appears in a high molecular weight form whose accumulation requires a specific chaperonin 60 isoform. Gel filtration analysis and blue native gels showed that endogenous and recombinant RAF1 are trimeric; however, following in vivo cross-linking, RAF1 copurifies with Rubisco large subunit, suggesting that they interact weakly or transiently. RAF1 is predominantly expressed in bundle sheath chloroplasts, consistent with a Rubisco accumulation function. Our results support the hypothesis that RAF1 acts during Rubisco assembly by releasing and/or sequestering the large subunit from chaperonins early in the assembly process. PMID:22942379

Characterization of a Novel Rieske-Type Alkane Monooxygenase System in Pusillimonas sp. Strain T7-7

PubMed Central

Li, Ping; Wang, Lei

2013-01-01

The cold-tolerant bacterium Pusillimonas sp. strain T7-7 is able to utilize diesel oils (C5 to C30 alkanes) as a sole carbon and energy source. In the present study, bioinformatics, proteomics, and real-time reverse transcriptase PCR approaches were used to identify the alkane hydroxylation system present in this bacterium. This system is composed of a Rieske-type monooxygenase, a ferredoxin, and an NADH-dependent reductase. The function of the monooxygenase, which consists of one large (46.711 kDa) and one small (15.355 kDa) subunit, was further studied using in vitro biochemical analysis and in vivo heterologous functional complementation tests. The purified large subunit of the monooxygenase was able to oxidize alkanes ranging from pentane (C5) to tetracosane (C24) using NADH as a cofactor, with greatest activity on the C15 substrate. The large subunit also showed activity on several alkane derivatives, including nitromethane and methane sulfonic acid, but it did not act on any aromatic hydrocarbons. The optimal reaction condition of the large subunit is pH 7.5 at 30°C. Fe2+ can enhance the activity of the enzyme evidently. This is the first time that an alkane monooxygenase system belonging to the Rieske non-heme iron oxygenase family has been identified in a bacterium. PMID:23417490

Structural changes of homodimers in the PDB.

PubMed

Koike, Ryotaro; Amemiya, Takayuki; Horii, Tatsuya; Ota, Motonori

2018-04-01

Protein complexes are involved in various biological phenomena. These complexes are intrinsically flexible, and structural changes are essential to their functions. To perform a large-scale automated analysis of the structural changes of complexes, we combined two original methods. An application, SCPC, compares two structures of protein complexes and decides the match of binding mode. Another application, Motion Tree, identifies rigid-body motions in various sizes and magnitude from the two structural complexes with the same binding mode. This approach was applied to all available homodimers in the Protein Data Bank (PDB). We defined two complex-specific motions: interface motion and subunit-spanning motion. In the former, each subunit of a complex constitutes a rigid body, and the relative movement between subunits occurs at the interface. In the latter, structural parts from distinct subunits constitute a rigid body, providing the relative movement spanning subunits. All structural changes were classified and examined. It was revealed that the complex-specific motions were common in the homodimers, detected in around 40% of families. The dimeric interfaces were likely to be small and flat for interface motion, while large and rugged for subunit-spanning motion. Interface motion was accompanied by a drastic change in contacts at the interface, while the change in the subunit-spanning motion was moderate. These results indicate that the interface properties of homodimers correlated with the type of complex-specific motion. The study demonstrates that the pipeline of SCPC and Motion Tree is useful for the massive analysis of structural change of protein complexes. Copyright © 2017 Elsevier Inc. All rights reserved.

A unique enhancer boundary complex on the mouse ribosomal RNA genes persists after loss of Rrn3 or UBF and the inactivation of RNA polymerase I transcription

PubMed Central

Stefanovsky, Victor Y.; Tremblay, Michel G.; Lindsay, Helen; Robinson, Mark D.

2017-01-01

Transcription of the several hundred of mouse and human Ribosomal RNA (rRNA) genes accounts for the majority of RNA synthesis in the cell nucleus and is the determinant of cytoplasmic ribosome abundance, a key factor in regulating gene expression. The rRNA genes, referred to globally as the rDNA, are clustered as direct repeats at the Nucleolar Organiser Regions, NORs, of several chromosomes, and in many cells the active repeats are transcribed at near saturation levels. The rDNA is also a hotspot of recombination and chromosome breakage, and hence understanding its control has broad importance. Despite the need for a high level of rDNA transcription, typically only a fraction of the rDNA is transcriptionally active, and some NORs are permanently silenced by CpG methylation. Various chromatin-remodelling complexes have been implicated in counteracting silencing to maintain rDNA activity. However, the chromatin structure of the active rDNA fraction is still far from clear. Here we have combined a high-resolution ChIP-Seq protocol with conditional inactivation of key basal factors to better understand what determines active rDNA chromatin. The data resolve questions concerning the interdependence of the basal transcription factors, show that preinitiation complex formation is driven by the architectural factor UBF (UBTF) independently of transcription, and that RPI termination and release corresponds with the site of TTF1 binding. They further reveal the existence of an asymmetric Enhancer Boundary Complex formed by CTCF and Cohesin and flanked upstream by phased nucleosomes and downstream by an arrested RNA Polymerase I complex. We find that the Enhancer Boundary Complex is the only site of active histone modification in the 45kbp rDNA repeat. Strikingly, it not only delimits each functional rRNA gene, but also is stably maintained after gene inactivation and the re-establishment of surrounding repressive chromatin. Our data define a poised state of rDNA chromatin and place the Enhancer Boundary Complex as the likely entry point for chromatin remodelling complexes. PMID:28715449

A unique enhancer boundary complex on the mouse ribosomal RNA genes persists after loss of Rrn3 or UBF and the inactivation of RNA polymerase I transcription.

PubMed

Herdman, Chelsea; Mars, Jean-Clement; Stefanovsky, Victor Y; Tremblay, Michel G; Sabourin-Felix, Marianne; Lindsay, Helen; Robinson, Mark D; Moss, Tom

2017-07-01

Transcription of the several hundred of mouse and human Ribosomal RNA (rRNA) genes accounts for the majority of RNA synthesis in the cell nucleus and is the determinant of cytoplasmic ribosome abundance, a key factor in regulating gene expression. The rRNA genes, referred to globally as the rDNA, are clustered as direct repeats at the Nucleolar Organiser Regions, NORs, of several chromosomes, and in many cells the active repeats are transcribed at near saturation levels. The rDNA is also a hotspot of recombination and chromosome breakage, and hence understanding its control has broad importance. Despite the need for a high level of rDNA transcription, typically only a fraction of the rDNA is transcriptionally active, and some NORs are permanently silenced by CpG methylation. Various chromatin-remodelling complexes have been implicated in counteracting silencing to maintain rDNA activity. However, the chromatin structure of the active rDNA fraction is still far from clear. Here we have combined a high-resolution ChIP-Seq protocol with conditional inactivation of key basal factors to better understand what determines active rDNA chromatin. The data resolve questions concerning the interdependence of the basal transcription factors, show that preinitiation complex formation is driven by the architectural factor UBF (UBTF) independently of transcription, and that RPI termination and release corresponds with the site of TTF1 binding. They further reveal the existence of an asymmetric Enhancer Boundary Complex formed by CTCF and Cohesin and flanked upstream by phased nucleosomes and downstream by an arrested RNA Polymerase I complex. We find that the Enhancer Boundary Complex is the only site of active histone modification in the 45kbp rDNA repeat. Strikingly, it not only delimits each functional rRNA gene, but also is stably maintained after gene inactivation and the re-establishment of surrounding repressive chromatin. Our data define a poised state of rDNA chromatin and place the Enhancer Boundary Complex as the likely entry point for chromatin remodelling complexes.

Characterization of copy numbers of 16S rDNA and 16S rRNA of Candidatus Liberibacter asiaticus and the implication in detection in planta using quantitative PCR.

PubMed

Kim, Jeong-Soon; Wang, Nian

2009-03-06

Citrus Huanglongbing (HLB) is one of the most devastating diseases on citrus and is associated with Candidatus Liberibacter spp.. The pathogens are phloem limited and have not been cultured in vitro. The current management strategy of HLB is to remove infected citrus trees and reduce psyllid populations with insecticides to prevent the spreading. This strategy requires sensitive and reliable diagnostic methods for early detection. We investigated the copy numbers of the 16S rDNA and 16S rRNA of the HLB pathogen and the implication of improving the diagnosis of HLB for early detection using Quantitative PCR. We compared the detection of HLB with different Quantitative PCR based methods with primers/probe targeting either 16S rDNA, beta-operon DNA, 16S rRNA, or beta-operon RNA. The 16S rDNA copy number of Ca. Liberibacter asiaticus was estimated to be three times of that of the beta-operon region, thus allowing detection of lower titer of Ca. L. asiaticus. Quantitative reverse transcriptional PCR (QRT-PCR) indicated that the 16S rRNA averaged 7.83 times more than that of 16S rDNA for the same samples. Dilution analysis also indicates that QRT-PCR targeting 16S rRNA is 10 time more sensitive than QPCR targeting 16S rDNA. Thus QRT-PCR was able to increase the sensitivity of detection by targeting 16S rRNA. Our result indicates that Candidatus Liberibacter asiaticus contains three copies of 16S rDNA. The copy number of 16S rRNA of Ca. L. asiaticus in planta averaged about 7.8 times of 16S rDNA for the same set of samples tested in this study. Detection sensitivity of HLB could be improved through the following approaches: using 16S rDNA based primers/probe in the QPCR assays; and using QRT-PCR assays targeting 16S rRNA.

Comparative physical mapping of 18S rDNA in the karyotypes of six leafcutter ant species of the genera Atta and Acromyrmex (Formicidae: Myrmicinae).

PubMed

Teixeira, Gisele Amaro; Barros, Luísa Antônia Campos; de Aguiar, Hilton Jeferson Alves Cardoso; das Graças Pompolo, Silvia

2017-10-01

Leafcutter ants of the Atta and Acromyrmex genera are important plagues in different cultures. Cytogenetic data on chromosome number, morphology, and chromosomal banding pattern are only available for 17 species of leafcutter ants. Molecular cytogenetic data for the detection of ribosomal genes by the FISH technique are scarce, and only 15 Neotropical ant species have been studied. This study aimed to physically map the 18S ribosomal RNA genes (rDNA) of six leafcutter ants belonging to the genera Atta and Acromyrmex using FISH. The results were compared with data on the fluorochrome CMA 3 currently available for these species. All analyzed species presented the 18S rDNA on one pair of chromosomes. In Acromyrmex subterraneus molestans and Ac. aspersus, FISH signals were observed in the terminal region of the short arm of the largest subtelocentric pair, while in Atta bisphaerica, A. laevigata, and A. sexdens, FISH signals were observed in the interstitial region of the long arm of the fourth metacentric pair. In Acromyrmex striatus, 18S rDNA was located in the interstitial region of the second metacentric pair. The karyotypic formula for Ac. aspersus was 2n = 38 (8m + 10sm + 16st + 4a), representing the first report in this species. The observed 18S rDNA regions in A. laevigata, A. sexdens, A. bisphaerica, Ac. aspersus, and Ac. subterraneus molestans corresponded to the CMA 3 + bands, while in Ac. striatus, several GC-rich bands and one pair of 18S rDNA bands were observed. No differential bands were visible using the DAPI fluorochrome. Karyotype uniformity with previously studied Atta spp. was also observed at the level of molecular cytogenetics using 18S rDNA FISH. A difference in the size of the chromosomal pair carrying the 18S rDNA gene was observed in Ac. striatus (2n = 22) and Atta spp. (2n = 22) highlighting the dissimilarity between these species. The results from the present study contribute to the description of 18S rDNA clusters in Neotropical ants.

Chromatin-Specific Regulation of Mammalian rDNA Transcription by Clustered TTF-I Binding Sites

PubMed Central

Diermeier, Sarah D.; Németh, Attila; Rehli, Michael; Grummt, Ingrid; Längst, Gernot

2013-01-01

Enhancers and promoters often contain multiple binding sites for the same transcription factor, suggesting that homotypic clustering of binding sites may serve a role in transcription regulation. Here we show that clustering of binding sites for the transcription termination factor TTF-I downstream of the pre-rRNA coding region specifies transcription termination, increases the efficiency of transcription initiation and affects the three-dimensional structure of rRNA genes. On chromatin templates, but not on free rDNA, clustered binding sites promote cooperative binding of TTF-I, loading TTF-I to the downstream terminators before it binds to the rDNA promoter. Interaction of TTF-I with target sites upstream and downstream of the rDNA transcription unit connects these distal DNA elements by forming a chromatin loop between the rDNA promoter and the terminators. The results imply that clustered binding sites increase the binding affinity of transcription factors in chromatin, thus influencing the timing and strength of DNA-dependent processes. PMID:24068958

Evolution of helotialean fungi (Leotiomycetes, Pezizomycotina): a nuclear rDNA phylogeny.

PubMed

Wang, Zheng; Binder, Manfred; Schoch, Conrad L; Johnston, Peter R; Spatafora, Joseph W; Hibbett, David S

2006-11-01

The highly divergent characters of morphology, ecology, and biology in the Helotiales make it one of the most problematic groups in traditional classification and molecular phylogeny. Sequences of three rDNA regions, SSU, LSU, and 5.8S rDNA, were generated for 50 helotialean fungi, representing 11 out of 13 families in the current classification. Data sets with different compositions were assembled, and parsimony and Bayesian analyses were performed. The phylogenetic distribution of lifestyle and ecological factors was assessed. Plant endophytism is distributed across multiple clades in the Leotiomycetes. Our results suggest that (1) the inclusion of LSU rDNA and a wider taxon sampling greatly improves resolution of the Helotiales phylogeny, however, the usefulness of rDNA in resolving the deep relationships within the Leotiomycetes is limited; (2) a new class Geoglossomycetes, including Geoglossum, Trichoglossum, and Sarcoleotia, is the basal lineage of the Leotiomyceta; (3) the Leotiomycetes, including the Helotiales, Erysiphales, Cyttariales, Rhytismatales, and Myxotrichaceae, is monophyletic; and (4) nine clades can be recognized within the Helotiales.

Karyotype divergence and spreading of 5S rDNA sequences between genomes of two species: darter and emerald gobies ( Ctenogobius , Gobiidae).

PubMed

Lima-Filho, P A; Bertollo, L A C; Cioffi, M B; Costa, G W W F; Molina, W F

2014-01-01

Karyotype analyses of the cryptobenthic marine species Ctenogobius boleosoma and C. smaragdus were performed by means of classical and molecular cytogenetics, including physical mapping of the multigene 18S and 5S rDNA families. C. boleosoma has 2n = 44 chromosomes (2 submetacentrics + 42 acrocentrics; FN = 46) with a single chromosome pair each carrying 18S and 5S ribosomal sites; whereas C. smaragdus has 2n = 48 chromosomes (2 submetacentrics + 46 acrocentrics; FN = 50), also with a single pair bearing 18S rDNA, but an extensive increase in the number of GC-rich 5S rDNA sites in 21 chromosome pairs. The highly divergent karyotypes among Ctenogobius species contrast with observations in several other marine fish groups, demonstrating an accelerated rate of chromosomal evolution mediated by both chromosomal rearrangements and the extensive dispersion of 5S rDNA sequences in the genome. © 2014 S. Karger AG, Basel.

Phylogenetic study on Shiraia bambusicola by rDNA sequence analyses.

PubMed

Cheng, Tian-Fan; Jia, Xiao-Ming; Ma, Xiao-Hang; Lin, Hai-Ping; Zhao, Yu-Hua

2004-01-01

In this study, 18S rDNA and ITS-5.8S rDNA regions of four Shiraia bambusicola isolates collected from different species of bamboos were amplified by PCR with universal primer pairs NS1/NS8 and ITS5/ITS4, respectively, and sequenced. Phylogenetic analyses were conducted on three selected datasets of rDNA sequences. Maximum parsimony, distance and maximum likelihood criteria were used to infer trees. Morphological characteristics were also observed. The positioning of Shiraia in the order Pleosporales was well supported by bootstrap, which agreed with the placement by Amano (1980) according to their morphology. We did not find significant inter-hostal differences among these four isolates from different species of bamboos. From the results of analyses and comparison of their rDNA sequences, we conclude that Shiraia should be classified into Pleosporales as Amano (1980) proposed and suggest that it might be positioned in the family Phaeosphaeriaceae. Copyright 2004 WILEY-VCH Verlag GmbH & Co.

Diverse Roles for Auxiliary Subunits in Phosphorylation-Dependent Regulation of Mammalian Brain Voltage-Gated Potassium Channels

PubMed Central

Vacher, Helene; Trimmer, James S.

2012-01-01

Voltage-gated ion channels are a diverse family of signaling proteins that mediate rapid electrical signaling events. Among these, voltage-gated potassium or Kv channels are the most diverse, in part due to the large number of principal (or α) subunits and auxiliary subunits that can assemble in different combinations to generate Kv channel complexes with distinct structures and functions. The diversity of Kv channels underlies much of the variability in the active properties between different mammalian central neurons, and the dynamic changes that lead to experience-dependent plasticity in intrinsic excitability. Recent studies have revealed that Kv channel α subunits and auxiliary subunits are extensively phosphorylated, contributing to additional structural and functional diversity. Here we highlight recent studies that show that auxiliary subunits exert some of their profound effects on dendritic Kv4 and axonal Kv1 channels through phosphorylation-dependent mechanisms, either due to phosphorylation on the auxiliary subunit itself, or by influencing the extent and/or impact of α subunit phosphorylation. The complex effects of auxiliary subunits and phosphorylation provide a potent mechanism to generate additional diversity in the structure and function of Kv4 and Kv1 channels, as well as allowing for dynamic reversible regulation of these important ion channels. PMID:21822597

Effects of 16S rDNA sampling on estimates of the number of endosymbiont lineages in sucking lice

PubMed Central

Burleigh, J. Gordon; Light, Jessica E.; Reed, David L.

2016-01-01

Phylogenetic trees can reveal the origins of endosymbiotic lineages of bacteria and detect patterns of co-evolution with their hosts. Although taxon sampling can greatly affect phylogenetic and co-evolutionary inference, most hypotheses of endosymbiont relationships are based on few available bacterial sequences. Here we examined how different sampling strategies of Gammaproteobacteria sequences affect estimates of the number of endosymbiont lineages in parasitic sucking lice (Insecta: Phthirapatera: Anoplura). We estimated the number of louse endosymbiont lineages using both newly obtained and previously sequenced 16S rDNA bacterial sequences and more than 42,000 16S rDNA sequences from other Gammaproteobacteria. We also performed parametric and nonparametric bootstrapping experiments to examine the effects of phylogenetic error and uncertainty on these estimates. Sampling of 16S rDNA sequences affects the estimates of endosymbiont diversity in sucking lice until we reach a threshold of genetic diversity, the size of which depends on the sampling strategy. Sampling by maximizing the diversity of 16S rDNA sequences is more efficient than randomly sampling available 16S rDNA sequences. Although simulation results validate estimates of multiple endosymbiont lineages in sucking lice, the bootstrap results suggest that the precise number of endosymbiont origins is still uncertain. PMID:27547523

Sequence data for two large-subunit rRNA genes from an Asian strain of Alexandrium catenella.

PubMed Central

Yeung, P K; Kong, K F; Wong, F T; Wong, J T

1996-01-01

PCR generated two distinct products from a toxic isolate of Alexandrium catenella, which had been taken from Dai Ya Bay (southern China), by using primers for large-subunit rRNA. This pattern is distinct from published data for North American Alexandrium species. Sequences of the two products suggest that the smaller was generated by a deletion event. Single-cell PCR generated the same pattern, confirming that the two products were not the results from different individuals. PMID:8900010

[Identification and phylogenetic analysis of one strain of Lactobacillus delbrueckii subsp. bulgaricus separated from yoghourt].

PubMed

Wang, Chuan; Zhang, Chaowu; Pei, Xiaofang; Liu, Hengchuan

2007-11-01

For being further applied and studied, one strain of Lactobacillus delbrueckii subsp. bulgaricus (wch9901) separated from yoghourt which had been identified by phenotype characteristic analysis was identified by 16S rDNA and phylogenetic analyzed. The 16S rDNA of wch9901 was amplified with the genomic DNA of wch9901 as template, and the conservative sequences of the 16S rDNA as primers. Inserted 16S rDNA amplified into clonal vector pGEM-T under the function of T4 DNA ligase to construct recombined plasmid pGEM-wch9901 16S rDNA. The recombined plasmid was identified by restriction enzyme digestion, and the eligible plasmid was presented to sequencing company for DNA sequencing. Nucleic acid sequence was blast in GenBank and phylogenetic tree was constructed using neighbor-joining method of distance methods by Mega3.1 soft. Results of blastn showed that the homology of 16S rDNA of wch9901 with the 16S rDNA of Lactobacillus delbrueckii subsp. bulgaricus strains was higher than 96%. On the phylogenetic tree, wch9901 formed a separate branch and located between Lactobacillus delbrueckii subsp. bulgaricus LGM2 evolution branch and another evolution branch which was composed of Lactobacillus delbrueckii subsp. bulgaricus DL2 evolution cluster and Lactobacillus delbrueckii subsp. bulgaricus JSQ evolution cluster. The distance between wch9901 evolution branch and Lactobacillus delbrueckii subsp. bulgaricus LGM2 evolution branch was the closest. wch9901 belonged to Lactobacillus delbrueckii subsp. bulgaricus. wch9901 showed the closest evolution relationship to Lactobacillus delbrueckii subsp. bulgaricus LGM2.

LEGO-NMR spectroscopy: a method to visualize individual subunits in large heteromeric complexes.

PubMed

Mund, Markus; Overbeck, Jan H; Ullmann, Janina; Sprangers, Remco

2013-10-18

Seeing the big picture: Asymmetric macromolecular complexes that are NMR active in only a subset of their subunits can be prepared, thus decreasing NMR spectral complexity. For the hetero heptameric LSm1-7 and LSm2-8 rings NMR spectra of the individual subunits of the complete complex are obtained, showing a conserved RNA binding site. This LEGO-NMR technique makes large asymmetric complexes accessible to detailed NMR spectroscopic studies. © 2013 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of Creative Commons the Attribution Non-Commercial NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Stopped-flow studies of changes in fluorescence of 8-anilino-1-naphthalene sulfonic acid caused by magnesium and salt binding to yeast enolase.

PubMed

Brewer, J M

1976-12-11

Stopped-flow studies of magnesium and salt (potassium chloride and acetate) effects on yeast enolase were carried out by following 8-anilino-1-naphthalenesulfonic acid fluorescence changes. The fluorescence changes appear to be largely caused by subunit association and dissociation, though there is evidence in some reactions for large changes in fluorescence occurring within the dead time of the stopped-flow measurements. These data are combined with measurements of initial enzyme activity after incubation in various solvents with or without magnesium to obtain subunit association and dissociation rates. From these, it is concluded that magnesium and the salts act by directly changing the affinities of the subunits for each other, apparently by producing a rapid change in protein conformation.

GABAA receptors: Various stoichiometrics of subunit arrangement in α1β3 and α1β3ε receptors.

PubMed

Has, Ahmad Tarmizi Che; Chebib, Mary

2018-05-15

GABAA receptors (GABAARs) are members of the Cys-loop ligand-gated ion channel (LGIC) superfamily, which includes nicotinic acetylcholine, glycine, and serotonin (5HT3) receptors [1,2,3,4]. LGICs typically mediate fast synaptic transmission via the movement of ions through channels gated by neurotransmitters, such as acetylcholine for nicotinic receptors and GABA for GABAARs [5]. The term Cys-loop receptors originates from the presence of a conserved disulphide bond (or bridge) which holds together two cysteine amino acids of the loop that forms from the structure of polypeptides in the extracellular domain of the receptor's subunit [6]. GABAARs are pentameric transmembrane protein complexes consisting of five subunits from a variety of polypeptide subunits [7,8]. All of these subunits are pseudo-symmetrically organized in the plane of the membrane, with a Cl--selective channel in the middle of the complex. To date, nineteen GABAAR subunits have been identified and categorized into eight classes, α1-6, β1-3, γ1-3, δ, ε, θ, π and ρ1-3, but their variety is further broadened by the existence of several splice forms for certain subunits (e.g., α6, β2 and γ2) [9,10,11,12]. The subunits within each class have an amino acid sequence homology of 70% or more, whereas those with a sequence homology of 30% or less are grouped into different classes [13,14]. A subunit consists of four transmembrane domains (TM1-TM4), each forming an α-helix; a large extracellular N-terminal domain that incorporates part of the orthosteric agonist/antagonist binding site; a large intracellular loop between the TM3 and TM4; a small intracellular loop between TM1 and TM2; a small extracellular loop between TM2 and TM3; and a C-terminal extracellular domain [15,16]. Each subunit is arranged in such a way as to create principal and complementary interfaces with the other subunits, and in a position such that the TM2 of each subunit forms the wall of the channel pore [17,18,19]. The major subunit combination found in the brain comprises α1, β2 and γ2 subunits with the stoichiometry 2α1:2β2:1γ2 [18,20]. For the GABAA α1β2γ2 receptors, the subunits form a specific arrangement in which α1 and β2 subunits alternate with each other and are connected by a γ2 subunit (Figure A) [16,20,21]. For minor subtypes, different α and β subunits have been detected to co-exist as proven by the existence of GABAARs containing α1α2, α1α3, α1α5, α2α3, α3α5, α4α1, α4α2 and α4α3 in the central nervous system [22,23]. Meanwhile, the same pattern has been detected with β and γ subunits, at least the co-occurrence of β in the same GABAAR as well as γ2 with γ3, indicating that these subunits have the capacity to co-exist with each other [24,25,26]. Since different subunits can actually occur in one receptor, GABAARs are considered to exist in a multi-subunit arrangement, leading to ambiguity in the determination of a receptor's stoichiometry. In this review, we first briefly discuss the different subunit arrangements of α1 and β3 subunits in the binary α1β3 receptors. Then we review the GABAA ε-containing receptors predominantly in terms of the ability of ε subunit to present in different position in the ternary α1β3ε receptors, which introduce distinct populations of receptor. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

In and out of the rRNA genes: characterization of Pokey elements in the sequenced Daphnia genome

PubMed Central

2013-01-01

Background Only a few transposable elements are known to exhibit site-specific insertion patterns, including the well-studied R-element retrotransposons that insert into specific sites within the multigene rDNA. The only known rDNA-specific DNA transposon, Pokey (superfamily: piggyBac) is found in the freshwater microcrustacean, Daphnia pulex. Here, we present a genome-wide analysis of Pokey based on the recently completed whole genome sequencing project for D. pulex. Results Phylogenetic analysis of Pokey elements recovered from the genome sequence revealed the presence of four lineages corresponding to two divergent autonomous families and two related lineages of non-autonomous miniature inverted repeat transposable elements (MITEs). The MITEs are also found at the same 28S rRNA gene insertion site as the Pokey elements, and appear to have arisen as deletion derivatives of autonomous elements. Several copies of the full-length Pokey elements may be capable of producing an active transposase. Surprisingly, both families of Pokey possess a series of 200 bp repeats upstream of the transposase that is derived from the rDNA intergenic spacer (IGS). The IGS sequences within the Pokey elements appear to be evolving in concert with the rDNA units. Finally, analysis of the insertion sites of Pokey elements outside of rDNA showed a target preference for sites similar to the specific sequence that is targeted within rDNA. Conclusions Based on the target site preference of Pokey elements and the concerted evolution of a segment of the element with the rDNA unit, we propose an evolutionary path by which the ancestors of Pokey elements have invaded the rDNA niche. We discuss how specificity for the rDNA unit may have evolved and how this specificity has played a role in the long-term survival of these elements in the subgenus Daphnia. PMID:24059783

First Molecular Identification and Phylogeny of Moroccan Anopheles sergentii (Diptera: Culicidae) Based on Second Internal Transcribed Spencer (ITS2) and Cytochrome c Oxidase I (COI) Sequences.

PubMed

Benabdelkrim Filali, Oumama; Kabine, Mostafa; El Hamouchi, Adil; Lemrani, Meryem; Debboun, Mustapha; Sarih, M'hammed

2018-06-05

Anopheles sergentii known as the "oasis vector" or the "desert malaria vector" is considered the main vector of malaria in the southern parts of Morocco. Its presence in Morocco is confirmed for the first time through sequencing of mitochondrial DNA (mDNA) cytochrome c oxidase subunit I (COI) barcodes and nuclear ribosomal DNA (rDNA) second internal transcribed spacer (ITS2) sequences and direct comparison with specimens of A. sergentii of other countries. The DNA barcodes (n = 39) obtained from A. sergentii collected in 2015 and 2016 showed more diversity with 10 haplotypes, compared with 3 haplotypes obtained from ITS2 sequences (n = 59). Moreover, the comparison using the ITS2 sequences showed closer evolutionary relationship between the Moroccan and Egyptian strains than the Iranian strain. Nevertheless, genetic differences due to geographical segregation were also observed. This study provides the first report on the sequence of rDNA-ITS2 and mtDNA COI, which could be used to better understand the biodiversity of A. sergentii.

Morphological and molecular characterization of Paramecium (Viridoparamecium nov. subgen.) chlorelligerum Kahl (Ciliophora).

PubMed

Kreutz, Martin; Stoeck, Thorsten; Foissner, Wilhelm

2012-01-01

We redescribe Paramecium chlorelligerum, a forgotten species, which Kahl (Tierwelt Dtl., 1935, 30:651) briefly but precisely described in the addendum to his ciliate monographs as a Paramecium with symbiotic green algae. The redescription is based on classical morphological methods and the analysis of the small subunit (SSU) rDNA. Morphologically, P. chlorelligerum differs from P. (C.) bursaria, the second green species in the genus, by having a special swimming shape, the length of the caudal cilia, the size of the micronucleus, the size of the symbiotic algae, the contractile vacuoles (with collecting vesicles vs. collecting canals), and the number of excretory pores/contractile vacuole (1 vs. 2-3). The molecular investigations show that P. chlorelligerum forms a distinct branch distant from the P. (Chloroparamecium) bursaria clade. Thus, we classify P. chlorelligerum in a new subgenus: Paramecium (Viridoparamecium) chlorelligerum. The symbiotic alga belongs to the little-known genus Meyerella, as yet recorded only from the plankton of a North American lake. © 2012 The Author(s) Journal of Eukaryotic Microbiology © 2012 International Society of Protistologists.

Comparative phylogenetic analyses of Halomonas variabilis and related organisms based on 16S rRNA, gyrB and ectBC gene sequences.

PubMed

Okamoto, Takuji; Maruyama, Akihiko; Imura, Satoshi; Takeyama, Haruko; Naganuma, Takeshi

2004-05-01

Halomonas variabilis and phylogenetically related organisms were isolated from various habitats such as Antarctic terrain and saline ponds, deep-sea sediment, deep-sea waters affected by hydrothermal plumes, and hydrothermal vent fluids. Ten strains were selected for physiological and phylogenetic characterization in detail. All of those strains were found to be piezotolerant and psychrotolerant, as well as euryhaline halophilic or halotolerant. Their stress tolerance may facilitate their wide occurrence, even in so-called extreme environments. The 16S rDNA-based phylogenetic relationship was complemented by analyses of the DNA gyrase subunit B gene (gyrB) and genes involved in the synthesis of the major compatible solute, ectoine: diaminobutyric acid aminotransferase gene (ectB) and ectoine synthase gene (ectC). The phylogenetic relationships of H. variabilis and related organisms were very similar in terms of 16S rDNA, gyrB, and ectB. The ectC-based tree was inconsistent with the other phylogenetic trees. For that reason, ectC was inferred to derive from horizontal transfer.

LIFE experiment: isolation of cryptoendolithic organisms from Antarctic colonized sandstone exposed to space and simulated Mars conditions on the international space station.

PubMed

Scalzi, Giuliano; Selbmann, Laura; Zucconi, Laura; Rabbow, Elke; Horneck, Gerda; Albertano, Patrizia; Onofri, Silvano

2012-06-01

Desiccated Antarctic rocks colonized by cryptoendolithic communities were exposed on the International Space Station (ISS) to space and simulated Mars conditions (LiFE-Lichens and Fungi Experiment). After 1.5 years in space samples were retrieved, rehydrated and spread on different culture media. Colonies of a green alga and a pink-coloured fungus developed on Malt-Agar medium; they were isolated from a sample exposed to simulated Mars conditions beneath a 0.1 % T Suprasil neutral density filter and from a sample exposed to space vacuum without solar radiation exposure, respectively. None of the other flight samples showed any growth after incubation. The two organisms able to grow were identified at genus level by Small SubUnit (SSU) and Internal Transcribed Spacer (ITS) rDNA sequencing as Stichococcus sp. (green alga) and Acarospora sp. (lichenized fungal genus) respectively. The data in the present study provide experimental information on the possibility of eukaryotic life transfer from one planet to another by means of rocks and of survival in Mars environment.

Three new species of Trichoderma with hyaline ascospores from China.

PubMed

Zhu, Z X; Zhuang, W Y

2015-01-01

Collections of Trichoderma having hyaline ascospores from different areas of China were examined. Using combined analyses of morphological data, culture characters and phylogenetic information based on rDNA sequences of partial nuc translation elongation factor 1-α encoding gene (TEF1-α) and the gene encoding the second largest nuc RNA polymerase subunit (RPB2), three new species, Trichoderma applanatum, T. oligosporum and T. sinoluteum, were discovered and are described. Trichoderma applanatum produces continuous flat to pulvinate, white to cream stromata with dense orange or pale brown ostioles, and simple acremonium-like to verticillium-like conidiophores, belongs to the Hypocreanum clade and is closely related to T. decipiens. Trichoderma oligosporum forms reddish brown stromata with a downy surface, hyaline conidia and gliocladium-like conidiophores, and is closely related to but distinct from T. crystalligenum in the Psychrophila clade. Trichoderma sinoluteum, as a member of the Polysporum clade, is characterized by pale yellow stromata, white pustulate conidiomata, pachybasium-like conidiophores, and hyaline conidia. Differences between the new species and their close relatives are discussed. © 2015 by The Mycological Society of America.

LIFE Experiment: Isolation of Cryptoendolithic Organisms from Antarctic Colonized Sandstone Exposed to Space and Simulated Mars Conditions on the International Space Station

NASA Astrophysics Data System (ADS)

Scalzi, Giuliano; Selbmann, Laura; Zucconi, Laura; Rabbow, Elke; Horneck, Gerda; Albertano, Patrizia; Onofri, Silvano

2012-06-01

Desiccated Antarctic rocks colonized by cryptoendolithic communities were exposed on the International Space Station (ISS) to space and simulated Mars conditions (LiFE— Lichens and Fungi Experiment). After 1.5 years in space samples were retrieved, rehydrated and spread on different culture media. Colonies of a green alga and a pink-coloured fungus developed on Malt-Agar medium; they were isolated from a sample exposed to simulated Mars conditions beneath a 0.1 % T Suprasil neutral density filter and from a sample exposed to space vacuum without solar radiation exposure, respectively. None of the other flight samples showed any growth after incubation. The two organisms able to grow were identified at genus level by Small SubUnit (SSU) and Internal Transcribed Spacer (ITS) rDNA sequencing as Stichococcus sp. (green alga) and Acarospora sp. (lichenized fungal genus) respectively. The data in the present study provide experimental information on the possibility of eukaryotic life transfer from one planet to another by means of rocks and of survival in Mars environment.

Morphological and molecular genetic diversity of Strongyluris calotis (Nematoda: Ascaridida: Heterakidae) in South East and East Asian lizards.

PubMed

Tran, Binh Thi; Ong, An Vinh; Luc, Pham Van; Sato, Hiroshi

2016-07-01

Strongyluris calotis is a heterakid nematode in the large intestine of agamid lizards (Reptilia: Sauria: Agamidae) from the Oriental Region. The standard light microscopic definition of the species counts the "caudal papillae" as 10 pairs on male worms. However, previous work from our group using scanning electron microscopy (SEM) on the heterakid from agamid lizards in Japan, Taiwan, and Singapore revealed that this counting contained a pair of phasmids and that two pairs of postcloacal papillae were completely fused to form a pair of united papillae, thus resulting in "10 pairs." In the present study, we examined S. calotis specimens from the Emma Gray's forest lizard, Calotes emma (Agamidae), living in the plain forest at low altitude, and the Vietnam false bloodsucker, Pseudocalotes brevipes (Agamidae), living in the mountainous forest at high altitude in the northern part of Vietnam. Using SEM, the arrangement of caudal papillae in male worms from an Emma Gray's forest lizard was found to be comparable to classical S. calotis specimens from agamid lizards collected in Japan, Taiwan, and Singapore. However, male worms from Vietnam false bloodsuckers did not have a pair of united papillae but had 10 pairs of independent caudal papillae with a pair of phasmids. Molecular genetic analyses of the ribosomal RNA gene (rDNA) of worms of the classical S. calotis morphotype from Japan and Singapore and two S. calotis morphotypes from Vietnam demonstrated absolutely identical nucleotide sequences of partial 18S rDNA (at least 1764 base pairs (bp)) and 5.8S rDNA (158 bp). However, intraspecific differences were detected in other regions of the rDNA, related to the geographical distribution of hosts regardless of morphotype: 97.8-98.5 % identity (443-446 bp/453 bp) in the internal transcribed spacer (ITS)-1 region, 96.6-98.0 % identity (425-431 bp/440 bp) in the ITS-2 region, and 99.6-99.7 % identity (1149-1151 bp/1154 bp) in the 28S rDNA. Thus, in the future, taxonomic relationships of S. calotis distributed widely in the Oriental Region as well as other nominal Oriental Strongyluris spp., currently six in number, need to be extensively explored based on molecular genetic analyses in addition to intensive morphological characterization.

Selectivity by host plants affects the distribution of arbuscular mycorrhizal fungi: evidence from ITS rDNA sequence metadata.

PubMed

Yang, Haishui; Zang, Yanyan; Yuan, Yongge; Tang, Jianjun; Chen, Xin

2012-04-12

Arbuscular mycorrhizal fungi (AMF) can form obligate symbioses with the vast majority of land plants, and AMF distribution patterns have received increasing attention from researchers. At the local scale, the distribution of AMF is well documented. Studies at large scales, however, are limited because intensive sampling is difficult. Here, we used ITS rDNA sequence metadata obtained from public databases to study the distribution of AMF at continental and global scales. We also used these sequence metadata to investigate whether host plant is the main factor that affects the distribution of AMF at large scales. We defined 305 ITS virtual taxa (ITS-VTs) among all sequences of the Glomeromycota by using a comprehensive maximum likelihood phylogenetic analysis. Each host taxonomic order averaged about 53% specific ITS-VTs, and approximately 60% of the ITS-VTs were host specific. Those ITS-VTs with wide host range showed wide geographic distribution. Most ITS-VTs occurred in only one type of host functional group. The distributions of most ITS-VTs were limited across ecosystem, across continent, across biogeographical realm, and across climatic zone. Non-metric multidimensional scaling analysis (NMDS) showed that AMF community composition differed among functional groups of hosts, and among ecosystem, continent, biogeographical realm, and climatic zone. The Mantel test showed that AMF community composition was significantly correlated with plant community composition among ecosystem, among continent, among biogeographical realm, and among climatic zone. The structural equation modeling (SEM) showed that the effects of ecosystem, continent, biogeographical realm, and climatic zone were mainly indirect on AMF distribution, but plant had strongly direct effects on AMF. The distribution of AMF as indicated by ITS rDNA sequences showed a pattern of high endemism at large scales. This pattern indicates high specificity of AMF for host at different scales (plant taxonomic order and functional group) and high selectivity from host plants for AMF. The effects of ecosystemic, biogeographical, continental and climatic factors on AMF distribution might be mediated by host plants.

Phylogenetic placement of the enigmatic parasite, Polypodium hydriforme, within the Phylum Cnidaria

PubMed Central

2008-01-01

Background Polypodium hydriforme is a parasite with an unusual life cycle and peculiar morphology, both of which have made its systematic position uncertain. Polypodium has traditionally been considered a cnidarian because it possesses nematocysts, the stinging structures characteristic of this phylum. However, recent molecular phylogenetic studies using 18S rDNA sequence data have challenged this interpretation, and have shown that Polypodium is a close relative to myxozoans and together they share a closer affinity to bilaterians than cnidarians. Due to the variable rates of 18S rDNA sequences, these results have been suggested to be an artifact of long-branch attraction (LBA). A recent study, using multiple protein coding markers, shows that the myxozoan Buddenbrockia, is nested within cnidarians. Polypodium was not included in this study. To further investigate the phylogenetic placement of Polypodium, we have performed phylogenetic analyses of metazoans with 18S and partial 28S rDNA sequences in a large dataset that includes Polypodium and a comprehensive sampling of cnidarian taxa. Results Analyses of a combined dataset of 18S and partial 28S sequences, and partial 28S alone, support the placement of Polypodium within Cnidaria. Removal of the long-branched myxozoans from the 18S dataset also results in Polypodium being nested within Cnidaria. These results suggest that previous reports showing that Polypodium and Myxozoa form a sister group to Bilateria were an artifact of long-branch attraction. Conclusion By including 28S rDNA sequences and a comprehensive sampling of cnidarian taxa, we demonstrate that previously conflicting hypotheses concerning the phylogenetic placement of Polypodium can be reconciled. Specifically, the data presented provide evidence that Polypodium is indeed a cnidarian and is either the sister taxon to Hydrozoa, or part of the hydrozoan clade, Leptothecata. The former hypothesis is consistent with the traditional view that Polypodium should be placed in its own cnidarian class, Polypodiozoa. PMID:18471296

Phylogenetic placement of the enigmatic parasite, Polypodium hydriforme, within the Phylum Cnidaria.

PubMed

Evans, Nathaniel M; Lindner, Alberto; Raikova, Ekaterina V; Collins, Allen G; Cartwright, Paulyn

2008-05-09

Polypodium hydriforme is a parasite with an unusual life cycle and peculiar morphology, both of which have made its systematic position uncertain. Polypodium has traditionally been considered a cnidarian because it possesses nematocysts, the stinging structures characteristic of this phylum. However, recent molecular phylogenetic studies using 18S rDNA sequence data have challenged this interpretation, and have shown that Polypodium is a close relative to myxozoans and together they share a closer affinity to bilaterians than cnidarians. Due to the variable rates of 18S rDNA sequences, these results have been suggested to be an artifact of long-branch attraction (LBA). A recent study, using multiple protein coding markers, shows that the myxozoan Buddenbrockia, is nested within cnidarians. Polypodium was not included in this study. To further investigate the phylogenetic placement of Polypodium, we have performed phylogenetic analyses of metazoans with 18S and partial 28S rDNA sequences in a large dataset that includes Polypodium and a comprehensive sampling of cnidarian taxa. Analyses of a combined dataset of 18S and partial 28S sequences, and partial 28S alone, support the placement of Polypodium within Cnidaria. Removal of the long-branched myxozoans from the 18S dataset also results in Polypodium being nested within Cnidaria. These results suggest that previous reports showing that Polypodium and Myxozoa form a sister group to Bilateria were an artifact of long-branch attraction. By including 28S rDNA sequences and a comprehensive sampling of cnidarian taxa, we demonstrate that previously conflicting hypotheses concerning the phylogenetic placement of Polypodium can be reconciled. Specifically, the data presented provide evidence that Polypodium is indeed a cnidarian and is either the sister taxon to Hydrozoa, or part of the hydrozoan clade, Leptothecata. The former hypothesis is consistent with the traditional view that Polypodium should be placed in its own cnidarian class, Polypodiozoa.

Implications of High Molecular Divergence of Nuclear rRNA and Phylogenetic Structure for the Dinoflagellate Prorocentrum (Dinophyceae, Prorocentrales).

PubMed

Boopathi, Thangavelu; Faria, Daphne Georgina; Cheon, Ju-Yong; Youn, Seok Hyun; Ki, Jang-Seu

2015-01-01

The small and large nuclear subunit molecular phylogeny of the genus Prorocentrum demonstrated that the species are dichotomized into two clades. These two clades were significantly different (one-factor ANOVA, p < 0.01) with patterns compatible for both small and large subunit Bayesian phylogenetic trees, and for a larger taxon sampled dinoflagellate phylogeny. Evaluation of the molecular divergence levels showed that intraspecies genetic variations were significantly low (t-test, p < 0.05), than those for interspecies variations (> 2.9% and > 26.8% dissimilarity in the small and large subunit [D1/D2], respectively). Based on the calculated molecular divergence, the genus comprises two genetically distinct groups that should be considered as two separate genera, thereby setting the pace for major systematic changes for the genus Prorocentrum sensu Dodge. Moreover, the information presented in this study would be useful for improving species identification, detection of novel clades from environmental samples. © 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists.

Human alpha 7 acetylcholine receptor: cloning of the alpha 7 subunit from the SH-SY5Y cell line and determination of pharmacological properties of native receptors and functional alpha 7 homomers expressed in Xenopus oocytes.

PubMed

Peng, X; Katz, M; Gerzanich, V; Anand, R; Lindstrom, J

1994-03-01

The alpha-bungarotoxin-binding acetylcholine receptors from the human neuroblastoma cell line SH-SY5Y were found to cross-react with some monoclonal antibodies to alpha 7 subunits of nicotinic acetylcholine receptors from chicken brain. The human alpha 7 subunit cDNA from SH-SY5Y was cloned, revealing 94% amino acid sequence identity to rat alpha 7 subunits and 92% identity to chicken alpha 7 subunits. Native human alpha 7 receptors showed affinities for some ligands similar to those previously observed with native chicken alpha 7 receptors, but for other ligands there were large species-specific differences in binding affinity. These results paralleled properties of alpha 7 homomers expressed in Xenopus oocytes. Human alpha 7 homomers exhibited rapidly desensitizing, inwardly rectifying, agonist-induced, cation currents that triggered Ca(2+)-sensitive Cl- channels in the oocytes. A change in efficacy from partial agonist for chicken alpha 7 homomers to full agonist for human alpha 7 homomers was exhibited by 1,1-dimethyl-4-phenylpiperazinium. This result reveals a large species-specific pharmacological difference, despite small differences in alpha 7 sequences. This is important for understanding the effects of these drugs in humans and for identifying amino acids that may contribute to the acetylcholine binding site, for analysis by in vitro mutagenesis. These results also characterize properties of native alpha 7 receptors and alpha 7 homomers that will provide criteria for functional properties expected of structural subunits, when these can be identified, cloned, and coexpressed with alpha 7 subunits.

The Purification of the Chlamydomonas reinhardtii chloroplast ClpP complex: additional subunits and structural features

PubMed Central

Derrien, Benoît; Majeran, Wojciech; Effantin, Grégory; Ebenezer, Joseph; Friso, Giulia; van Wijk, Klaas J.; Steven, Alasdair C.; Maurizi, Michael R.; Vallon, Olivier

2012-01-01

The ClpP peptidase is a major constituent of the proteolytic machinery of bacteria and organelles. The chloroplast ClpP complex is unusual, in that it associates a large number of subunits, one of which (ClpP1) is encoded in the chloroplast, the others in the nucleus. The complexity of these large hetero-oligomeric complexes has been a major difficulty in their overproduction and biochemical characterization. In this paper, we describe the purification of native chloroplast ClpP complex from the green alga Chlamydomonas reinhardtii, using a strain that carries the Strep-tag II at the C-terminus of the ClpP1 subunit. Similar to land plants, the algal complex comprises active and inactive subunits (3 ClpP and 5 ClpR, respectively). Evidence is presented that a sub-complex can be produced by dissociation, comprising ClpP1 and ClpR1, 2, 3 and 4, similar to the ClpR-ring described in land plants. Our Chlamydomonas ClpP preparation also contains two ClpT subunits, ClpT3 and ClpT4, which like the land plant ClpT1 and ClpT2 show 2 Clp-N domains. ClpTs are believed to function in substrate binding and/or assembly of the two heptameric rings. Phylogenetic analysis indicates that ClpT subunits have appeared independently in Chlorophycean algae, in land plants and in dispersed cyanobacterial genomes. Negative staining electron microscopy shows that the Chlamydomonas complex retains the barrel-like shape of homo-oligomeric ClpPs, with 4 additional peripheral masses that we speculate represent either the additional IS1 domain of ClpP1 (a feature unique to algae) or ClpTs or extensions of ClpR subunits PMID:22772861

Variation of 45S rDNA intergenic spacers in Arabidopsis thaliana.

PubMed

Havlová, Kateřina; Dvořáčková, Martina; Peiro, Ramon; Abia, David; Mozgová, Iva; Vansáčová, Lenka; Gutierrez, Crisanto; Fajkus, Jiří

2016-11-01

Approximately seven hundred 45S rRNA genes (rDNA) in the Arabidopsis thaliana genome are organised in two 4 Mbp-long arrays of tandem repeats arranged in head-to-tail fashion separated by an intergenic spacer (IGS). These arrays make up 5 % of the A. thaliana genome. IGS are rapidly evolving sequences and frequent rearrangements inside the rDNA loci have generated considerable interspecific and even intra-individual variability which allows to distinguish among otherwise highly conserved rRNA genes. The IGS has not been comprehensively described despite its potential importance in regulation of rDNA transcription and replication. Here we describe the detailed sequence variation in the complete IGS of A. thaliana WT plants and provide the reference/consensus IGS sequence, as well as genomic DNA analysis. We further investigate mutants dysfunctional in chromatin assembly factor-1 (CAF-1) (fas1 and fas2 mutants), which are known to have a reduced number of rDNA copies, and plant lines with restored CAF-1 function (segregated from a fas1xfas2 genetic background) showing major rDNA rearrangements. The systematic rDNA loss in CAF-1 mutants leads to the decreased variability of the IGS and to the occurrence of distinct IGS variants. We present for the first time a comprehensive and representative set of complete IGS sequences, obtained by conventional cloning and by Pacific Biosciences sequencing. Our data expands the knowledge of the A. thaliana IGS sequence arrangement and variability, which has not been available in full and in detail until now. This is also the first study combining IGS sequencing data with RFLP analysis of genomic DNA.

i-rDNA: alignment-free algorithm for rapid in silico detection of ribosomal gene fragments from metagenomic sequence data sets.

PubMed

Mohammed, Monzoorul Haque; Ghosh, Tarini Shankar; Chadaram, Sudha; Mande, Sharmila S

2011-11-30

Obtaining accurate estimates of microbial diversity using rDNA profiling is the first step in most metagenomics projects. Consequently, most metagenomic projects spend considerable amounts of time, money and manpower for experimentally cloning, amplifying and sequencing the rDNA content in a metagenomic sample. In the second step, the entire genomic content of the metagenome is extracted, sequenced and analyzed. Since DNA sequences obtained in this second step also contain rDNA fragments, rapid in silico identification of these rDNA fragments would drastically reduce the cost, time and effort of current metagenomic projects by entirely bypassing the experimental steps of primer based rDNA amplification, cloning and sequencing. In this study, we present an algorithm called i-rDNA that can facilitate the rapid detection of 16S rDNA fragments from amongst millions of sequences in metagenomic data sets with high detection sensitivity. Performance evaluation with data sets/database variants simulating typical metagenomic scenarios indicates the significantly high detection sensitivity of i-rDNA. Moreover, i-rDNA can process a million sequences in less than an hour on a simple desktop with modest hardware specifications. In addition to the speed of execution, high sensitivity and low false positive rate, the utility of the algorithmic approach discussed in this paper is immense given that it would help in bypassing the entire experimental step of primer-based rDNA amplification, cloning and sequencing. Application of this algorithmic approach would thus drastically reduce the cost, time and human efforts invested in all metagenomic projects. A web-server for the i-rDNA algorithm is available at http://metagenomics.atc.tcs.com/i-rDNA/

Changes in 5S rDNA Chromatin Organization and Transcription during Heterochromatin Establishment in Arabidopsis

PubMed Central

Mathieu, Olivier; Jasencakova, Zuzana; Vaillant, Isabelle; Gendrel, Anne-Valérie; Colot, Vincent; Schubert, Ingo; Tourmente, Sylvette

2003-01-01

In the Arabidopsis accession Columbia, 5S rDNA is located in the pericentromeric heterochromatin of chromosomes 3, 4, and 5. Both a major and some minor 5S rRNA species are expressed from chromosomes 4 and 5, whereas the genes on chromosome 3 are not transcribed. Here, we show that 5S rDNA methylation is reduced in 2-day-old seedlings versus 4-day-old or older aerial plant tissues, and the minor 5S rRNA species are expressed most abundantly at this stage. Similarly, when 5S rDNA is demethylated by 5-azacytidine treatment or via the decrease in DNA methylation1 (ddm1) mutation, the expression of minor 5S rRNA species is increased. We also show that in leaf nuclei of mature wild-type plants, the transcribed fraction of 5S rDNA forms loops that emanate from chromocenters. These loops, which are enlarged in nuclei of mature ddm1 plants, are enriched for histone H3 acetylated at Lys-9 and methylated at Lys-4 compared with the heterochromatic chromocenters. Up to 4 days after germination, heterochromatin is not fully developed: the 5S rDNA resides in prechromocenters, does not form conspicuous loops, and shows the lowest transcription level. Our results indicate that the expression and chromatin organization of 5S rRNA genes change during heterochromatin establishment. PMID:14630972

The 5S rDNA in two Abracris grasshoppers (Ommatolampidinae: Acrididae): molecular and chromosomal organization.

PubMed

Bueno, Danilo; Palacios-Gimenez, Octavio Manuel; Martí, Dardo Andrea; Mariguela, Tatiane Casagrande; Cabral-de-Mello, Diogo Cavalcanti

2016-08-01

The 5S ribosomal DNA (rDNA) sequences are subject of dynamic evolution at chromosomal and molecular levels, evolving through concerted and/or birth-and-death fashion. Among grasshoppers, the chromosomal location for this sequence was established for some species, but little molecular information was obtained to infer evolutionary patterns. Here, we integrated data from chromosomal and nucleotide sequence analysis for 5S rDNA in two Abracris species aiming to identify evolutionary dynamics. For both species, two arrays were identified, a larger sequence (named type-I) that consisted of the entire 5S rDNA gene plus NTS (non-transcribed spacer) and a smaller (named type-II) with truncated 5S rDNA gene plus short NTS that was considered a pseudogene. For type-I sequences, the gene corresponding region contained the internal control region and poly-T motif and the NTS presented partial transposable elements. Between the species, nucleotide differences for type-I were noticed, while type-II was identical, suggesting pseudogenization in a common ancestor. At chromosomal point to view, the type-II was placed in one bivalent, while type-I occurred in multiple copies in distinct chromosomes. In Abracris, the evolution of 5S rDNA was apparently influenced by the chromosomal distribution of clusters (single or multiple location), resulting in a mixed mechanism integrating concerted and birth-and-death evolution depending on the unit.

Secondary structure prediction for complete rDNA sequences (18S, 5.8S, and 28S rDNA) of Demodex folliculorum, and comparison of divergent domains structures across Acari.

PubMed

Zhao, Ya-E; Wang, Zheng-Hang; Xu, Yang; Wu, Li-Ping; Hu, Li

2013-10-01

According to base pairing, the rRNA folds into corresponding secondary structures, which contain additional phylogenetic information. On the basis of sequencing for complete rDNA sequences (18S, ITS1, 5.8S, ITS2 and 28S rDNA) of Demodex, we predicted the secondary structure of the complete rDNA sequence (18S, 5.8S, and 28S rDNA) of Demodex folliculorum, which was in concordance with that of the main arthropod lineages in past studies. And together with the sequence data from GenBank, we also predicted the secondary structures of divergent domains in SSU rRNA of 51 species and in LSU rRNA of 43 species from four superfamilies in Acari (Cheyletoidea, Tetranychoidea, Analgoidea and Ixodoidea). The multiple alignment among the four superfamilies in Acari showed that, insertions from Tetranychoidea SSU rRNA formed two newly proposed helixes, and helix c3-2b of LSU rRNA was absent in Demodex (Cheyletoidea) taxa. Generally speaking, LSU rRNA presented more remarkable differences than SSU rRNA did, mainly in D2, D3, D5, D7a, D7b, D8 and D10. Copyright © 2013 Elsevier Inc. All rights reserved.

Pnc1p-Mediated Nicotinamide Clearance Modifies the Epigenetic Properties of rDNA Silencing in Saccharomyces cerevisiae

PubMed Central

McClure, Julie M.; Gallo, Christopher M.; Smith, Daniel L.; Matecic, Mirela; Hontz, Robert D.; Buck, Stephen W.; Racette, Frances G.; Smith, Jeffrey S.

2008-01-01

The histone deacetylase activity of Sir2p is dependent on NAD+ and inhibited by nicotinamide (NAM). As a result, Sir2p-regulated processes in Saccharomyces cerevisiae such as silencing and replicative aging are susceptible to alterations in cellular NAD+ and NAM levels. We have determined that high concentrations of NAM in the growth medium elevate the intracellular NAD+ concentration through a mechanism that is partially dependent on NPT1, an important gene in the Preiss–Handler NAD+ salvage pathway. Overexpression of the nicotinamidase, Pnc1p, prevents inhibition of Sir2p by the excess NAM while maintaining the elevated NAD+ concentration. This growth condition alters the epigenetics of rDNA silencing, such that repression of a URA3 reporter gene located at the rDNA induces growth on media that either lacks uracil or contains 5-fluoroorotic acid (5-FOA), an unusual dual phenotype that is reminiscent of telomeric silencing (TPE) of URA3. Despite the similarities to TPE, the modified rDNA silencing phenotype does not require the SIR complex. Instead, it retains key characteristics of typical rDNA silencing, including RENT and Pol I dependence, as well as a requirement for the Preiss–Handler NAD+ salvage pathway. Exogenous nicotinamide can therefore have negative or positive impacts on rDNA silencing, depending on the PNC1 expression level. PMID:18780747

RNAi drives nonreciprocal translocations at eroding chromosome ends to establish telomere-free linear chromosomes.

PubMed

Begnis, Martina; Apte, Manasi S; Masuda, Hirohisa; Jain, Devanshi; Wheeler, David Lee; Cooper, Julia Promisel

2018-04-01

The identification of telomerase-negative HAATI (heterochromatin amplification-mediated and telomerase-independent) cells, in which telomeres are superseded by nontelomeric heterochromatin tracts, challenged the idea that canonical telomeres are essential for chromosome linearity and raised crucial questions as to how such tracts translocate to eroding chromosome ends and confer end protection. Here we show that HAATI arises when telomere loss triggers a newly recognized illegitimate translocation pathway that requires RNAi factors. While RNAi is necessary for the translocation events that mobilize ribosomal DNA (rDNA) tracts to all chromosome ends (forming "HAATI rDNA " chromosomes), it is dispensable for HAATI rDNA maintenance. Surprisingly, Dicer (Dcr1) plays a separate, RNAi-independent role in preventing formation of the rare HAATI subtype in which a different repetitive element (the subtelomeric element) replaces telomeres. Using genetics and fusions between shelterin components and rDNA-binding proteins, we mapped the mechanism by which rDNA loci engage crucial end protection factors-despite the absence of telomere repeats-and secure end protection. Sequence analysis of HAATI rDNA genomes allowed us to propose RNA and DNA polymerase template-switching models for the mechanism of RNAi-triggered rDNA translocations. Collectively, our results reveal unforeseen roles for noncoding RNAs (ncRNAs) in assembling a telomere-free chromosome end protection device. © 2018 Begnis et al.; Published by Cold Spring Harbor Laboratory Press.

Preferential cleavage sites for Sau3A restriction endonuclease in human ribosomal DNA.

PubMed

Kupriyanova, N S; Kirilenko, P M; Netchvolodov, K K; Ryskov, A P

2000-07-21

Previous studies of cloned ribosomal DNA (rDNA) variants isolated from the cosmid library of human chromosome 13 have revealed some disproportion in representativity of different rDNA regions (N. S. Kupriyanova, K. K. Netchvolodov, P. M. Kirilenko, B. I. Kapanadze, N. K. Yankovsky, and A. P. Ryskov, Mol. Biol. 30, 51-60, 1996). Here we show nonrandom cleavage of human rDNA with Sau3A or its isoshizomer MboI under mild hydrolysis conditions. The hypersensitive cleavage sites were found to be located in the ribosomal intergenic spacer (rIGS), especially in the regions of about 5-5.5 and 11 kb upstream of the rRNA transcription start point. This finding is based on sequencing mapping of the rDNA insert ends in randomly selected cosmid clones of human chromosome 13 and on the data of digestion kinetics of cloned and noncloned human genomic rDNA with Sau3A and MboI. The results show that a methylation status and superhelicity state of the rIGS have no effect on cleavage site sensitivity. It is interesting that all primary cleavage sites are adjacent to or entering into Alu or Psi cdc 27 retroposons of the rIGS suggesting a possible role of neighboring sequences in nuclease accessibility. The results explain nonequal representation of rDNA sequences in the human genomic DNA library used for this study. Copyright 2000 Academic Press.

Conopeptide Vt3.1 preferentially inhibits BK potassium channels containing β4 subunits via electrostatic interactions.

PubMed

Li, Min; Chang, Shan; Yang, Longjin; Shi, Jingyi; McFarland, Kelli; Yang, Xiao; Moller, Alyssa; Wang, Chunguang; Zou, Xiaoqin; Chi, Chengwu; Cui, Jianmin

2014-02-21

BK channel β subunits (β1-β4) modulate the function of channels formed by slo1 subunits to produce tissue-specific phenotypes. The molecular mechanism of how the homologous β subunits differentially alter BK channel functions and the role of different BK channel functions in various physiologic processes remain unclear. By studying channels expressed in Xenopus laevis oocytes, we show a novel disulfide-cross-linked dimer conopeptide, Vt3.1 that preferentially inhibits BK channels containing the β4 subunit, which is most abundantly expressed in brain and important for neuronal functions. Vt3.1 inhibits the currents by a maximum of 71%, shifts the G-V relation by 45 mV approximately half-saturation concentrations, and alters both open and closed time of single channel activities, indicating that the toxin alters voltage dependence of the channel. Vt3.1 contains basic residues and inhibits voltage-dependent activation by electrostatic interactions with acidic residues in the extracellular loops of the slo1 and β4 subunits. These results suggest a large interaction surface between the slo1 subunit of BK channels and the β4 subunit, providing structural insight into the molecular interactions between slo1 and β4 subunits. The results also suggest that Vt3.1 is an excellent tool for studying β subunit modulation of BK channels and for understanding the physiological roles of BK channels in neurophysiology.

Detection of a variable number of ribosomal DNA loci by fluorescent in situ hybridization in Populus species.

PubMed

Prado, E A; Faivre-Rampant, P; Schneider, C; Darmency, M A

1996-10-01

Fluorescent in situ hybridization (FISH) was applied to related Populus species (2n = 19) in order to detect rDNA loci. An interspecific variability in the number of hybridization sites was revealed using as probe an homologous 25S clone from Populus deltoides. The application of image analysis methods to measure fluorescence intensity of the hybridization signals has enabled us to characterize major and minor loci in the 18S-5.8S-25S rDNA. We identified one pair of such rDNA clusters in Populus alba; two pairs, one major and one minor, in both Populus nigra and P. deltoides; and three pairs in Populus balsamifera, (two major and one minor) and Populus euroamericana (one major and two minor). FISH results are in agreement with those based on RFLP analysis. The pBG13 probe containing 5S sequence from flax detected two separate clusters corresponding to the two size classes of units that coexist within 5S rDNA of most Populus species. Key words : Populus spp., fluorescent in situ hybridization, FISH, rDNA variability, image analysis.

Wnt5a Signals through DVL1 to Repress Ribosomal DNA Transcription by RNA Polymerase I.

PubMed

Dass, Randall A; Sarshad, Aishe A; Carson, Brittany B; Feenstra, Jennifer M; Kaur, Amanpreet; Obrdlik, Ales; Parks, Matthew M; Prakash, Varsha; Love, Damon K; Pietras, Kristian; Serra, Rosa; Blanchard, Scott C; Percipalle, Piergiorgio; Brown, Anthony M C; Vincent, C Theresa

2016-08-01

Ribosome biogenesis is essential for cell growth and proliferation and is commonly elevated in cancer. Accordingly, numerous oncogene and tumor suppressor signaling pathways target rRNA synthesis. In breast cancer, non-canonical Wnt signaling by Wnt5a has been reported to antagonize tumor growth. Here, we show that Wnt5a rapidly represses rDNA gene transcription in breast cancer cells and generates a chromatin state with reduced transcription of rDNA by RNA polymerase I (Pol I). These effects were specifically dependent on Dishevelled1 (DVL1), which accumulates in nucleolar organizer regions (NORs) and binds to rDNA regions of the chromosome. Upon DVL1 binding, the Pol I transcription activator and deacetylase Sirtuin 7 (SIRT7) releases from rDNA loci, concomitant with disassembly of Pol I transcription machinery at the rDNA promoter. These findings reveal that Wnt5a signals through DVL1 to suppress rRNA transcription. This provides a novel mechanism for how Wnt5a exerts tumor suppressive effects and why disruption of Wnt5a signaling enhances mammary tumor growth in vivo.

Subunit architecture and functional modular rearrangements of the transcriptional Mediator complex

PubMed Central

Tsai, Kuang-Lei; Tomomori-Sato, Chieri; Sato, Shigeo; Conaway, Ronald C.; Conaway, Joan W.; Asturias, Francisco J.

2014-01-01

SUMMARY The multisubunit Mediator comprising ~30 distinct proteins, plays an essential role in gene expression regulation by acting as a bridge between DNA binding transcription factors and the RNA polymerase II (RNAPII) transcription machinery. Efforts to uncover the Mediator mechanism have been hindered by a poor understanding of its structure, subunit organization, and conformational rearrangements. By overcoming biochemical and image analysis hurdles, we obtained accurate EM structures of yeast and human Mediators. Subunit localization experiments, docking of partial X-ray structures, and biochemical analyses resulted in comprehensive mapping of yeast Mediator subunits and a complete reinterpretation of our previous Mediator organization model. Large-scale Mediator rearrangements depend on changes at the interfaces between previously described Mediator modules, which appear to be facilitated by factors conducive to transcription initiation. Conservation across eukaryotes of Mediator structure, subunit organization, and RNA polymerase II interaction suggest conservation of fundamental aspects of the Mediator mechanism. PMID:24882805

Exploring the uncultured microeukaryote majority in the oceans: reevaluation of ribogroups within stramenopiles.

PubMed

Massana, Ramon; del Campo, Javier; Sieracki, Michael E; Audic, Stéphane; Logares, Ramiro

2014-04-01

Molecular surveys in planktonic marine systems have unveiled a large novel diversity of small protists. A large part of this diversity belongs to basal heterotrophic stramenopiles and is distributed in a set of polyphyletic ribogroups (described from rDNA sequences) collectively named as MAST (MArine STramenopiles). In the few groups investigated, MAST cells are globally distributed and abundant bacterial grazers, therefore having a putatively large impact on marine ecosystem functioning. The main aim of this study is to reevaluate the MAST ribogroups described so far and to determine whether additional groups can be found. For this purpose, we used traditional and state-of-the-art molecular tools, combining 18S rDNA sequences from publicly available clone libraries, single amplified genomes (SAGs) of planktonic protists, and a pyrosequencing survey from coastal waters and sediments. Our analysis indicated a final set of 18 MAST groups plus 5 new ribogroups within Ochrophyta (named as MOCH). The MAST ribogroups were then analyzed in more detail. Seven were typical of anoxic systems and one of oxic sediments. The rest were clearly members of oxic marine picoplankton. We characterized the genetic diversity within each MAST group and defined subclades for the more diverse (46 subclades in 8 groups). The analyses of sequences within subclades revealed further ecological specializations. Our data provide a renovated framework for phylogenetic classification of the numerous MAST ribogroups and support the notion of a tight link between phylogeny and ecological distribution. These diverse and largely uncultured protists are widespread and ecologically relevant members of marine microbial assemblages.

Genetic Diversity and Phylogenetic Relationships of Coevolving Symbiont-Harboring Insect Trypanosomatids, and Their Neotropical Dispersal by Invader African Blowflies (Calliphoridae)

PubMed Central

Borghesan, Tarcilla C.; Campaner, Marta; Matsumoto, Tania E.; Espinosa, Omar A.; Razafindranaivo, Victor; Paiva, Fernando; Carranza, Julio C.; Añez, Nestor; Neves, Luis; Teixeira, Marta M. G.; Camargo, Erney P.

2018-01-01

This study is about the inter- and intra-specific genetic diversity of trypanosomatids of the genus Angomonas, and their association with Calliphoridae (blowflies) in Neotropical and Afrotropical regions. Microscopic examination of 3,900 flies of various families, mostly Calliphoridae, revealed that 31% of them harbored trypanosomatids. Small subunit rRNA (SSU rRNA) barcoding showed that Angomonas predominated (46%) over the other common trypanosomatids of blowflies of genera Herpetomonas and Wallacemonas. Among Angomonas spp., A. deanei was much more common than the two-other species, A. desouzai and A. ambiguus. Phylogenetic analyses based on SSU rRNA, glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) and internal transcribed spacer rDNA (ITS rDNA) sequences revealed a marked genetic diversity within A. deanei, which comprised four infraspecific genotypes (Dea1–Dea4), and four corresponding symbiont genotypes (Kcr1–Kcr4). Host and symbiont phylogenies were highly congruent corroborating their co-divergence, consistent with host-symbiont interdependent metabolism and symbiont reduced genomes shaped by a long coevolutionary history. We compared the diversity of Angomonas/symbionts from three genera of blowflies, Lucilia, Chrysomya and Cochliomyia. A. deanei, A. desouzai, and A. ambiguus were found in the three genera of blowflies in South America. In Africa, A. deanei and A. ambiguus were identified in Chrysomya. The absence of A. desouzai in Africa and its presence in Neotropical Cochliomyia and Lucilia suggests parasite spillback of A. desouzai into Chrysomya, which was most likely introduced four decades ago from Africa into the Neotropic. The absence of correlation between parasite diversity and geographic and genetic distances, with identical genotypes of A. deanei found in the Neotropic and Afrotropic, is consistent with disjunct distribution due to the recent human-mediated transoceanic dispersal of Angomonas by Chrysomya. This study provides the most comprehensive data gathered so far on the genetic repertoires of a genus of trypanosomatids found in flies from a wide geographical range. PMID:29467742

Human Babesiosis in Japan: Epizootiologic Survey of Rodent Reservoir and Isolation of New Type of Babesia microti-Like Parasite

PubMed Central

Tsuji, Masayoshi; Wei, Qiang; Zamoto, Aya; Morita, Chiharu; Arai, Satoru; Shiota, Tsunezo; Fujimagari, Masato; Itagaki, Asao; Fujita, Hiromi; Ishihara, Chiaki

2001-01-01

We have carried out epizootiologic surveys at various sites in Japan to investigate wild animals that serve as reservoirs for the agents of human babesiosis in the country. Small mammals comprising six species, Apodemus speciosus, Apodemus argenteus, Clethrionomys rufocanus, Eothenomys smithii, Crocidura dsinezumi, and Sorex unguiculatus, were trapped at various places, including Hokkaido, Chiba, Shiga, Hyogo, Shimane, and Tokushima Prefectures. Animals harboring Babesia microti-like parasites were detected in all six prefectures. Inoculation of their blood samples into hamsters gave rise to a total of 20 parasite isolates; 19 were from A. speciosus, and the other 1 was from C. rufocanus. Sequencing of the parasite small-subunit rRNA gene (rDNA) sequence revealed that 2 of the 20 isolates were classified as Kobe type because their rDNAs were identical to that of the Kobe strain (the strain from the Japanese index case). The other 18 isolates were classified as a new type, designated the Hobetsu type, because they all shared an identical rDNA sequence which differed significantly from both that of Kobe-type isolates and that of northeastern United States B. microti (U.S. type). The parasites with Kobe-, Hobetsu- and U.S.-type rDNAs were phylogenetically closely related to each other but clearly different from each other antigenically. The isolates from rodents were demonstrated to be infective for human erythrocytes by inoculation into SCID mice whose erythrocytes had been replaced with human erythrocytes. The results suggest that a new type of B. microti-like parasite, namely, the Hobetsu type, is the major one which is prevalent among Japanese wild rodents, that A. speciosus serves as a major reservoir for both Kobe- and Hobetsu-type B. microti-like parasites, and that C. rufocanus may also be an additional reservoir on Hokkaido Island. PMID:11724838

Molecular identification of Blastocystis sp. subtypes in water samples collected from Black sea, Turkey.

PubMed

Koloren, Zeynep; Gulabi, Berivan Basak; Karanis, Panagiotis

2018-04-01

The aim of this study was to identify the subtypes of Blastocystis sp. and complete a phylogenetic analysis of 268 water samples that were collected from the Samsun, Amasya and Sinop Provinces of the Black Sea in Turkey, between the years 2011 and 2014. Blastocystis sp. was investigated in 48 uncultured sea water samples that were collected from 4 sites within the Sinop Province. A total of 100 river water samples were collected from 37 sites in the Samsun Province and 120 river water samples were collected from 10 sampling sites within the Amasya Province. The small subunit (SSU) rDNA gene Polymerase chain reaction (PCR) were performed for the detection of Blastocytis sp. and the PCR-positive samples were sequenced. Subsequently, the (SSU) rDNA sequences were aligned by Bioedit and phylogenetic trees were constructed for Blastocystis with reference to the genotypes from GenBank. Blastocystis sp. were found in 3 out of the 75 (4%) river water samples that were collected from the Samsun Province. Six of the 120 (5%) river water samples and 1 out of the 48 (2%) seawater samples were positive for Blastocystis in the Amasya and Sinop Provinces. There were two different subtypes (ST; 1 and 3) found from sequencing all of the samples from the investigated sites. Two and one PCR products were found to be positive for ST1 and ST3 from the different samples collected within the Samsun Province. Two and 4 PCR products from the Amasya Province were ST1 and ST3, respectively and only one sample from the Sinop Province was found to be positive for ST1. This is the first report to identify and report the occurrence of Blastocystis subtypes within the Black Sea of Turkey. Copyright © 2018 Elsevier B.V. All rights reserved.

Molecular genetic diversity of Gongylonema neoplasticum (Fibiger & Ditlevsen, 1914) (Spirurida: Gongylonematidae) from rodents in Southeast Asia.

PubMed

Setsuda, Aogu; Ribas, Alexis; Chaisiri, Kittipong; Morand, Serge; Chou, Monidarin; Malbas, Fidelino; Yunus, Muchammad; Sato, Hiroshi

2018-03-01

More than a dozen Gongylonema spp. (Spirurida: Spiruroidea: Gongylonematidae) have been described from a variety of rodent hosts worldwide. Gongylonema neoplasticum (Fibiger & Ditlevsen, 1914), which dwells in the gastric mucosa of rats such as Rattus norvegicus (Berkenhout) and Rattus rattus (Linnaeus), is currently regarded as a cosmopolitan nematode in accordance with global dispersion of its definitive hosts beyond Asia. To facilitate the reliable specific differentiation of local rodent Gongylonema spp. from the cosmopolitan congener, the genetic characterisation of G. neoplasticum from Asian Rattus spp. in the original endemic area should be considered since the morphological identification of Gongylonema spp. is often difficult due to variations of critical phenotypical characters, e.g. spicule lengths and numbers of caudal papillae. In the present study, morphologically identified G. neoplasticum from 114 rats of seven species from Southeast Asia were selected from archived survey materials from almost 4,500 rodents: Thailand (58 rats), Cambodia (52 rats), Laos (three rats) and Philippines (one rat). In addition, several specimens from four rats in Indonesia were used in the study. Nucleotide sequences of the ribosomal RNA gene (rDNA) (5,649 bp) and the cytochrome c oxidase subunit 1 gene (cox1) (818 bp) were characterised. The rDNA showed little nucleotide variation, including the internal transcribed spacer (ITS) regions. The cox1 showed 24 haplotypes, with up to 15 (1.83%) nucleotide substitutions regardless of parasite origin. Considering that Rattus spp. have been shown to originate from the southern region of Asia and G. neoplasticum is their endogenous parasite, it is reasonable to propose that the present study covers a wide spectrum of the genetic diversity of G. neoplasticum, useful for both the molecular genetic speculation of the species and the molecular genetic differentiation of other local rodent Gongylonema spp. from the cosmopolitan congener.

Non-concerted ITS evolution in Mammillaria (Cactaceae).

PubMed

Harpke, Doerte; Peterson, Angela

2006-12-01

Molecular studies of 21 species of the large Cactaceae genus Mammillaria representing a variety of intrageneric taxonomic levels revealed a high degree of intra-individual polymorphism of the internal transcribed spacer region (ITS1, 5.8S rDNA, ITS2). Only a few of these ITS copies belong to apparently functional genes, whereas most are probably non-functional (pseudogenes). As a multiple gene family, the ITS region is subjected to concerted evolution. However, the high degree of intra-individual polymorphism of up to 36% in ITS1 and up to 35% in ITS2 suggests a non-concerted evolution of these loci in Mammillaria. Conserved angiosperm motifs of ITS1 and ITS2 were compared between genomic and cDNA ITS clones of Mammillaria. Some of these motifs (e.g., ITS1 motif 1, 'TGGT' within ITS2) in combination with the determination of GC-content, length comparisons of the spacers and ITS2 secondary structure (helices II and III) are helpful in the identification of pseudogene rDNA regions.

Enrichment of dynamic chromosomal crosslinks drive phase separation of the nucleolus

PubMed Central

Hult, Caitlin; Adalsteinsson, David; Vasquez, Paula A.; Lawrimore, Josh; Bennett, Maggie; York, Alyssa; Cook, Diana; Yeh, Elaine; Forest, Mark Gregory

2017-01-01

Abstract Regions of highly repetitive DNA, such as those found in the nucleolus, show a self-organization that is marked by spatial segregation and frequent self-interaction. The mechanisms that underlie the sequestration of these sub-domains are largely unknown. Using a stochastic, bead-spring representation of chromatin in budding yeast, we find enrichment of protein-mediated, dynamic chromosomal cross-links recapitulates the segregation, morphology and self-interaction of the nucleolus. Rates and enrichment of dynamic crosslinking have profound consequences on domain morphology. Our model demonstrates the nucleolus is phase separated from other chromatin in the nucleus and predicts that multiple rDNA loci will form a single nucleolus independent of their location within the genome. Fluorescent labeling of budding yeast nucleoli with CDC14-GFP revealed that a split rDNA locus indeed forms a single nucleolus. We propose that nuclear sub-domains, such as the nucleolus, result from phase separations within the nucleus, which are driven by the enrichment of protein-mediated, dynamic chromosomal crosslinks. PMID:28977453

Intramolecular electron transport in quinoprotein alcohol dehydrogenase of Acetobacter methanolicus: a redox-titration study

PubMed

Frébortova; Matsushita; Arata; Adachi

1998-01-27

Quinohemoprotein-cytochrome c complex alcohol dehydrogenase (ADH) of acetic acid bacteria consists of three subunits, of which subunit I contains pyrroloquinoline quinone (PQQ) and heme c, and subunit II contains three heme c components. The PQQ and heme c components are believed to be involved in the intramolecular electron transfer from ethanol to ubiquinone. To study the intramolecular electron transfer in ADH of Acetobacter methanolicus, the redox potentials of heme c components were determined with ADH complex and the isolated subunits I and II of A. methanolicus, as well as hybrid ADH consisting of the subunit I/III complex of Gluconobacter suboxydans ADH and subunit II of A. methanolicus ADH. The redox potentials of hemes c in ADH complex were -130, 49, 188, and 188 mV at pH 7.0 and 24, 187, 190, and 255 mV at pH 4.5. In hybrid ADH, one of these heme c components was largely changed in the redox potential. Reduced ADH was fully oxidized with potassium ferricyanide, while ubiquinone oxidized the enzyme partially. The results indicate that electrons extracted from ethanol at PQQ site are transferred to ubiquinone via heme c in subunit I and two of the three hemes c in subunit II. Copyright 1998 Elsevier Science B.V.

Early-life nutrition modulates the epigenetic state of specific rDNA genetic variants in mice.

PubMed

Holland, Michelle L; Lowe, Robert; Caton, Paul W; Gemma, Carolina; Carbajosa, Guillermo; Danson, Amy F; Carpenter, Asha A M; Loche, Elena; Ozanne, Susan E; Rakyan, Vardhman K

2016-07-29

A suboptimal early-life environment, due to poor nutrition or stress during pregnancy, can influence lifelong phenotypes in the progeny. Epigenetic factors are thought to be key mediators of these effects. We show that protein restriction in mice from conception until weaning induces a linear correlation between growth restriction and DNA methylation at ribosomal DNA (rDNA). This epigenetic response remains into adulthood and is restricted to rDNA copies associated with a specific genetic variant within the promoter. Related effects are also found in models of maternal high-fat or obesogenic diets. Our work identifies environmentally induced epigenetic dynamics that are dependent on underlying genetic variation and establishes rDNA as a genomic target of nutritional insults. Copyright © 2016, American Association for the Advancement of Science.

Basic cytogenetics and physical mapping of 5S and 18S ribosomal genes in Hoplias malabaricus (Osteichthyes, Characiformes, Erythrinidae) from isolated natural lagoons: a conserved karyomorph along the Iguaçu river basin.

PubMed

Gemi, Gisele; Lui, Roberto Laridondo; Treco, Fernando Rodrigo; Paiz, Leonardo Marcel; Moresco, Rafaela Maria; Margarido, Vladimir Pavan

2014-01-01

Erythrinidae include Neotropical teleost fish that are widely distributed in South America. Hoplias Gill, 1903 include two large groups: H. malabaricus Bloch, 1794 and H. lacerdae Miranda Ribeiro, 1908. Hoplias malabaricus is characterized by remarkable karyotype diversity, with some karyomorphs widely distributed geographically while others are more restricted to certain river basins. Cytogenetic analyzes were performed in a population of Hoplias malabaricus from the Wildlife Refuge of Campos de Palmas, the Iguaçu River basin. The specimens showed diploid number of 42 chromosomes (24m+18sm) without differentiated sex chromosomes system. The impregnation by silver nitrate showed multiple AgNORs. Seven pairs (4, 7, 10, 13, 16, 20 and 21) carrying 18S rDNA were detected by FISH. Heterochromatin was verified in the centromeric and pericentromeric region of most chromosomes and the terminal region of some pairs. FISH with 5S rDNA probes showed two chromosome pairs carrying these sites in the interstitial region (8 and 14). The data obtained in this study are similar to those found for two other populations of H. malabaricus already studied in the basin of the Iguaçu River, confirming the hypothesis that this species is natural, not having been introduced, as well as having an intrinsic characteristic, such as the largest number of sites of 18S rDNA.

Bacterial 16S rRNA gene analysis revealed that bacteria related to Arcobacter spp. constitute an abundant and common component of the oyster microbiota (Tiostrea chilensis).

PubMed

Romero, J; García-Varela, M; Laclette, J P; Espejo, R T

2002-11-01

To explore the bacterial microbiota in Chilean oyster (Tiostrea chilensis), a molecular approach that permits detection of different bacteria, independently of their capacity to grow in culture media, was used. Bacterial diversity was assessed by analysis of both the 16S rDNA and the 16S-23S intergenic region, obtained by PCR amplifications of DNA extracted from depurated oysters. RFLP of the PCR amplified 16S rDNA showed a prevailing pattern in most of the individuals analyzed, indicating that a few bacterial species were relatively abundant and common in oysters. Cloning and sequencing of the 16S rDNA with the prevailing RFLP pattern indicated that this rRNA was most closely related to Arcobacter spp. However, analysis by the size of the amplified 16S-23S rRNA intergenic regions revealed not Arcobacter spp. but Staphylococcus spp. related bacteria as a major and common component in oyster. These different results may be caused by the absence of target for one of the primers employed for amplification of the intergenic region. Neither of the two bacteria species found in large abundance was recovered after culturing under aerobic, anaerobic, or microaerophilic conditions. This result, however, is expected because the number of bacteria recovered after cultivation was less than 0.01% of the total. All together, these observations suggest that Arcobacter-related strains are probably abundant and common in the Chilean oyster bacterial microbiota.

UBF-binding site arrays form pseudo-NORs and sequester the RNA polymerase I transcription machinery

PubMed Central

Mais, Christine; Wright, Jane E.; Prieto, José-Luis; Raggett, Samantha L.; McStay, Brian

2005-01-01

Human ribosomal genes (rDNA) are located in nucleolar organizer regions (NORs) on the short arms of acrocentric chromosomes. Metaphase NORs that were transcriptionally active in the previous cell cycle appear as prominent chromosomal features termed secondary constrictions that are achromatic in chromosome banding and positive in silver staining. The architectural RNA polymerase I (pol I) transcription factor UBF binds extensively across rDNA throughout the cell cycle. To determine if UBF binding underpins NOR structure, we integrated large arrays of heterologous UBF-binding sequences at ectopic sites on human chromosomes. These arrays efficiently recruit UBF even to sites outside the nucleolus and, during metaphase, form novel silver stainable secondary constrictions, termed pseudo-NORs, morphologically similar to NORs. We demonstrate for the first time that in addition to UBF the other components of the pol I machinery are found associated with sequences across the entire human rDNA repeat. Remarkably, a significant fraction of these same pol I factors are sequestered by pseudo-NORs independent of both transcription and nucleoli. Because of the heterologous nature of the sequence employed, we infer that sequestration is mediated primarily by protein–protein interactions with UBF. These results suggest that extensive binding of UBF is responsible for formation and maintenance of the secondary constriction at active NORs. Furthermore, we propose that UBF mediates recruitment of the pol I machinery to nucleoli independently of promoter elements. PMID:15598984

Profiling Nematode Communities in Unmanaged Flowerbed and Agricultural Field Soils in Japan by DNA Barcode Sequencing

PubMed Central

Morise, Hisashi; Miyazaki, Erika; Yoshimitsu, Shoko; Eki, Toshihiko

2012-01-01

Soil nematodes play crucial roles in the soil food web and are a suitable indicator for assessing soil environments and ecosystems. Previous nematode community analyses based on nematode morphology classification have been shown to be useful for assessing various soil environments. Here we have conducted DNA barcode analysis for soil nematode community analyses in Japanese soils. We isolated nematodes from two different environmental soils of an unmanaged flowerbed and an agricultural field using the improved flotation-sieving method. Small subunit (SSU) rDNA fragments were directly amplified from each of 68 (flowerbed samples) and 48 (field samples) isolated nematodes to determine the nucleotide sequence. Sixteen and thirteen operational taxonomic units (OTUs) were obtained by multiple sequence alignment from the flowerbed and agricultural field nematodes, respectively. All 29 SSU rDNA-derived OTUs (rOTUs) were further mapped onto a phylogenetic tree with 107 known nematode species. Interestingly, the two nematode communities examined were clearly distinct from each other in terms of trophic groups: Animal predators and plant feeders were markedly abundant in the flowerbed soils, in contrast, bacterial feeders were dominantly observed in the agricultural field soils. The data from the flowerbed nematodes suggests a possible food web among two different trophic nematode groups and plants (weeds) in the closed soil environment. Finally, DNA sequences derived from the mitochondrial cytochrome oxidase c subunit 1 (COI) gene were determined as a DNA barcode from 43 agricultural field soil nematodes. These nematodes were assigned to 13 rDNA-derived OTUs, but in the COI gene analysis were assigned to 23 COI gene-derived OTUs (cOTUs), indicating that COI gene-based barcoding may provide higher taxonomic resolution than conventional SSU rDNA-barcoding in soil nematode community analysis. PMID:23284767

Exobasidium maculosum, a new species causing leaf and fruit spots on blueberry in the southeastern USA and its relationship with other Exobasidium spp. parasitic to blueberry and cranberry.

PubMed

Brewer, Marin Talbot; Turner, Ashley N; Brannen, Phillip M; Cline, William O; Richardson, Elizabeth A

2014-01-01

Exobasidium leaf and fruit spot of blueberry (Vaccinium section Cyanococcus) is an emerging disease that has rapidly increased in prevalence throughout the southeastern USA. To determine whether this disease is caused by a new species of Exobasidium, we studied the morphology and phylogenetic relationship of the causal fungus compared with other members of the genus, including the type species E. vaccinii and other species that parasitize blueberry and cranberry (V. macrocarpon). Both scanning electron microscopy and light microscopy were used for morphological characterization. For phylogenetic analyses, we sequenced the large subunit of the rDNA (LSU) from 10 isolates collected from leaf or fruit spots of rabbiteye blueberry (V. virgatum), highbush blueberry (V. corymbosum) and southern highbush blueberry (Vaccinium interspecific hybrid) from Georgia and North Carolina and six isolates from leaf spots of lowbush blueberry (V. angustifolium) from Maine and Nova Scotia, Canada. LSU was sequenced from isolates causing red leaf disease of lowbush blueberry and red leaf spot (E. rostrupii) and red shoot (E. perenne) of cranberry. In addition, LSU sequences from GenBank, including sequences with high similarity to the emerging parasite and from Exobasidium spp. parasitizing other Vaccinium spp. and related hosts, were obtained. All sequences were aligned and subjected to phylogenetic analyses. Results indicated that the emerging parasite in the southeastern USA differs morphologically and phylogenetically from other described species and is described herein as Exobasidium maculosum. Within the southeastern USA, clustering based on host species, host tissue type (leaf or fruit) or geographic region was not detected; however, leaf spot isolates from lowbush blueberry were genetically different and likely represent a unique species. © 2014 by The Mycological Society of America.

Genetic analyses place most Spanish isolates of Beauveria bassiana in a molecular group with word-wide distribution

PubMed Central

2011-01-01

Background The entomopathogenic anamorphic fungus Beauveria bassiana is currently used as a biocontrol agent (BCA) of insects. Fifty-seven Beauveria bassiana isolates -53 from Spain- were characterized, integrating group I intron insertion patterns at the 3'-end of the nuclear large subunit ribosomal gene (LSU rDNA) and elongation factor 1-alpha (EF1-α) phylogenetic information, in order to assess the genetic structure and diversity of this Spanish collection of B. bassiana. Results Group I intron genotype analysis was based on the four highly conserved insertion sites of the LSU (Ec2653, Ec2449, Ec2066, Ec1921). Of the 16 possible combinations/genotypes, only four were detected, two of which were predominant, containing 44 and 9 members out of 57 isolates, respectively. Interestingly, the members of the latter two genotypes showed unique differences in their growth temperatures. In follow, EF1-α phylogeny served to classify most of the strains in the B. bassiana s.s. (sensu stricto) group and separate them into 5 molecular subgroups, all of which contained a group I intron belonging to the IC1 subtype at the Ec1921 position. A number of parameters such as thermal growth or origin (host, geographic location and climatic conditions) were also examined but in general no association could be found. Conclusion Most Spanish B. bassiana isolates (77.2%) are grouped into a major phylogenetic subgroup with word-wide distribution. However, high phylogenetic diversity was also detected among Spanish isolates from close geographic zones with low climatic variation. In general, no correlation was observed between the molecular distribution and geographic origin or climatic characteristics where the Spanish B. bassiana isolates were sampled. PMID:21521527

Phylogenetic Diversity Analysis of Subterranean Hot Springs in Iceland

PubMed Central

Marteinsson, Viggó Thór; Hauksdóttir, Sigurbjörg; Hobel, Cédric F. V.; Kristmannsdóttir, Hrefna; Hreggvidsson, Gudmundur Oli; Kristjánsson, Jakob K.

2001-01-01

Geothermal energy has been harnessed and used for domestic heating in Iceland. In wells that are typically drilled to a depth of 1,500 to 2,000 m, the temperature of the source water is 50 to 130°C. The bottoms of the boreholes can therefore be regarded as subterranean hot springs and provide a unique opportunity to study the subterranean biosphere. Large volumes of geothermal fluid from five wells and a mixture of geothermal water from 50 geothermal wells (hot tap water) were sampled and concentrated through a 0.2-μm-pore-size filter. Cells were observed in wells RG-39 (91.4°C) and MG-18 (71.8°C) and in hot tap water (76°C), but no cells were detected in wells SN-4, SN-5 (95 to 117°C), and RV-5 (130°C). Archaea and Bacteria were detected by whole-cell fluorescent in situ hybridization. DNAs were extracted from the biomass, and small-subunit rRNA genes (16S rDNAs) were amplified by PCR using primers specific for the Archaea and Bacteria domains. The PCR products were cloned and sequenced. The sequence analysis showed 11 new operational taxonomic units (OTUs) out of 14, 3 of which were affiliated with known surface OTUs. Samples from RG-39 and hot tap water were inoculated into enrichment media and incubated at 65 and 85°C. Growth was observed only in media based on geothermal water. 16S rDNA analysis showed enrichments dominated with Desulfurococcales relatives. Two strains belonging to Desulfurococcus mobilis and to the Thermus/Deinococcus group were isolated from borehole RG-39. The results indicate that subsurface volcanic zones are an environment that provides a rich subsurface for novel thermophiles. PMID:11526029

Emendation and new species of Hapalorhynchus Stunkard, 1922 (Digenea: Schistosomatoidea) from musk turtles (Kinosternidae: Sternotherus) in Alabama and Florida rivers.

PubMed

Roberts, Jackson R; Halanych, Kenneth M; Arias, Cova R; Folt, Brian; Goessling, Jeffrey M; Bullard, Stephen A

2017-12-01

Hapalorhynchus Stunkard, 1922 is emended based on morphological study of existing museum specimens (type and voucher specimens) and newly-collected specimens infecting musk turtles (Testudines: Kinosternidae: Sternotherus spp.) from rivers in Alabama and Florida (USA). Hapalorhynchus conecuhensis n. sp. is described from an innominate musk turtle, Sternotherus cf. minor, (type host) from Blue Spring (31°5'27.64″N, 86°30'53.21″W; Pensacola Bay Basin, Alabama) and the loggerhead musk turtle, Sternotherus minor (Agassiz, 1857) from the Wacissa River (30°20'24.73″N, 83°59'27.56″W; Apalachee Bay Basin, Florida). It differs from congeners by lacking a body constriction at level of the ventral sucker, paired anterior caeca, and a transverse ovary as well as by having a small ventral sucker, proportionally short posterior caeca, nearly equally-sized anterior and posterior testes, a small cirrus sac, and a uterus extending dorsal to the ovary and the anterior testis. Specimens of Hapalorhynchus reelfooti Byrd, 1939 infected loggerhead musk turtles, stripe-necked musk turtles (Sternotherus peltifer Smith and Glass, 1947), Eastern musk turtles (Sternotherus odoratus [Latreille in Sonnini and Latreille, 1801]), and S. cf. minor. Those of Hapalorhynchus cf. stunkardi infected S. minor and S. odoratus. Sternothorus minor, S. peltifer, and S. cf. minor plus S. minor and S. odoratus are new host records for H. reelfooti and H. cf. stunkardi, respectively. This is the first report of an infected musk turtle from the Coosa and Tallapoosa Rivers (Mobile-Tensaw River Basin), Pensacola Bay Basin, or Apalachee Bay Basin. Sequence analysis of the large subunit rDNA (28S) showed a strongly-supported clade for Hapalorhynchus. Copyright © 2017 Elsevier B.V. All rights reserved.

Phylogenetic diversity analysis of subterranean hot springs in Iceland.

PubMed

Marteinsson, V T; Hauksdóttir, S; Hobel, C F; Kristmannsdóttir, H; Hreggvidsson, G O; Kristjánsson, J K

2001-09-01

Geothermal energy has been harnessed and used for domestic heating in Iceland. In wells that are typically drilled to a depth of 1,500 to 2,000 m, the temperature of the source water is 50 to 130 degrees C. The bottoms of the boreholes can therefore be regarded as subterranean hot springs and provide a unique opportunity to study the subterranean biosphere. Large volumes of geothermal fluid from five wells and a mixture of geothermal water from 50 geothermal wells (hot tap water) were sampled and concentrated through a 0.2-microm-pore-size filter. Cells were observed in wells RG-39 (91.4 degrees C) and MG-18 (71.8 degrees C) and in hot tap water (76 degrees C), but no cells were detected in wells SN-4, SN-5 (95 to 117 degrees C), and RV-5 (130 degrees C). Archaea and Bacteria were detected by whole-cell fluorescent in situ hybridization. DNAs were extracted from the biomass, and small-subunit rRNA genes (16S rDNAs) were amplified by PCR using primers specific for the Archaea and Bacteria domains. The PCR products were cloned and sequenced. The sequence analysis showed 11 new operational taxonomic units (OTUs) out of 14, 3 of which were affiliated with known surface OTUs. Samples from RG-39 and hot tap water were inoculated into enrichment media and incubated at 65 and 85 degrees C. Growth was observed only in media based on geothermal water. 16S rDNA analysis showed enrichments dominated with Desulfurococcales relatives. Two strains belonging to Desulfurococcus mobilis and to the Thermus/Deinococcus group were isolated from borehole RG-39. The results indicate that subsurface volcanic zones are an environment that provides a rich subsurface for novel thermophiles.

Mycorrhizal fungi of Vanilla: diversity, specificity and effects on seed germination and plant growth.

PubMed

Porras-Alfaro, Andrea; Bayman, Paul

2007-01-01

Mycorrhizal fungi are essential for the germination of orchid seeds. However, the specificity of orchids for their mycorrhizal fungi and the effects of the fungi on orchid growth are controversial. Mycorrhizal fungi have been studied in some temperate and tropical, epiphytic orchids, but the symbionts of tropical, terrestrial orchids are still unknown. Here we study diversity, specificity and function of mycorrhizal fungi in Vanilla, a pantropical genus that is both terrestrial and epiphytic. Mycorrhizal roots were collected from four Vanilla species in Puerto Rico, Costa Rica and Cuba. Cultured and uncultured mycorrhizal fungi were identified by sequencing the internal transcribed spacer region of nuclear rDNA (nrITS) and part of the mitochondrial ribosomal large subunit (mtLSU), and by counting number of nuclei in hyphae. Vanilla spp. were associated with a wide range of mycorrhizal fungi: Ceratobasidium, Thanatephorus and Tulasnella. Related fungi were found in different species of Vanilla, although at different relative frequencies. Ceratobasidium was more common in roots in soil and Tulasnella was more common in roots on tree bark, but several clades of fungi included strains from both substrates. Relative frequencies of genera of mycorrhizal fungi differed significantly between cultured fungi and those detected by direct amplification. Ceratobasidium and Tulasnella were tested for effects on seed germination of Vanilla and effects on growth of Vanilla and Dendrobium plants. We found significant differences among fungi in effects on seed germination and plant growth. Effects of mycorrhizal fungi on Vanilla and Dendrobium were similar: a clade of Ceratobasidium had a consistently positive effect on plant growth and seed germination. This clade has potential use in germination and propagation of orchids. Results confirmed that a single orchid species can be associated with several mycorrhizal fungi with different functional consequences for the plant.

Transcript levels, alternative splicing and proteolytic cleavage of TFIIIA control 5S rRNA accumulation during Arabidopsis thaliana development.

PubMed

Layat, Elodie; Cotterell, Sylviane; Vaillant, Isabelle; Yukawa, Yasushi; Tutois, Sylvie; Tourmente, Sylvette

2012-07-01

Ribosome biogenesis is critical for eukaryotic cells and requires coordinated synthesis of the protein and rRNA moieties of the ribosome, which are therefore highly regulated. 5S ribosomal RNA, an essential component of the large ribosomal subunit, is transcribed by RNA polymerase III and specifically requires transcription factor IIIA (TFIIIA). To obtain insight into the regulation of 5S rRNA transcription, we have investigated the expression of 5S rRNA and the exon-skipped (ES) and exon-including (EI) TFIIIA transcripts, two transcript isoforms that result from alternative splicing of the TFIIIA gene, and TFIIIA protein amounts with respect to requirements for 5S rRNA during development. We show that 5S rRNA quantities are regulated through distinct but complementary mechanisms operating through transcriptional and post-transcriptional control of TFIIIA transcripts as well as at the post-translational level through proteolytic cleavage of the TFIIIA protein. During the reproductive phase, high expression of the TFIIIA gene together with low proteolytic cleavage contributes to accumulation of functional, full-length TFIIIA protein, and results in 5S rRNA accumulation in the seed. In contrast, just after germination, the levels of TFIIIA-encoding transcripts are low and stable. Full-length TFIIIA protein is undetectable, and the level of 5S rRNA stored in the embryo progressively decreases. After day 4, in correlation with the reorganization of 5S rDNA chromatin to a mature state, full-length TFIIIA protein with transcriptional activity accumulates and permits de novo transcription of 5S rRNA. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

An integrated approach to fast and informative morphological vouchering of nematodes for applications in molecular barcoding

PubMed Central

De Ley, Paul; De Ley, Irma Tandingan; Morris, Krystalynne; Abebe, Eyualem; Mundo-Ocampo, Manuel; Yoder, Melissa; Heras, Joseph; Waumann, Dora; Rocha-Olivares, Axayácatl; Jay Burr, A.H; Baldwin, James G; Thomas, W. Kelley

2005-01-01

Molecular surveys of meiofaunal diversity face some interesting methodological challenges when it comes to interstitial nematodes from soils and sediments. Morphology-based surveys are greatly limited in processing speed, while barcoding approaches for nematodes are hampered by difficulties of matching sequence data with traditional taxonomy. Intermediate technology is needed to bridge the gap between both approaches. An example of such technology is video capture and editing microscopy, which consists of the recording of taxonomically informative multifocal series of microscopy images as digital video clips. The integration of multifocal imaging with sequence analysis of the D2D3 region of large subunit (LSU) rDNA is illustrated here in the context of a combined morphological and barcode sequencing survey of marine nematodes from Baja California and California. The resulting video clips and sequence data are made available online in the database NemATOL (http://nematol.unh.edu/). Analyses of 37 barcoded nematodes suggest that these represent at least 32 species, none of which matches available D2D3 sequences in public databases. The recorded multifocal vouchers allowed us to identify most specimens to genus, and will be used to match specimens with subsequent species identifications and descriptions of preserved specimens. Like molecular barcodes, multifocal voucher archives are part of a wider effort at structuring and changing the process of biodiversity discovery. We argue that data-rich surveys and phylogenetic tools for analysis of barcode sequences are an essential component of the exploration of phyla with a high fraction of undiscovered species. Our methods are also directly applicable to other meiofauna such as for example gastrotrichs and tardigrades. PMID:16214752

Zoanthid diversity (Anthozoa: Hexacorallia) in the Galapagos Islands: a molecular examination

NASA Astrophysics Data System (ADS)

Reimer, J. D.; Sinniger, F.; Hickman, C. P.

2008-09-01

Although the Galapagos are famous for their unique biodiversity, many groups of marine invertebrates from this isolated archipelago remain understudied or not investigated. One such group is the zoanthids (Order Zoantharia, =Zoanthidea, =Zoanthiniaria), anthozoans (Cnidaria) found in marine ecosystems worldwide. Zoanthid taxonomy has been in a state of disorganization and neglect due in large part to the morphological plasticity within species and questions about the accuracy of traditionally used morphological and ecological characteristics. However, recent studies utilizing molecular methodology combined with morphology have proven to be very useful in understanding zoanthid diversity. The results of a survey of zoanthids from the Galapagos and the east Pacific are reported in this study. Shallow water (<35 m) zoanthid specimens were identified using the molecular markers mitochondrial 16S ribosomal DNA (mt 16S rDNA), cytochrome oxidase subunit I (COI) gene, and the internal transcribed spacer of ribosomal DNA (ITS-rDNA). From the collected specimens seven putative zoanthid species-level clades from three known genera ( Zoanthus, Palythoa, Parazoanthus) were identified at the molecular level. These identifications were further supported by morphological and ecological data. While almost all specimens belonged to known zoanthid genera, based on unique molecular and ecological data one group of specimens (designated unknown zoanthid sp. “03-103”) is potentially a novel undescribed genus. Additionally, the remaining three azooxanthellate Parazoanthus clades may also be undescribed new species, but due to the overall lack of zoanthid research and descriptions from neighboring areas (East Pacific, west coast of South America) further research is needed to clearly ascertain this. Additionally, notes on the four observed nominal azooxanthellate zoanthid species and a key to all eight nominal (seven from known genera, one from a potentially new genus) shallow water zoanthid species found thus far in the Galapagos Islands are provided.

Introduction of a novel 18S rDNA gene arrangement along with distinct ITS region in the saline water microalga Dunaliella

PubMed Central

2010-01-01

Comparison of 18S rDNA gene sequences is a very promising method for identification and classification of living organisms. Molecular identification and discrimination of different Dunaliella species were carried out based on the size of 18S rDNA gene and, number and position of introns in the gene. Three types of 18S rDNA structure have already been reported: the gene with a size of ~1770 bp lacking any intron, with a size of ~2170 bp consisting one intron near 5' terminus, and with a size of ~2570 bp harbouring two introns near 5' and 3' termini. Hereby, we report a new 18S rDNA gene arrangement in terms of intron localization and nucleotide sequence in a Dunaliella isolated from Iranian salt lakes (ABRIINW-M1/2). PCR amplification with genus-specific primers resulted in production of a ~2170 bp DNA band, which is similar to that of D. salina 18S rDNA gene containing only one intron near 5' terminus. Whilst, sequence composition of the gene revealed the lack of any intron near 5' terminus in our isolate. Furthermore, another alteration was observed due to the presence of a 440 bp DNA fragment near 3' terminus. Accordingly, 18S rDNA gene of the isolate is clearly different from those of D. salina and any other Dunaliella species reported so far. Moreover, analysis of ITS region sequence showed the diversity of this region compared to the previously reported species. 18S rDNA and ITS sequences of our isolate were submitted with accesion numbers of EU678868 and EU927373 in NCBI database, respectively. The optimum growth rate of this isolate occured at the salinity level of 1 M NaCl. The maximum carotenoid content under stress condition of intense light (400 μmol photon m-2 s-1), high salinity (4 M NaCl) and deficiency of nitrate and phosphate nutritions reached to 240 ng/cell after 15 days. PMID:20377865

Diagnostics of Neisseriaceae and Moraxellaceae by Ribosomal DNA Sequencing: Ribosomal Differentiation of Medical Microorganisms

PubMed Central

Harmsen, Dag; Singer, Christian; Rothgänger, Jörg; Tønjum, Tone; Sybren de Hoog, Gerrit; Shah, Haroun; Albert, Jürgen; Frosch, Matthias

2001-01-01

Fast and reliable identification of microbial isolates is a fundamental goal of clinical microbiology. However, in the case of some fastidious gram-negative bacterial species, classical phenotype identification based on either metabolic, enzymatic, or serological methods is difficult, time-consuming, and/or inadequate. 16S or 23S ribosomal DNA (rDNA) bacterial sequencing will most often result in accurate speciation of isolates. Therefore, the objective of this study was to find a hypervariable rDNA stretch, flanked by strongly conserved regions, which is suitable for molecular species identification of members of the Neisseriaceae and Moraxellaceae. The inter- and intrageneric relationships were investigated using comparative sequence analysis of PCR-amplified partial 16S and 23S rDNAs from a total of 94 strains. When compared to the type species of the genera Acinetobacter, Moraxella, and Neisseria, an average of 30 polymorphic positions was observed within the partial 16S rDNA investigated (corresponding to Escherichia coli positions 54 to 510) for each species and an average of 11 polymorphic positions was observed within the 202 nucleotides of the 23S rDNA gene (positions 1400 to 1600). Neisseria macacae and Neisseria mucosa subsp. mucosa (ATCC 19696) had identical 16S and 23S rDNA sequences. Species clusters were heterogeneous in both genes in the case of Acinetobacter lwoffii, Moraxella lacunata, and N. mucosa. Neisseria meningitidis isolates failed to cluster only in the 23S rDNA subset. Our data showed that the 16S rDNA region is more suitable than the partial 23S rDNA for the molecular diagnosis of Neisseriaceae and Moraxellaceae and that a reference database should include more than one strain of each species. All sequence chromatograms and taxonomic and disease-related information are available as part of our ribosomal differentiation of medical microorganisms (RIDOM) web-based service (http://www.ridom.hygiene.uni-wuerzburg.de/). Users can submit a sequence and conduct a similarity search against the RIDOM reference database for microbial identification purposes. PMID:11230407

Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi

PubMed Central

Schoch, Conrad L.; Seifert, Keith A.; Huhndorf, Sabine; Robert, Vincent; Spouge, John L.; Levesque, C. André; Chen, Wen; Bolchacova, Elena; Voigt, Kerstin; Crous, Pedro W.; Miller, Andrew N.; Wingfield, Michael J.; Aime, M. Catherine; An, Kwang-Deuk; Bai, Feng-Yan; Barreto, Robert W.; Begerow, Dominik; Bergeron, Marie-Josée; Blackwell, Meredith; Boekhout, Teun; Bogale, Mesfin; Boonyuen, Nattawut; Burgaz, Ana R.; Buyck, Bart; Cai, Lei; Cai, Qing; Cardinali, G.; Chaverri, Priscila; Coppins, Brian J.; Crespo, Ana; Cubas, Paloma; Cummings, Craig; Damm, Ulrike; de Beer, Z. Wilhelm; de Hoog, G. Sybren; Del-Prado, Ruth; Dentinger, Bryn; Diéguez-Uribeondo, Javier; Divakar, Pradeep K.; Douglas, Brian; Dueñas, Margarita; Duong, Tuan A.; Eberhardt, Ursula; Edwards, Joan E.; Elshahed, Mostafa S.; Fliegerova, Katerina; Furtado, Manohar; García, Miguel A.; Ge, Zai-Wei; Griffith, Gareth W.; Griffiths, K.; Groenewald, Johannes Z.; Groenewald, Marizeth; Grube, Martin; Gryzenhout, Marieka; Guo, Liang-Dong; Hagen, Ferry; Hambleton, Sarah; Hamelin, Richard C.; Hansen, Karen; Harrold, Paul; Heller, Gregory; Herrera, Cesar; Hirayama, Kazuyuki; Hirooka, Yuuri; Ho, Hsiao-Man; Hoffmann, Kerstin; Hofstetter, Valérie; Högnabba, Filip; Hollingsworth, Peter M.; Hong, Seung-Beom; Hosaka, Kentaro; Houbraken, Jos; Hughes, Karen; Huhtinen, Seppo; Hyde, Kevin D.; James, Timothy; Johnson, Eric M.; Johnson, Joan E.; Johnston, Peter R.; Jones, E.B. Gareth; Kelly, Laura J.; Kirk, Paul M.; Knapp, Dániel G.; Kõljalg, Urmas; Kovács, Gábor M.; Kurtzman, Cletus P.; Landvik, Sara; Leavitt, Steven D.; Liggenstoffer, Audra S.; Liimatainen, Kare; Lombard, Lorenzo; Luangsa-ard, J. Jennifer; Lumbsch, H. Thorsten; Maganti, Harinad; Maharachchikumbura, Sajeewa S. N.; Martin, María P.; May, Tom W.; McTaggart, Alistair R.; Methven, Andrew S.; Meyer, Wieland; Moncalvo, Jean-Marc; Mongkolsamrit, Suchada; Nagy, László G.; Nilsson, R. Henrik; Niskanen, Tuula; Nyilasi, Ildikó; Okada, Gen; Okane, Izumi; Olariaga, Ibai; Otte, Jürgen; Papp, Tamás; Park, Duckchul; Petkovits, Tamás; Pino-Bodas, Raquel; Quaedvlieg, William; Raja, Huzefa A.; Redecker, Dirk; Rintoul, Tara L.; Ruibal, Constantino; Sarmiento-Ramírez, Jullie M.; Schmitt, Imke; Schüßler, Arthur; Shearer, Carol; Sotome, Kozue; Stefani, Franck O.P.; Stenroos, Soili; Stielow, Benjamin; Stockinger, Herbert; Suetrong, Satinee; Suh, Sung-Oui; Sung, Gi-Ho; Suzuki, Motofumi; Tanaka, Kazuaki; Tedersoo, Leho; Telleria, M. Teresa; Tretter, Eric; Untereiner, Wendy A.; Urbina, Hector; Vágvölgyi, Csaba; Vialle, Agathe; Vu, Thuy Duong; Walther, Grit; Wang, Qi-Ming; Wang, Yan; Weir, Bevan S.; Weiß, Michael; White, Merlin M.; Xu, Jianping; Yahr, Rebecca; Yang, Zhu L.; Yurkov, Andrey; Zamora, Juan-Carlos; Zhang, Ning; Zhuang, Wen-Ying; Schindel, David

2012-01-01

Six DNA regions were evaluated as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life, by a multinational, multilaboratory consortium. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it is difficult to amplify in fungi, often includes large introns, and can be insufficiently variable. Three subunits from the nuclear ribosomal RNA cistron were compared together with regions of three representative protein-coding genes (largest subunit of RNA polymerase II, second largest subunit of RNA polymerase II, and minichromosome maintenance protein). Although the protein-coding gene regions often had a higher percent of correct identification compared with ribosomal markers, low PCR amplification and sequencing success eliminated them as candidates for a universal fungal barcode. Among the regions of the ribosomal cistron, the internal transcribed spacer (ITS) region has the highest probability of successful identification for the broadest range of fungi, with the most clearly defined barcode gap between inter- and intraspecific variation. The nuclear ribosomal large subunit, a popular phylogenetic marker in certain groups, had superior species resolution in some taxonomic groups, such as the early diverging lineages and the ascomycete yeasts, but was otherwise slightly inferior to the ITS. The nuclear ribosomal small subunit has poor species-level resolution in fungi. ITS will be formally proposed for adoption as the primary fungal barcode marker to the Consortium for the Barcode of Life, with the possibility that supplementary barcodes may be developed for particular narrowly circumscribed taxonomic groups. PMID:22454494

Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi.

PubMed

Schoch, Conrad L; Seifert, Keith A; Huhndorf, Sabine; Robert, Vincent; Spouge, John L; Levesque, C André; Chen, Wen

2012-04-17

Six DNA regions were evaluated as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life, by a multinational, multilaboratory consortium. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it is difficult to amplify in fungi, often includes large introns, and can be insufficiently variable. Three subunits from the nuclear ribosomal RNA cistron were compared together with regions of three representative protein-coding genes (largest subunit of RNA polymerase II, second largest subunit of RNA polymerase II, and minichromosome maintenance protein). Although the protein-coding gene regions often had a higher percent of correct identification compared with ribosomal markers, low PCR amplification and sequencing success eliminated them as candidates for a universal fungal barcode. Among the regions of the ribosomal cistron, the internal transcribed spacer (ITS) region has the highest probability of successful identification for the broadest range of fungi, with the most clearly defined barcode gap between inter- and intraspecific variation. The nuclear ribosomal large subunit, a popular phylogenetic marker in certain groups, had superior species resolution in some taxonomic groups, such as the early diverging lineages and the ascomycete yeasts, but was otherwise slightly inferior to the ITS. The nuclear ribosomal small subunit has poor species-level resolution in fungi. ITS will be formally proposed for adoption as the primary fungal barcode marker to the Consortium for the Barcode of Life, with the possibility that supplementary barcodes may be developed for particular narrowly circumscribed taxonomic groups.

Three new anascosporic genera of the Saccharomycotina: Danielozyma gen. nov., Deakozyma gen. nov. and Middelhovenomyces gen. nov.

USDA-ARS?s Scientific Manuscript database

Three new non-ascosporic, ascomycetous yeast genera are proposed based on their isolation from currently described species and genera. Phylogenetic placement of the genera was determined from analysis of nuclear gene sequences for D1/D2 large subunit rRNA, small subunit rRNA, translation elongation...

Cryo-EM structure of the large subunit of the spinach chloroplast ribosome

PubMed Central

Ahmed, Tofayel; Yin, Zhan; Bhushan, Shashi

2016-01-01

Protein synthesis in the chloroplast is mediated by the chloroplast ribosome (chloro-ribosome). Overall architecture of the chloro-ribosome is considerably similar to the Escherichia coli (E. coli) ribosome but certain differences are evident. The chloro-ribosome proteins are generally larger because of the presence of chloroplast-specific extensions in their N- and C-termini. The chloro-ribosome harbours six plastid-specific ribosomal proteins (PSRPs); four in the small subunit and two in the large subunit. Deletions and insertions occur throughout the rRNA sequence of the chloro-ribosome (except for the conserved peptidyl transferase center region) but the overall length of the rRNAs do not change significantly, compared to the E. coli. Although, recent advancements in cryo-electron microscopy (cryo-EM) have provided detailed high-resolution structures of ribosomes from many different sources, a high-resolution structure of the chloro-ribosome is still lacking. Here, we present a cryo-EM structure of the large subunit of the chloro-ribosome from spinach (Spinacia oleracea) at an average resolution of 3.5 Å. High-resolution map enabled us to localize and model chloro-ribosome proteins, chloroplast-specific protein extensions, two PSRPs (PSRP5 and 6) and three rRNA molecules present in the chloro-ribosome. Although comparable to E. coli, the polypeptide tunnel and the tunnel exit site show chloroplast-specific features. PMID:27762343

Kinetic pathway of 40S ribosomal subunit recruitment to hepatitis C virus internal ribosome entry site.

PubMed

Fuchs, Gabriele; Petrov, Alexey N; Marceau, Caleb D; Popov, Lauren M; Chen, Jin; O'Leary, Seán E; Wang, Richard; Carette, Jan E; Sarnow, Peter; Puglisi, Joseph D

2015-01-13

Translation initiation can occur by multiple pathways. To delineate these pathways by single-molecule methods, fluorescently labeled ribosomal subunits are required. Here, we labeled human 40S ribosomal subunits with a fluorescent SNAP-tag at ribosomal protein eS25 (RPS25). The resulting ribosomal subunits could be specifically labeled in living cells and in vitro. Using single-molecule Förster resonance energy transfer (FRET) between RPS25 and domain II of the hepatitis C virus (HCV) internal ribosome entry site (IRES), we measured the rates of 40S subunit arrival to the HCV IRES. Our data support a single-step model of HCV IRES recruitment to 40S subunits, irreversible on the initiation time scale. We furthermore demonstrated that after binding, the 40S:HCV IRES complex is conformationally dynamic, undergoing slow large-scale rearrangements. Addition of translation extracts suppresses these fluctuations, funneling the complex into a single conformation on the 80S assembly pathway. These findings show that 40S:HCV IRES complex formation is accompanied by dynamic conformational rearrangements that may be modulated by initiation factors.