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Sample records for 18s small subunit

  1. The widely used small subunit 18S rDNA molecule greatly underestimates true diversity in biodiversity surveys of the meiofauna.

    PubMed

    Tang, Cuong Q; Leasi, Francesca; Obertegger, Ulrike; Kieneke, Alexander; Barraclough, Timothy G; Fontaneto, Diego

    2012-10-02

    Molecular tools have revolutionized the exploration of biodiversity, especially in organisms for which traditional taxonomy is difficult, such as for microscopic animals (meiofauna). Environmental (eDNA) metabarcode surveys of DNA extracted from sediment samples are increasingly popular for surveying biodiversity. Most eDNA surveys use the nuclear gene-encoding small-subunit rDNA gene (18S) as a marker; however, different markers and metrics used for delimiting species have not yet been evaluated against each other or against morphologically defined species (morphospecies). We assessed more than 12,000 meiofaunal sequences of 18S and of the main alternatively used marker [Cytochrome c oxidase subunit I (COI) mtDNA] belonging to 55 datasets covering three taxonomic ranks. Our results show that 18S reduced diversity estimates by a factor of 0.4 relative to morphospecies, whereas COI increased diversity estimates by a factor of 7.6. Moreover, estimates of species richness using COI were robust among three of four commonly used delimitation metrics, whereas estimates using 18S varied widely with the different metrics. We show that meiofaunal diversity has been greatly underestimated by 18S eDNA surveys and that the use of COI provides a better estimate of diversity. The suitability of COI is supported by cross-mating experiments in the literature and evolutionary analyses of discreteness in patterns of genetic variation. Furthermore its splitting of morphospecies is expected from documented levels of cryptic taxa in exemplar meiofauna. We recommend against using 18S as a marker for biodiversity surveys and suggest that use of COI for eDNA surveys could provide more accurate estimates of species richness in the future.

  2. The phylogenetic position of eriophyoid mites (superfamily Eriophyoidea) in Acariformes inferred from the sequences of mitochondrial genomes and nuclear small subunit (18S) rRNA gene.

    PubMed

    Xue, Xiao-Feng; Dong, Yan; Deng, Wei; Hong, Xiao-Yue; Shao, Renfu

    2017-04-01

    Eriophyoid mites (superfamily Eriophyoidea) comprise >4400 species worldwide. Despite over a century of study, the phylogenetic position of these mites within Acariformes is still poorly resolved. Currently, Eriophyoidea is placed in the order Trombidiformes. We inferred the high-level phylogeny of Acari with the mitochondrial (mt) genome sequences of 110 species including four eriophyoid species, and the nuclear small subunit (18S) rRNA gene sequences of 226 species including 25 eriophyoid species. Maximum likelihood (ML), Bayesian inference (BI) and Maximum parsimony (MP) methods were used to analyze the sequence data. Divergence times were estimated for major lineages of Acari using Bayesian approaches. Our analyses consistently recovered the monophyly of Eriophyoidea but rejected the monophyly of Trombidiformes. The eriophyoid mites were grouped with the sarcoptiform mites, or were the sister group of sarcoptiform mites+non-eriophyoid trombidiform mites, depending on data partition strategies. Eriophyoid mites diverged from other mites in the Devonian (384Mya, 95% HPD, 352-410Mya). The origin of eriophyoid mites was dated to the Permian (262Mya, 95% HPD 230-307Mya), mostly prior to the radiation of gymnosperms (Triassic-Jurassic) and angiosperms (early Cretaceous). We propose that the placement of Eriophyoidea in the order Trombidiformes under the current classification system should be reviewed.

  3. Prevalence, Genetic Characterization, and 18S Small Subunit Ribosomal RNA Diversity of Trypanosoma rangeli in Triatomine and Mammal Hosts in Endemic Areas for Chagas Disease in Ecuador

    PubMed Central

    Ocaña-Mayorga, Sofia; Aguirre-Villacis, Fernanda; Pinto, C. Miguel; Vallejo, Gustavo A.

    2015-01-01

    Abstract Trypanosoma rangeli is a nonpathogenic parasite for humans; however, its medical importance relies in its similarity and overlapping distribution with Trypanosoma cruzi, causal agent of Chagas disease in the Americas. The genetic diversity of T. rangeli and its association with host species (triatomines and mammals) has been identified along Central and the South America; however, it has not included data of isolates from Ecuador. This study reports infection with T. rangeli in 18 genera of mammal hosts and five species of triatomines in three environments (domestic, peridomestic, and sylvatic). Higher infection rates were found in the sylvatic environment, in close association with Rhodnius ecuadoriensis. The results of this study extend the range of hosts infected with this parasite and the geographic range of the T. rangeli genotype KP1(−)/lineage C in South America. It was not possible to detect variation on T. rangeli from the central coastal region and southern Ecuador with the analysis of the small subunit ribosomal RNA (SSU-rRNA) gene, even though these areas are ecologically different and a phenotypic subdivision of R. ecuadoriensis has been found. R. ecuadoriensis is considered one of the most important vectors for Chagas disease transmission in Ecuador due to its wide distribution and adaptability to diverse environments. An extensive knowledge of the trypanosomes circulating in this species of triatomine, and associated mammal hosts, is important for delineating transmission dynamics and preventive measures in the endemic areas of Ecuador and Northern Peru. PMID:26645579

  4. Prevalence, Genetic Characterization, and 18S Small Subunit Ribosomal RNA Diversity of Trypanosoma rangeli in Triatomine and Mammal Hosts in Endemic Areas for Chagas Disease in Ecuador.

    PubMed

    Ocaña-Mayorga, Sofia; Aguirre-Villacis, Fernanda; Pinto, C Miguel; Vallejo, Gustavo A; Grijalva, Mario J

    2015-12-01

    Trypanosoma rangeli is a nonpathogenic parasite for humans; however, its medical importance relies in its similarity and overlapping distribution with Trypanosoma cruzi, causal agent of Chagas disease in the Americas. The genetic diversity of T. rangeli and its association with host species (triatomines and mammals) has been identified along Central and the South America; however, it has not included data of isolates from Ecuador. This study reports infection with T. rangeli in 18 genera of mammal hosts and five species of triatomines in three environments (domestic, peridomestic, and sylvatic). Higher infection rates were found in the sylvatic environment, in close association with Rhodnius ecuadoriensis. The results of this study extend the range of hosts infected with this parasite and the geographic range of the T. rangeli genotype KP1(-)/lineage C in South America. It was not possible to detect variation on T. rangeli from the central coastal region and southern Ecuador with the analysis of the small subunit ribosomal RNA (SSU-rRNA) gene, even though these areas are ecologically different and a phenotypic subdivision of R. ecuadoriensis has been found. R. ecuadoriensis is considered one of the most important vectors for Chagas disease transmission in Ecuador due to its wide distribution and adaptability to diverse environments. An extensive knowledge of the trypanosomes circulating in this species of triatomine, and associated mammal hosts, is important for delineating transmission dynamics and preventive measures in the endemic areas of Ecuador and Northern Peru.

  5. Megraft: A software package to graft ribosomal small subunit (16S/18S) fragments onto full-length sequences for accurate species richness and sequencing depth analysis in pyrosequencing-length metagenomes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Metagenomic libraries represent subsamples of the total DNA found at a study site and offer unprecedented opportunities to study ecological and functional aspects of microbial communities. To examine the depth of the sequencing effort, rarefaction analysis of the ribosomal small sub-unit (SSU/16S/18...

  6. Metaxa: a software tool for automated detection and discrimination among ribosomal small subunit (12S/16S/18S) sequences of archaea, bacteria, eukaryotes, mitochondria, and chloroplasts in metagenomes and environmental sequencing datasets.

    PubMed

    Bengtsson, Johan; Eriksson, K Martin; Hartmann, Martin; Wang, Zheng; Shenoy, Belle Damodara; Grelet, Gwen-Aëlle; Abarenkov, Kessy; Petri, Anna; Rosenblad, Magnus Alm; Nilsson, R Henrik

    2011-10-01

    The ribosomal small subunit (SSU) rRNA gene has emerged as an important genetic marker for taxonomic identification in environmental sequencing datasets. In addition to being present in the nucleus of eukaryotes and the core genome of prokaryotes, the gene is also found in the mitochondria of eukaryotes and in the chloroplasts of photosynthetic eukaryotes. These three sets of genes are conceptually paralogous and should in most situations not be aligned and analyzed jointly. To identify the origin of SSU sequences in complex sequence datasets has hitherto been a time-consuming and largely manual undertaking. However, the present study introduces Metaxa ( http://microbiology.se/software/metaxa/ ), an automated software tool to extract full-length and partial SSU sequences from larger sequence datasets and assign them to an archaeal, bacterial, nuclear eukaryote, mitochondrial, or chloroplast origin. Using data from reference databases and from full-length organelle and organism genomes, we show that Metaxa detects and scores SSU sequences for origin with very low proportions of false positives and negatives. We believe that this tool will be useful in microbial and evolutionary ecology as well as in metagenomics.

  7. Heteroduplex mobility assay-guided sequence discovery: elucidation of the small subunit (18S) rDNA sequences of Pfiesteria piscicida and related dinoflagellates from complex algal culture and environmental sample DNA pools.

    PubMed

    Oldach, D W; Delwiche, C F; Jakobsen, K S; Tengs, T; Brown, E G; Kempton, J W; Schaefer, E F; Bowers, H A; Glasgow, H B; Burkholder, J M; Steidinger, K A; Rublee, P A

    2000-04-11

    The newly described heterotrophic estuarine dinoflagellate Pfiesteria piscicida has been linked with fish kills in field and laboratory settings, and with a novel clinical syndrome of impaired cognition and memory disturbance among humans after presumptive toxin exposure. As a result, there is a pressing need to better characterize the organism and these associations. Advances in Pfiesteria research have been hampered, however, by the absence of genomic sequence data. We employed a sequencing strategy directed by heteroduplex mobility assay to detect Pfiesteria piscicida 18S rDNA "signature" sequences in complex pools of DNA and used those data as the basis for determination of the complete P. piscicida 18S rDNA sequence. Specific PCR assays for P. piscicida and other estuarine heterotrophic dinoflagellates were developed, permitting their detection in algal cultures and in estuarine water samples collected during fish kill and fish lesion events. These tools should enhance efforts to characterize these organisms and their ecological relationships. Heteroduplex mobility assay-directed sequence discovery is broadly applicable, and may be adapted for the detection of genomic sequence data of other novel or nonculturable organisms in complex assemblages.

  8. 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.

  9. Role of the Rubisco Small Subunit

    SciTech Connect

    Spreitzer, Robert Joseph

    2016-11-05

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of CO2 fixation in photosynthesis. However, it is a slow enzyme, and O2 competes with CO2 at the active site. Oxygenation initiates the photorespiratory pathway, which also results in the loss of CO2. If carboxylation could be increased or oxygenation decreased, an increase in net CO2 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, 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 CO2/O2 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

  10. Direct chemical probing of the conformation of the 3' functional domain of rabbit 18S rRNA in 40S subunits, 80S ribosomes and polyribosomes

    SciTech Connect

    Rubino, H.M.; Rairkar, A.; Lockard, R.E.

    1987-05-01

    Recent evidence suggests that the 3' minor domain of eukaryotic 18S rRNA, as in prokaryotes, is directly involved in protein biosynthesis. To determine regions of possible functional importance, they have probed the higher order structure of rabbit 18S rRNA in both 40S subunits and 80S ribosomes, as well as polyribosomes using the single-strand specific chemical probes dimethyl sulfate (DMS) and diethyl pyrocarbonate (DEPC) which react with unpaired guanosine and adenosine residues, respectively. The modified 18S rRNA was isolated from these particles and the resultant modified nucleotides identified on polyacrylamide sequencing gels upon either aniline-induced strand scission of /sup 32/P-end-labeled intact rRNA or by DNA primer extension using sequence specific deoxyoligomers with reverse transcriptase. Their results indicate a decreased reactivity of residue C-1692 in rabbit 18S rRNA (corresponding to C-1400 E. coli) within the putative tRNA contact site in polyribosomes as compared with 40S subunits and 80S ribosomes. They have also determined varying reactivities of a number of other residues within specific regions of the 3' functional domain when 40S, 80S, and polyribosomes are compared, which may be important for both subunit association as well as mRNA binding.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  12. gar2 is a nucleolar protein from Schizosaccharomyces pombe required for 18S rRNA and 40S ribosomal subunit accumulation.

    PubMed Central

    Gulli, M P; Girard, J P; Zabetakis, D; Lapeyre, B; Melese, T; Caizergues-Ferrer, M

    1995-01-01

    Several nucleolar proteins, such as nucleolin, NOP1/fibrillarin, SSB1, NSR1 and GAR1 share a common glycine and arginine rich structural motif called the GAR domain. To identify novel nucleolar proteins from fission yeast we screened Schizosaccharomyces pombe genomic DNA libraries with a probe encompassing the GAR structural motif. Here we report the identification and characterization of a S.pombe gene coding for a novel nucleolar protein, designated gar2. The structure of the fission yeast gar2 is reminiscent of that of nucleolin from vertebrates and NSR1 from Saccharomyces cerevisiae. In addition, like these proteins, gar2 has a nucleolar localisation. The disruption of the gar2+ gene affects normal cell growth, leads to an accumulation of 35S pre-rRNA and a decrease of mature 18S rRNA steady state levels. Moreover, ribosomal profiles of the mutant show an increase of free 60S ribosomal subunits and an absence of free 40S ribosomal subunits. gar2 is able to rescue a S.cerevisiae mutant lacking NSR1, thus establishing gar2 as a functional homolog of NSR1. We propose that gar2 helps the assembly of pre-ribosomal particles containing 18S rRNA. Images PMID:7596817

  13. A small subunit processome protein promotes cancer by altering translation.

    PubMed

    Yang, H W; Kim, T-M; Song, S S; Menon, L; Jiang, X; Huang, W; Black, P M; Park, P J; Carroll, R S; Johnson, M D

    2015-08-20

    Dysregulation of ribosome biogenesis or translation can promote cancer, but the underlying mechanisms remain unclear. UTP18 is a component of the small subunit processome, a nucleolar multi-protein complex whose only known function is to cleave pre-ribosomal RNA to yield the 18S ribosomal RNA component of 40S ribosomal subunits. Here, we show that UTP18 also alters translation to promote stress resistance and growth, and that UTP18 is frequently gained and overexpressed in cancer. We observed that UTP18 localizes to the cytoplasm in a subset of cells, and that serum withdrawal increases cytoplasmic UTP18 localization. Cytoplasmic UTP18 associates with the translation complex and Hsp90 to upregulate the translation of IRES-containing transcripts such as HIF1a, Myc and VEGF, thereby inducing stress resistance. Hsp90 inhibition decreases cytoplasmic UTP18 and UTP18-induced increases in translation. Importantly, elevated UTP18 expression correlates with increased aggressiveness and decreased survival in numerous cancers. Enforced UTP18 overexpression promotes transformation and tumorigenesis, whereas UTP18 knockdown inhibits these processes. This stress adaptation mechanism is thus co-opted for growth by cancers, and its inhibition may represent a promising new therapeutic target.

  14. Phylogenetic classification of the frog pathogen Amphibiothecum (Dermosporidium) penneri based on small ribosomal subunit sequencing

    USGS Publications Warehouse

    Feldman, S.H.; Wimsatt, J.H.; Green, D.E.

    2005-01-01

    We determined 1,600 base pairs of DNA sequence in the 18S small ribosomal subunit from two geographically distinct isolates of Dermosporidium penneri. Maximum likelihood and parsimony analysis of these sequences place D. penneri in the order Dermocystida of the class Mesomycetozoea. The 18S rRNA sequences from these two isolates only differ within a single region of 16 contiguous nucleotides. Based on the distant phylogenetic relationship of these organisms to Amphibiocystidium ranae and similarity to Sphaerothecum destruens we propose the organism be renamed Amphibiothecum penneri.

  15. Compilation of small ribosomal subunit RNA structures.

    PubMed Central

    Neefs, J M; Van de Peer, Y; De Rijk, P; Chapelle, S; De Wachter, R

    1993-01-01

    The database on small ribosomal subunit RNA structure contained 1804 nucleotide sequences on April 23, 1993. This number comprises 365 eukaryotic, 65 archaeal, 1260 bacterial, 30 plastidial, and 84 mitochondrial sequences. These are stored in the form of an alignment in order to facilitate the use of the database as input for comparative studies on higher-order structure and for reconstruction of phylogenetic trees. The elements of the postulated secondary structure for each molecule are indicated by special symbols. The database is available on-line directly from the authors by ftp and can also be obtained from the EMBL nucleotide sequence library by electronic mail, ftp, and on CD ROM disk. PMID:8332525

  16. Rubisco small subunit gene family in cassava.

    PubMed

    Yeo, T W; Mak, Y M; Ho, K K

    1999-01-01

    Cassava leaves of two different cultivars, Brazil and Buloh, were used to isolate mRNA. The mRNA isolated was successfully used in the construction of cDNA libraries for each of the cultivars. The cDNA libraries were screened for members of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit gene family and positive clones were sequenced. A total of seven different SSU genes, of which five were from cultivar Brazil and two were from cultivar Buloh, were isolated. Comparison results show that even though all the sequences are highly similar, they can be classified into three subfamilies. Homology between members of the same subfamily is higher than homology between members from the same cultivar.

  17. 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

  18. Nuclear export of the small ribosomal subunit requires the Ran–GTPase cycle and certain nucleoporins

    PubMed Central

    Moy, Terence I.; Silver, Pamela A.

    1999-01-01

    After their assembly in the nucleolus, ribosomal subunits are exported from the nucleus to the cytoplasm. After export, the 20S rRNA in the small ribosomal subunit is cleaved to yield 18S rRNA and the small 5′ ITS1 fragment. The 5′ ITS1 RNA is normally degraded by the cytoplasmic Xrn1 exonuclease, but in strains lacking XRN1, the 5′ ITS1 fragment accumulates in the cytoplasm. Using the cytoplasmic localization of the 5′ ITS1 fragment as an indicator for the export of the small ribosomal subunit, we have identified genes that are required for ribosome export. Ribosome export is dependent on the Ran–GTPase as mutations in Ran or its regulators caused 5′ ITS1 to accumulate in the nucleoplasm. Mutations in the genes encoding the nucleoporin Nup82 and in the NES exporter Xpo1/Crm1 also caused the nucleoplasmic accumulation of 5′ ITS1. Mutants in a subset of nucleoporins and in the nuclear transport factors Srp1, Kap95, Pse1, Cse1, and Mtr10 accumulate the 5′ ITS1 in the nucleolus and affect ribosome assembly. In contrast, we did not detect nuclear accumulation of 5′ ITS1 in 28 yeast strains that have mutations in other genes affecting nuclear trafficking. PMID:10465789

  19. Genetic characterization and phylogenetic relationships based on 18S rRNA and ITS1 region of small form of canine Babesia spp. from India.

    PubMed

    Mandal, M; Banerjee, P S; Garg, Rajat; Ram, Hira; Kundu, K; Kumar, Saroj; Kumar, G V P P S Ravi

    2014-10-01

    Canine babesiosis is a vector borne disease caused by intra-erythrocytic apicomplexan parasites Babesia canis (large form) and Babesia gibsoni (small form), throughout the globe. Apart from few sporadic reports on the occurrence of B. gibsoni infection in dogs, no attempt has been made to characterize Babesia spp. of dogs in India. Fifteen canine blood samples, positive for small form of Babesia, collected from northern to eastern parts of India, were used for amplification of 18S rRNA gene (∼1665bp) of Babesia sp. and partial ITS1 region (∼254bp) of B. gibsoni Asian genotype. Cloning and sequencing of the amplified products of each sample was performed separately. Based on sequences and phylogenetic analysis of 18S rRNA and ITS1 sequences, 13 were considered to be B. gibsoni. These thirteen isolates shared high sequence identity with each other and with B. gibsoni Asian genotype. The other two isolates could not be assigned to any particular species because of the difference(s) in 18S rRNA sequence with B. gibsoni and closer identity with Babesiaoccultans and Babesiaorientalis. In the phylogenetic tree, all the isolates of B. gibsoni Asian genotype formed a separate major clade named as Babesia spp. sensu stricto clade with high bootstrap support. The two unnamed Babesia sp. (Malbazar and Ludhiana isolates) clustered close together with B. orientalis, Babesia sp. (Kashi 1 isolate) and B. occultans of bovines. It can be inferred from this study that 18S rRNA gene and ITS1 region are highly conserved among 13 B. gibsoni isolates from India. It is the maiden attempt of genetic characterization by sequencing of 18S rRNA gene and ITS1 region of B. gibsoni from India and is also the first record on the occurrence of an unknown Babesia sp. of dogs from south and south-east Asia.

  20. Amino-terminal truncations of the ribulose-bisphosphate carboxylase small subunit influence catalysis and subunit interactions.

    PubMed Central

    Paul, K; Morell, M K; Andrews, T J

    1993-01-01

    The first 20 residues at the amino terminus of the small subunit of spinach ribulose-1,5-bisphosphate carboxylase form an irregular arm that makes extensive contacts with the large subunit and also with another small subunit (S. Knight, I. Andersson, and C.-I. Brändén [1990] J Mol Biol 215: 113-160). The influence of these contacts on subunit binding and, indirectly, on catalysis was investigated by constructing truncations from the amino terminus of the small subunit of the highly homologous enzyme from Synechococcus PCC 6301 expressed in Escherichia coli. Removal of the first six residues (and thus the region of contact with a neighboring small subunit) affected neither the affinity with which the small subunits bound to the large subunits nor the catalytic properties of the assembled holoenzyme. Extending the truncation to include the first 12 residues (which encroaches into a highly conserved region that interacts with the large subunit) also did not weaken intersubunit binding appreciably, but it reduced the catalytic activity of the holoenzyme nearly 5-fold. Removal of an additional single residue (i.e. removal of a total of 13 residues) weakened intersubunit binding approximately 80-fold. Paradoxically, this partially restored catalytic activity to approximately 40% of that of the wild-type holoenzyme. None of these truncations materially affected the Km values for ribulose-1,5-bisphosphate or CO2. Removal of all 20 residues of the irregular arm (thereby deleting the conserved region of contact with large subunits) totally abolished the small subunit's ability to bind to large subunits to form a stable holoenzyme. However, this truncated small subunit was still synthesized by the E. coli cells. These data are interpreted in terms of the role of the amino-terminal arm of the small subunit in maintaining the structure of the holoenzyme. PMID:8278544

  1. Structure of the archaeal Cascade subunit Csa5: relating the small subunits of CRISPR effector complexes.

    PubMed

    Reeks, Judith; Graham, Shirley; Anderson, Linzi; Liu, Huanting; White, Malcolm F; Naismith, James H

    2013-05-01

    The Cascade complex for CRISPR-mediated antiviral immunity uses CRISPR RNA (crRNA) to target invading DNA species from mobile elements such as viruses, leading to their destruction. The core of the Cascade effector complex consists of the Cas5 and Cas7 subunits, which are widely conserved in prokaryotes. Cas7 binds crRNA and forms the helical backbone of Cascade. Many archaea encode a version of the Cascade complex (denoted Type I-A) that includes a Csa5 (or small) subunit, which interacts weakly with the core proteins. Here, we report the crystal structure of the Csa5 protein from Sulfolobus solfataricus. Csa5 comprises a conserved α-helical domain with a small insertion consisting of a weakly conserved β-strand domain. In the crystal, the Csa5 monomers have multimerized into infinite helical threads. At each interface is a strictly conserved intersubunit salt bridge, deletion of which disrupts multimerization. Structural analysis indicates a shared evolutionary history among the small subunits of the CRISPR effector complexes. The same α-helical domain is found in the C-terminal domain of Cse2 (from Type I-E Cascade), while the N-terminal domain of Cse2 is found in Cmr5 of the CMR (Type III-B) effector complex. As Cmr5 shares no match with Csa5, two possibilities present themselves: selective domain loss from an ancestral Cse2 to create two new subfamilies or domain fusion of two separate families to create a new Cse2 family. A definitive answer awaits structural studies of further small subunits from other CRISPR effector complexes.

  2. Conformation of yeast 18S rRNA. Direct chemical probing of the 5' domain in ribosomal subunits and in deproteinized RNA by reverse transcriptase mapping of dimethyl sulfate-accessible.

    PubMed Central

    Lempereur, L; Nicoloso, M; Riehl, N; Ehresmann, C; Ehresmann, B; Bachellerie, J P

    1985-01-01

    The structure of the 5' domain of yeast 18S rRNA has been probed by dimethyl sulfate (DMS), either in "native" deproteinized molecules or in the 40S ribosomal subunits. DMS-reacted RNA has been used as a template for reverse transcription and a large number of reactive sites, corresponding to all types of bases have been mapped by a primer extension procedure, taking advantage of blocks in cDNA elongation immediately upstream from bases methylated at atom positions involved in the base-pair recognition of the template. Since the same atom positions are protected from DMS in base-paired nucleotides, the secondary structure status of each nucleotide can be directly assessed in this procedure, thus allowing to evaluate the potential contribution of proteins in modulating subunit rRNA conformation. While the DMS probing of deproteinized rRNA confirms a number of helical stems predicted by phylogenetic comparisons, it is remarkable that a few additional base-pairings, while proven by the comparative analysis, appear to require the presence of the bound ribosomal subunit proteins to be stabilized. Images PMID:2417197

  3. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Caers, A; De Rijk, P; De Wachter, R

    1998-01-01

    About 8600 complete or nearly complete sequences are now available from the Antwerp database on small ribosomal subunit RNA. All these sequences are aligned with one another on the basis of the adopted secondary structure model, which is corroborated by the observation of compensating substitutions in the alignment. Literature references, accession numbers and detailed taxonomic information are also compiled. The database can be consulted via the World Wide Web at URL http://rrna.uia.ac.be/ssu/ PMID:9399829

  4. Ribosomal small subunit domains radiate from a central core

    PubMed Central

    Gulen, Burak; Petrov, Anton S.; Okafor, C. Denise; Vander Wood, Drew; O’Neill, Eric B.; Hud, Nicholas V.; Williams, Loren Dean

    2016-01-01

    The domain architecture of a large RNA can help explain and/or predict folding, function, biogenesis and evolution. We offer a formal and general definition of an RNA domain and use that definition to experimentally characterize the rRNA of the ribosomal small subunit. Here the rRNA comprising a domain is compact, with a self-contained system of molecular interactions. A given rRNA helix or stem-loop must be allocated uniquely to a single domain. Local changes such as mutations can give domain-wide effects. Helices within a domain have interdependent orientations, stabilities and interactions. With these criteria we identify a core domain (domain A) of small subunit rRNA. Domain A acts as a hub, linking the four peripheral domains and imposing orientational and positional restraints on the other domains. Experimental characterization of isolated domain A, and mutations and truncations of it, by methods including selective 2′OH acylation analyzed by primer extension and circular dichroism spectroscopy are consistent with our architectural model. The results support the utility of the concept of an RNA domain. Domain A, which exhibits structural similarity to tRNA, appears to be an essential core of the small ribosomal subunit. PMID:26876483

  5. Differential accumulation of ribonucleotide reductase subunits in clam oocytes: the large subunit is stored as a polypeptide, the small subunit as untranslated mRNA

    PubMed Central

    1986-01-01

    Within minutes of fertilization of clam oocytes, translation of a set of maternal mRNAs is activated. One of the most abundant of these stored mRNAs encodes the small subunit of ribonucleotide reductase (Standart, N. M., S. J. Bray, E. L. George, T. Hunt, and J. V. Ruderman, 1985, J. Cell Biol., 100:1968-1976). Unfertilized oocytes do not contain any ribonucleotide reductase activity; such activity begins to appear shortly after fertilization. In virtually all organisms, this enzyme is composed of two dissimilar subunits with molecular masses of approximately 44 and 88 kD, both of which are required for activity. This paper reports the identification of the large subunit of clam ribonucleotide reductase isolated by dATP-Sepharose chromatography as a relatively abundant 86-kD polypeptide which is already present in oocytes, and whose level remains constant during early development. The enzyme activity of this large subunit was established in reconstitution assays using the small subunit isolated from embryos by virtue of its binding to the anti-tubulin antibody YL 1/2. Thus the two components of clam ribonucleotide reductase are differentially stored in the oocyte: the small subunit in the form of untranslated mRNA and the large subunit as protein. When fertilization triggers the activation of translation of the maternal mRNA, the newly synthesized small subunit combines with the preformed large subunit to generate active ribonucleotide reductase. PMID:3536960

  6. Identification of Egyptian Fasciola species by PCR and restriction endonucleases digestion of the nuclear small subunit ribosomal RNA gene.

    PubMed

    El-Gozamy, Bothina R; Shoukry, Nahla M

    2009-08-01

    Fascioliasis is one of the familiar zoonotic health problems of worldwide distribution including Egypt. In this study, a simple and rapid polymerase chain reaction/restriction fragment length polymorphisms (PCR/RFLPs) assay, using the common restriction endonucleases Aval, EcoRI, Eael, Sac11 and Avail was applied to differentiate between both Fasciola gigantica and F. hepatica. The five restriction endonucleases were used to differentiate between the two species of Fasciola based on -1950 bp long sequence of the 18S nuclear small subunit ribosomal RNA gene. Aval and EcoRI restriction endonucleases failed to differentiate between the two Fasciola species when each restriction enzyme gave the same restriction patterns in both of them. However, F. gigantica and F. hepatica were well-differentiated when their small subunit ribosomal DNA were digested with Eael and Sac 11 restriction endonucleases.

  7. Genetic characterization of clinical acanthamoeba isolates from Japan using nuclear and mitochondrial small subunit ribosomal RNA.

    PubMed

    Rahman, Md Moshiur; Yagita, Kenji; Kobayashi, Akira; Oikawa, Yosaburo; Hussein, Amjad I A; Matsumura, Takahiro; Tokoro, Masaharu

    2013-08-01

    Because of an increased number of Acanthamoeba keratitis (AK) along with associated disease burdens, medical professionals have become more aware of this pathogen in recent years. In this study, by analyzing both the nuclear 18S small subunit ribosomal RNA (18S rRNA) and mitochondrial 16S rRNA gene loci, 27 clinical Acanthamoeba strains that caused AK in Japan were classified into 3 genotypes, T3 (3 strains), T4 (23 strains), and T5 (one strain). Most haplotypes were identical to the reference haplotypes reported from all over the world, and thus no specificity of the haplotype distribution in Japan was found. The T4 sub-genotype analysis using the 16S rRNA gene locus also revealed a clear sub-conformation within the T4 cluster, and lead to the recognition of a new sub-genotype T4i, in addition to the previously reported sub-genotypes T4a-T4h. Furthermore, 9 out of 23 strains in the T4 genotype were identified to a specific haplotype (AF479533), which seems to be a causal haplotype of AK. While heterozygous nuclear haplotypes were observed from 2 strains, the mitochondrial haplotypes were homozygous as T4 genotype in the both strains, and suggested a possibility of nuclear hybridization (mating reproduction) between different strains in Acanthamoeba. The nuclear 18S rRNA gene and mitochondrial 16S rRNA gene loci of Acanthamoeba spp. possess different unique characteristics usable for the genotyping analyses, and those specific features could contribute to the establishment of molecular taxonomy for the species complex of Acanthamoeba.

  8. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Nicolaï, S; De Rijk, P; De Wachter, R

    1996-01-01

    The Antwerp database on small ribosomal subunit RNA offers over 4300 nucleotide sequences (August 1995). All these sequences are stored in the form of an alignment based on the adopted secondary structure model, which in turn is corroborated by the observation of compensating substitutions in the alignment. Besides the primary and secondary structure information, literature references, accession numbers and detailed taxonomic information are also compiled. The complete database is made available to the scientific community through anonymous ftp and World Wide Web(WWW). PMID:8594609

  9. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Van den Broeck, I; De Rijk, P; De Wachter, R

    1994-01-01

    The database on small ribosomal subunit RNA structure contains (June 1994) 2824 nucleotide sequences. All these sequences are stored in the form of an alignment based on the adopted secondary structure model, which in turn is corroborated by the observation of compensating substitutions in the alignment. The complete database is made available to the scientific community through anonymous ftp on our server in Antwerp. A special effort was made to improve electronic retrieval and a program is supplied that allows to create different file formats. The database can also be obtained from the EMBL nucleotide sequence library. PMID:7524022

  10. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Jansen, J; De Rijk, P; De Wachter, R

    1997-01-01

    The Antwerp database on small ribosomal subunit RNA now offers more than 6000 nucleotide sequences (August 1996). All these sequences are stored in the form of an alignment based on the adopted secondary structure model, which is corroborated by the observation of compensating substitutions in the alignment. Besides the primary and secondary structure information, literature references, accession numbers and detailed taxonomic information are also compiled. For ease of use, the complete database is made available to the scientific community via World Wide Web at URL http://rrna.uia.ac.be/ssu/ . PMID:9016516

  11. The discovery of the two types of small subunit ribosomal RNA gene in Eimeria mitis contests the existence of E. mivati as an independent species.

    PubMed

    Vrba, Vladimir; Poplstein, Martin; Pakandl, Michal

    2011-12-29

    Although the validity of the coccidian species, Eimeria mivati, has been questioned by many researchers for a long time there has not been any molecular analysis that would help resolve this issue. Here we report on the discovery of the two types of small ribosomal subunit (18S) gene within the Eimeria mitis genome that correspond to the known 18S sequences of E. mitis and E. mivati, and this is in conflict with the existence of E. mivati as an independent species. We have carried out five single oocyst isolations to obtain five single-oocyst-derived strains of E. mitis and these were analyzed by the sequencing of 18S and mitochondrial cytochrome c oxidase subunit I genes. The two types of 18S gene were found to be present in each strain in roughly equal ratios. This indicates that if the strains carrying only one or the other 18S type exist, they will likely cross-breed and still represent a single species. However, the more probable explanation is that all strains of E. mitis contain two types of 18S gene and that the occasional detection of only one or the other type by sequencing might be caused by insufficient sampling. This is also the first report of the two types of 18S gene in Eimeria, which has already been described in some other apicomplexan species, most notably Plasmodium. We also found that these two types of ribosomal RNA differ significantly in their secondary structure. The biological significance of the two 18S gene variants in E. mitis is not known, however, we hypothesize that these variants might be used in different stages of the parasite's life-cycle as it is in other apicomplexan species investigated so far.

  12. Seasonal and geographical distribution of near-surface small photosynthetic eukaryotes in the western North Pacific determined by pyrosequencing of 18S rDNA.

    PubMed

    Kataoka, Takafumi; Yamaguchi, Haruyo; Sato, Mayumi; Watanabe, Tsuyoshi; Taniuchi, Yukiko; Kuwata, Akira; Kawachi, Masanobu

    2017-02-01

    In this study, we investigated the distribution of small photosynthetic eukaryotes in the near-surface layer of the western North Pacific at four stations, including two oceanic stations where the subarctic Oyashio and subtropical Kuroshio currents influence a transition region and the bay mouth and head of the Sendai Bay, from April 2012 to May 2013. Flow cytometry was applied to sort small photosynthetic eukaryotes (<5 μm), and high-throughput sequencing of 18S rDNA was performed. Our taxonomic analysis showed that 19/195 operational taxonomic units (OTUs) were frequently distributed among all sites. Composition analysis showed that the OTUs had characteristic patterns and were divided into four main groups. Two groups reflected the low-saline water and winter season, with the characteristic OTUs belonging to diatoms; Chaetoceros and Leptocylindrus were characteristic of low saline water, and two diatom genera (Minidiscus and Minutocellus) and Cryptomonadales-related OTUs were prevalent in the winter. Our results indicate that the community composition of small photosynthetic eukaryotes seasonally changes in a dynamic manner according to variations in water properties.

  13. Hybrid Rubisco of tomato large subunits and tobacco small subunits is functional in tobacco plants.

    PubMed

    Zhang, Xing-Hai; Webb, James; Huang, Yi-Hong; Lin, Li; Tang, Ri-Sheng; Liu, Aimin

    2011-03-01

    Biogenesis of functional ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in plants requires specific assembly in the chloroplast of the imported, cytosol-synthesized small subunits (SS) with the chloroplast-made large subunits (LS). Accumulating evidence indicates that chloroplasts (plastids) generally have a low tolerance for assembling foreign or modified Rubisco. To explore Rubisco engineering, we created two lines of transplastomic tobacco plants whose rbcL gene was replaced by tomato-derived rbcL: plant LLS2 with Rubisco composed of tobacco SS and Q437R LS and plant LLS4 with a hybrid Rubisco of tobacco SS and tomato LS (representing four substitutions of Y226F, A230T, S279T and Q437R from tobacco LS). Plant LLS2 exhibited similar phenotypes as the wild type. Plant LLS4 showed lower chlorophyll and Rubisco levels particularly in young emerging leaves, lower photosynthesis rates and biomass during early stages of development, but was able to reach reproductive maturity and somewhat wild type-like phenotype under ambient CO₂ condition. In vitro assays detected similar carboxylase activity and RuBP affinity in LLS2 and LLS4 plants as in wild type. Our studies demonstrated that tomato LS was sufficiently assembled with tobacco SS into functional Rubisco. The hybrid Rubisco of tomato LS and tobacco SS can drive photosynthesis that supports photoautotrophic growth and reproduction of tobacco plants under ambient CO₂ and light conditions. We discuss the effect of these residue substitutions on Rubisco activity and the possible attribution of chlorophyll deficiency to the in planta photosynthesis performance in the hybrid Rubisco plants.

  14. An overview of the secondary structure of the V4 region of eukaryotic small-subunit ribosomal RNA.

    PubMed Central

    Nickrent, D L; Sargent, M L

    1991-01-01

    The V4 region of the small subunit (18S) ribosomal RNA was examined in 72 different sequences representing a broad sample eukaryotic diversity. This domain is the most variable region of the 18S rRNA molecule and ranges in length from ca. 230 to over 500 bases. Based upon comparative analysis, secondary structural models were constructed for all sequences and the resulting generalized model shows that most organisms possess seven helices for this region. The protists and two insects show from one to as many as four helices in addition to the above seven. In this report, we summarize secondary structure information presented elsewhere for the V4 region, describe the general features for helical and apical regions, and identify signature sequences useful in helix identification. Our model generally agrees with other current concepts; however, we propose modifications or alternative structures for the start of the V4 region, the large protist inserts, and the sector that may possibly contain a pseudoknot. PMID:2014163

  15. Prevalent ciliate symbiosis on copepods: high genetic diversity and wide distribution detected using small subunit ribosomal RNA gene.

    PubMed

    Guo, Zhiling; Liu, Sheng; Hu, Simin; Li, Tao; Huang, Yousong; Liu, Guangxing; Zhang, Huan; Lin, Senjie

    2012-01-01

    Toward understanding the genetic diversity and distribution of copepod-associated symbiotic ciliates and the evolutionary relationships with their hosts in the marine environment, we developed a small subunit ribosomal RNA gene (18S rDNA)-based molecular method and investigated the genetic diversity and genotype distribution of the symbiotic ciliates on copepods. Of the 10 copepod species representing six families collected from six locations of Pacific and Atlantic Oceans, 9 were found to harbor ciliate symbionts. Phylogenetic analysis of the 391 ciliate 18S rDNA sequences obtained revealed seven groups (ribogroups), six (containing 99% of all the sequences) belonging to subclass Apostomatida, the other clustered with peritrich ciliate Vorticella gracilis. Among the Apostomatida groups, Group III were essentially identical to Vampyrophrya pelagica, and the other five groups represented the undocumented ciliates that were close to Vampyrophrya/Gymnodinioides/Hyalophysa. Group VI ciliates were found in all copepod species but one (Calanus sinicus), and were most abundant among all ciliate sequences obtained, indicating that they are the dominant symbiotic ciliates universally associated with copepods. In contrast, some ciliate sequences were found only in some of the copepods examined, suggesting the host selectivity and geographic differentiation of ciliates, which requires further verification by more extensive sampling. Our results reveal the wide occurrence and high genetic diversity of symbiotic ciliates on marine copepods and highlight the need to systematically investigate the host- and geography-based genetic differentiation and ecological roles of these ciliates globally.

  16. Prevalent Ciliate Symbiosis on Copepods: High Genetic Diversity and Wide Distribution Detected Using Small Subunit Ribosomal RNA Gene

    PubMed Central

    Guo, Zhiling; Liu, Sheng; Hu, Simin; Li, Tao; Huang, Yousong; Liu, Guangxing; Zhang, Huan; Lin, Senjie

    2012-01-01

    Toward understanding the genetic diversity and distribution of copepod-associated symbiotic ciliates and the evolutionary relationships with their hosts in the marine environment, we developed a small subunit ribosomal RNA gene (18S rDNA)-based molecular method and investigated the genetic diversity and genotype distribution of the symbiotic ciliates on copepods. Of the 10 copepod species representing six families collected from six locations of Pacific and Atlantic Oceans, 9 were found to harbor ciliate symbionts. Phylogenetic analysis of the 391 ciliate 18S rDNA sequences obtained revealed seven groups (ribogroups), six (containing 99% of all the sequences) belonging to subclass Apostomatida, the other clustered with peritrich ciliate Vorticella gracilis. Among the Apostomatida groups, Group III were essentially identical to Vampyrophrya pelagica, and the other five groups represented the undocumented ciliates that were close to Vampyrophrya/Gymnodinioides/Hyalophysa. Group VI ciliates were found in all copepod species but one (Calanus sinicus), and were most abundant among all ciliate sequences obtained, indicating that they are the dominant symbiotic ciliates universally associated with copepods. In contrast, some ciliate sequences were found only in some of the copepods examined, suggesting the host selectivity and geographic differentiation of ciliates, which requires further verification by more extensive sampling. Our results reveal the wide occurrence and high genetic diversity of symbiotic ciliates on marine copepods and highlight the need to systematically investigate the host- and geography-based genetic differentiation and ecological roles of these ciliates globally. PMID:23024768

  17. METAXA2: improved identification and taxonomic classification of small and large subunit rRNA in metagenomic data.

    PubMed

    Bengtsson-Palme, Johan; Hartmann, Martin; Eriksson, Karl Martin; Pal, Chandan; Thorell, Kaisa; Larsson, Dan Göran Joakim; Nilsson, Rolf Henrik

    2015-11-01

    The ribosomal rRNA genes are widely used as genetic markers for taxonomic identification of microbes. Particularly the small subunit (SSU; 16S/18S) rRNA gene is frequently used for species- or genus-level identification, but also the large subunit (LSU; 23S/28S) rRNA gene is employed in taxonomic assignment. The METAXA software tool is a popular utility for extracting partial rRNA sequences from large sequencing data sets and assigning them to an archaeal, bacterial, nuclear eukaryote, mitochondrial or chloroplast origin. This study describes a comprehensive update to METAXA - METAXA2 - that extends the capabilities of the tool, introducing support for the LSU rRNA gene, a greatly improved classifier allowing classification down to genus or species level, as well as enhanced support for short-read (100 bp) and paired-end sequences, among other changes. The performance of METAXA2 was compared to other commonly used taxonomic classifiers, showing that METAXA2 often outperforms previous methods in terms of making correct predictions while maintaining a low misclassification rate. METAXA2 is freely available from http://microbiology.se/software/metaxa2/.

  18. Differential Targeting of Gβγ-Subunit Signaling with Small Molecules

    NASA Astrophysics Data System (ADS)

    Bonacci, Tabetha M.; Mathews, Jennifer L.; Yuan, Chujun; Lehmann, David M.; Malik, Sundeep; Wu, Dianqing; Font, Jose L.; Bidlack, Jean M.; Smrcka, Alan V.

    2006-04-01

    G protein βγ subunits have potential as a target for therapeutic treatment of a number of diseases. We performed virtual docking of a small-molecule library to a site on Gβγ subunits that mediates protein interactions. We hypothesized that differential targeting of this surface could allow for selective modulation of Gβγ subunit functions. Several compounds bound to Gβγ subunits with affinities from 0.1 to 60 μM and selectively modulated functional Gβγ-protein-protein interactions in vitro, chemotactic peptide signaling pathways in HL-60 leukocytes, and opioid receptor-dependent analgesia in vivo. These data demonstrate an approach for modulation of G protein-coupled receptor signaling that may represent an important therapeutic strategy.

  19. DEAD-box RNA helicase Dbp4 is required for small-subunit processome formation and function.

    PubMed

    Soltanieh, Sahar; Osheim, Yvonne N; Spasov, Krasimir; Trahan, Christian; Beyer, Ann L; Dragon, François

    2015-03-01

    DEAD-box RNA helicase Dbp4 is required for 18S rRNA synthesis: cellular depletion of Dbp4 impairs the early cleavage reactions of the pre-rRNA and causes U14 small nucleolar RNA (snoRNA) to remain associated with pre-rRNA. Immunoprecipitation experiments (IPs) carried out with whole-cell extracts (WCEs) revealed that hemagglutinin (HA)-tagged Dbp4 is associated with U3 snoRNA but not with U14 snoRNA. IPs with WCEs also showed association with the U3-specific protein Mpp10, which suggests that Dbp4 interacts with the functionally active U3 RNP; this particle, called the small-subunit (SSU) processome, can be observed at the 5' end of nascent pre-rRNA. Electron microscopy analyses indicated that depletion of Dbp4 compromised SSU processome formation and cotranscriptional cleavage of the pre-rRNA. Sucrose density gradient analyses revealed that depletion of U3 snoRNA or the Mpp10 protein inhibited the release of U14 snoRNA from pre-rRNA, just as was seen with Dbp4-depleted cells, indicating that alteration of SSU processome components has significant consequences for U14 snoRNA dynamics. We also found that the C-terminal extension flanking the catalytic core of Dbp4 plays an important role in the release of U14 snoRNA from pre-rRNA.

  20. DEAD-Box RNA Helicase Dbp4 Is Required for Small-Subunit Processome Formation and Function

    PubMed Central

    Soltanieh, Sahar; Osheim, Yvonne N.; Spasov, Krasimir; Trahan, Christian; Beyer, Ann L.

    2014-01-01

    DEAD-box RNA helicase Dbp4 is required for 18S rRNA synthesis: cellular depletion of Dbp4 impairs the early cleavage reactions of the pre-rRNA and causes U14 small nucleolar RNA (snoRNA) to remain associated with pre-rRNA. Immunoprecipitation experiments (IPs) carried out with whole-cell extracts (WCEs) revealed that hemagglutinin (HA)-tagged Dbp4 is associated with U3 snoRNA but not with U14 snoRNA. IPs with WCEs also showed association with the U3-specific protein Mpp10, which suggests that Dbp4 interacts with the functionally active U3 RNP; this particle, called the small-subunit (SSU) processome, can be observed at the 5′ end of nascent pre-rRNA. Electron microscopy analyses indicated that depletion of Dbp4 compromised SSU processome formation and cotranscriptional cleavage of the pre-rRNA. Sucrose density gradient analyses revealed that depletion of U3 snoRNA or the Mpp10 protein inhibited the release of U14 snoRNA from pre-rRNA, just as was seen with Dbp4-depleted cells, indicating that alteration of SSU processome components has significant consequences for U14 snoRNA dynamics. We also found that the C-terminal extension flanking the catalytic core of Dbp4 plays an important role in the release of U14 snoRNA from pre-rRNA. PMID:25535329

  1. Group I introns in small subunit ribosomal DNA of several Phaeosphaeria species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In a study of small subunit ribosomal RNA (SSU-rRNA) gene sequences in Phaeosphaeria species, group I introns were found in 9 of 10 P. avenaria f.sp. avenaria (Paa) isolates, 1 of 2 Phaeosphaeria sp. (P-rye) isolates from Polish rye (Sn48-1), 1 Phaeosphaeria sp. from dallis grass (P-dg) (S-93-48) an...

  2. Recognition of chimeric small-subunit ribosomal DNAs composed of genes from uncultivated microorganisms

    NASA Technical Reports Server (NTRS)

    Kopczynski, E. D.; Bateson, M. M.; Ward, D. M.

    1994-01-01

    When PCR was used to recover small-subunit (SSU) rRNA genes from a hot spring cyanobacterial mat community, chimeric SSU rRNA sequences which exhibited little or no secondary structural abnormality were recovered. They were revealed as chimeras of SSU rRNA genes of uncultivated species through separate phylogenetic analysis of short sequence domains.

  3. Expression of a foreign Rubisco small subunit in tobacco with reduced levels of the native protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cDNA, ArRbcS3, for the small subunit of Rubisco from Amaranthus retroflexus (pigweed) was expressed in tobacco (Nicotiana tabacum) under the control of a strong leaf-specific Lhcb promoter. The coding region of the ArRbcS3 was fused to the plastid targeting sequence of the native tobacco rbcS to...

  4. Inheritance behavior of information coding for small subunit polypeptides of fraction 1 protein.

    PubMed

    Chen, K; Wildman, S G

    1980-12-01

    In various genera of plants, the small subunit of fraction 1 protein is often composed of more than one kind of polypeptide; these differ in isoelectric points and amino acid composition. Previous analysis of numerous individual progeny of Nicotiana tabacum (two kinds of polypeptides), N. glauca + N. langsdorffii parasexual hybrids (three kinds) and other examples showed no change in F-1 protein composition as a consequence of alternation of generations. Experiments reported here show that absence of one number of each of the 24 different pairs of chromosomes in an N. tabacum monosomic series and also absence of the "S" pair in a nullisome did not affect F-1 protein composition. Absence of the "E" pair caused reduction in the amount of the least acidic of the two kinds of N. tabacum small subunit polypeptides. The question of how many individual progeny of self-fertile hybrids would have to be analyzed to detect segregation of genes coding for F-1 protein small subunit polypeptides, if segregation occurs, was answered by analysis of F1 hybrids between N. otophora and N. tomentosiformis, and two subspecies of N. suaveolens, together with their F2 progeny. In both cases, analysis of 16 progeny was sufficient to demonstrate a segregation pattern of two F1 hybrid type to one each of the two parental types. Therefore, in the absence of segregation, it is likely that coding information for different kinds of F-1 protein small subunit polypeptides is sequestered on heterologous chromosomes, as postulated in previous reports.

  5. Molecular phylogeny of Stentor (Ciliophora: Heterotrichea) based on small subunit ribosomal RNA sequences.

    PubMed

    Gong, Ying-Chun; Yu, Yu-He; Zhu, Fei-Yun; Feng, Wei-Song

    2007-01-01

    To determine the phylogenetic position of Stentor within the Class Heterotrichea, the complete small subunit rRNA genes of three Stentor species, namely Stentor polymorphus, Stentor coeruleus, and Stentor roeseli, were sequenced and used to construct phylogenetic trees using the maximum parsimony, neighbor joining, and Bayesian analysis. With all phylogenetic methods, the genus Stentor was monophyletic, with S. roeseli branching basally.

  6. Functional hybrid rubisco enzymes with plant small subunits and algal large subunits: engineered rbcS cDNA for expression in chlamydomonas.

    PubMed

    Genkov, Todor; Meyer, Moritz; Griffiths, Howard; Spreitzer, Robert J

    2010-06-25

    There has been much interest in the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) as a target for engineering an increase in net CO(2) fixation in photosynthesis. Improvements in the enzyme would lead to an increase in the production of food, fiber, and renewable energy. Although the large subunit contains the active site, a family of rbcS nuclear genes encodes the Rubisco small subunits, which can also influence the carboxylation catalytic efficiency and CO(2)/O(2) specificity of the enzyme. To further define the role of the small subunit in Rubisco function, small subunits from spinach, Arabidopsis, and sunflower were assembled with algal large subunits by transformation of a Chlamydomonas reinhardtii mutant that lacks the rbcS gene family. Foreign rbcS cDNAs were successfully expressed in Chlamydomonas by fusing them to a Chlamydomonas rbcS transit peptide sequence engineered to contain rbcS introns. Although plant Rubisco generally has greater CO(2)/O(2) specificity but a lower carboxylation V(max) than Chlamydomonas Rubisco, the hybrid enzymes have 3-11% increases in CO(2)/O(2) specificity and retain near normal V(max) values. Thus, small subunits may make a significant contribution to the overall catalytic performance of Rubisco. Despite having normal amounts of catalytically proficient Rubisco, the hybrid mutant strains display reduced levels of photosynthetic growth and lack chloroplast pyrenoids. It appears that small subunits contain the structural elements responsible for targeting Rubisco to the algal pyrenoid, which is the site where CO(2) is concentrated for optimal photosynthesis.

  7. The gene for the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit relocated to the plastid genome of tobacco directs the synthesis of small subunits that assemble into Rubisco.

    PubMed

    Whitney, S M; Andrews, T J

    2001-01-01

    To assess the extent to which a nuclear gene for a chloroplast protein retained the ability to be expressed in its presumed preendosymbiotic location, we relocated the RbcS gene for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to the tobacco plastid genome. Plastid RbcS transgenes, both with and without the transit presequence, were equipped with 3' hepta-histidine-encoding sequences and psbA promoter and terminator elements. Both transgenes were transcribed abundantly, and their products were translated into small subunit polypeptides that folded correctly and assembled into the Rubisco hexadecamer. When present, either the transit presequence was not translated or the transit peptide was cleaved completely. After assembly into Rubisco, transplastomic small subunits were relatively stable. The hepta-histidine sequence fused to the C terminus of a single small subunit was sufficient for isolation of the whole Rubisco hexadecamer by Ni(2)+ chelation. Small subunits produced by the plastid transgenes were not abundant, never exceeding approximately 1% of the total small subunits, and they differed from cytoplasmically synthesized small subunits in their N-terminal modifications. The scarcity of transplastomic small subunits might be caused by inefficient translation or assembly.

  8. Mammalian mitochondrial ribosomal small subunit (MRPS) genes: A putative role in human disease.

    PubMed

    Gopisetty, Gopal; Thangarajan, Rajkumar

    2016-09-01

    Mitochondria are prominently understood as power houses producing ATP the primary energy currency of the cell. However, mitochondria are also known to play an important role in apoptosis and autophagy, and mitochondrial dysregulation can lead to pathological outcomes. Mitochondria are known to contain 1500 proteins of which only 13 are coded by mitochondrial DNA and the rest are coded by nuclear genes. Protein synthesis in mitochondria involves mitochondrial ribosomes which are 55-60S particles and are composed of small 28S and large 39S subunits. A feature of mammalian mitoribosome which differentiate it from bacterial ribosomes is the increased protein content. The human mitochondrial ribosomal protein (MRP) gene family comprises of 30 genes which code for mitochondrial ribosomal small subunit and 50 genes for the large subunit. The present review focuses on the mitochondrial ribosomal small subunit genes (MRPS), presents an overview of the literature and data gleaned from publicly available gene and protein expression databases. The survey revealed aberrations in MRPS gene expression patterns in varied human diseases indicating a putative role in their etiology.

  9. In Search of Small Molecule Inhibitors Targeting the Flexible CK2 Subunit Interface

    PubMed Central

    Bestgen, Benoît; Belaid-Choucair, Zakia; Lomberget, Thierry; Le Borgne, Marc; Filhol, Odile; Cochet, Claude

    2017-01-01

    Protein kinase CK2 is a tetrameric holoenzyme composed of two catalytic (α and/or α’) subunits and two regulatory (β) subunits. Crystallographic data paired with fluorescence imaging techniques have suggested that the formation of the CK2 holoenzyme complex within cells is a dynamic process. Although the monomeric CK2α subunit is endowed with a constitutive catalytic activity, many of the plethora of CK2 substrates are exclusively phosphorylated by the CK2 holoenzyme. This means that the spatial and high affinity interaction between CK2α and CK2β subunits is critically important and that its disruption may provide a powerful and selective way to block the phosphorylation of substrates requiring the presence of CK2β. In search of compounds inhibiting this critical protein–protein interaction, we previously designed an active cyclic peptide (Pc) derived from the CK2β carboxy-terminal domain that can efficiently antagonize the CK2 subunit interaction. To understand the functional significance of this interaction, we generated cell-permeable versions of Pc, exploring its molecular mechanisms of action and the perturbations of the signaling pathways that it induces in intact cells. The identification of small molecules inhibitors of this critical interaction may represent the first-choice approach to manipulate CK2 in an unconventional way. PMID:28165359

  10. In Search of Small Molecule Inhibitors Targeting the Flexible CK2 Subunit Interface.

    PubMed

    Bestgen, Benoît; Belaid-Choucair, Zakia; Lomberget, Thierry; Le Borgne, Marc; Filhol, Odile; Cochet, Claude

    2017-02-03

    Protein kinase CK2 is a tetrameric holoenzyme composed of two catalytic (α and/or α') subunits and two regulatory (β) subunits. Crystallographic data paired with fluorescence imaging techniques have suggested that the formation of the CK2 holoenzyme complex within cells is a dynamic process. Although the monomeric CK2α subunit is endowed with a constitutive catalytic activity, many of the plethora of CK2 substrates are exclusively phosphorylated by the CK2 holoenzyme. This means that the spatial and high affinity interaction between CK2α and CK2β subunits is critically important and that its disruption may provide a powerful and selective way to block the phosphorylation of substrates requiring the presence of CK2β. In search of compounds inhibiting this critical protein-protein interaction, we previously designed an active cyclic peptide (Pc) derived from the CK2β carboxy-terminal domain that can efficiently antagonize the CK2 subunit interaction. To understand the functional significance of this interaction, we generated cell-permeable versions of Pc, exploring its molecular mechanisms of action and the perturbations of the signaling pathways that it induces in intact cells. The identification of small molecules inhibitors of this critical interaction may represent the first-choice approach to manipulate CK2 in an unconventional way.

  11. Role of the Rubisco small subunit. Final report for period May 1, 1997--April 30,2000

    SciTech Connect

    Spreitzer, Robert J.

    2000-10-04

    CO{sub 2} and O{sub 2} are mutually competitive at the active site of ribulose-1,5-biphosphate (RuBP) carboxylase/oxygenase (Rubisco). Rubisco contains two subunits, each present in eight copies. The 15-kD small subunit is coded by a family of nuclear RbcS genes. Until now, the role of the small subunit in Rubisco structure or catalytic efficiency is not known. Because of other work in eliminating the two RbcS genes in the green algo Chlamydomonas reinhardtii, it is now possible to address questions about the structure-function relationships of the eukaryotic small subunit. There are three specific aims in this project: (1) Alanine scanning mutagenesis is being used to dissect the importance of the {beta}A/{beta}B loop, a feature unique to the eukaryotic small subunit. (2) Random mutagenesis is being used to identify additional residues or regions of the small subunit that are important for holoenzyme assembly and function. (3) Attempts are being made to express foreign small subunits in Chlamydomonas to examine the contribution of small subunits to holoenzyme assembly, catalytic efficiency, and CO{sub 2}/O{sub 2} specificity.

  12. Substitution rate calibration of small subunit ribosomal RNA identifies chlorarachniophyte endosymbionts as remnants of green algae.

    PubMed Central

    Van de Peer, Y; Rensing, S A; Maier, U G; De Wachter, R

    1996-01-01

    Chlorarachniophytes are amoeboid algae with chlorophyll a and b containing plastids that are surrounded by four membranes instead of two as in plants and green algae. These extra membranes form important support for the hypothesis that chlorarachniophytes have acquired their plastids by the ingestion of another eukaryotic plastid-containing alga. Chlorarachniophytes also contain a small nucleus-like structure called the nucleomorph situated between the two inner and the two outer membranes surrounding the plastid. This nucleomorph is a remnant of the endosymbiont's nucleus and encodes, among other molecules, small subunit ribosomal RNA. Previous phylogenetic analyses on the basis of this molecule provided unexpected and contradictory evidence for the origin of the chlorarachniophyte endosymbiont. We developed a new method for measuring the substitution rates of the individual nucleotides of small subunit ribosomal RNA. From the resulting substitution rate distribution, we derived an equation that gives a more realistic relationship between sequence dissimilarity and evolutionary distance than equations previously available. Phylogenetic trees constructed on the basis of evolutionary distances computed by this new method clearly situate the chlorarachniophyte nucleomorphs among the green algae. Moreover, this relationship is confirmed by transversion analysis of the Chlorarachnion plastid small subunit ribosomal RNA. PMID:8755544

  13. Role of Small Subunit in Mediating Assembly of Red-type Form I Rubisco

    PubMed Central

    Joshi, Jidnyasa; Mueller-Cajar, Oliver; Tsai, Yi-Chin C.; Hartl, F. Ulrich; Hayer-Hartl, Manajit

    2015-01-01

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the key enzyme involved in photosynthetic carbon fixation, converting atmospheric CO2 to organic compounds. Form I Rubisco is a cylindrical complex composed of eight large (RbcL) subunits that are capped by four small subunits (RbcS) at the top and four at the bottom. Form I Rubiscos are phylogenetically divided into green- and red-type. Some red-type enzymes have catalytically superior properties. Thus, understanding their folding and assembly is of considerable biotechnological interest. Folding of the green-type RbcL subunits in cyanobacteria is mediated by the GroEL/ES chaperonin system, and assembly to holoenzyme requires specialized chaperones such as RbcX and RAF1. Here, we show that the red-type RbcL subunits in the proteobacterium Rhodobacter sphaeroides also fold with GroEL/ES. However, assembly proceeds in a chaperone-independent manner. We find that the C-terminal β-hairpin extension of red-type RbcS, which is absent in green-type RbcS, is critical for efficient assembly. The β-hairpins of four RbcS subunits form an eight-stranded β-barrel that protrudes into the central solvent channel of the RbcL core complex. The two β-barrels stabilize the complex through multiple interactions with the RbcL subunits. A chimeric green-type RbcS carrying the C-terminal β-hairpin renders the assembly of a cyanobacterial Rubisco independent of RbcX. Our results may facilitate the engineering of crop plants with improved growth properties expressing red-type Rubisco. PMID:25371207

  14. Eukaryote-specific extensions in ribosomal proteins of the small subunit: Structure and function

    PubMed Central

    Ghosh, Arnab; Komar, Anton A

    2015-01-01

    High-resolution structures of yeast ribosomes have improved our understanding of the architecture and organization of eukaryotic rRNA and proteins, as well as eukaryote-specific extensions present in some conserved ribosomal proteins. Despite this progress, assignment of specific functions to individual proteins and/or eukaryote-specific protein extensions remains challenging. It has been suggested that eukaryote-specific extensions of conserved proteins from the small ribosomal subunit may facilitate eukaryote-specific reactions in the initiation phase of protein synthesis. This review summarizes emerging data describing the structural and functional significance of eukaryote-specific extensions of conserved small ribosomal subunit proteins, particularly their possible roles in recruitment and spatial organization of eukaryote-specific initiation factors. PMID:26779416

  15. Rubisco small and large subunit N-methyltransferases. Bi- and mono-functional methyltransferases that methylate the small and large subunits of Rubisco.

    PubMed

    Ying, Z; Mulligan, R M; Janney, N; Houtz, R L

    1999-12-17

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)is methylated at the alpha-amino group of the N-terminal methionine of the processed form of the small subunit (SS), and at the epsilon-amino group of lysine-14 of the large subunit (LS) in some species. The Rubisco LS methyltransferase (LSMT) gene has been cloned and expressed from pea and specifically methylates lysine-14 of the LS of Rubisco. We determine here that both pea and tobacco Rubisco LSMT also exhibit (alpha)N-methyltransferase activity toward the SS of Rubisco, suggesting that a single gene product can produce a bifunctional protein methyltransferase capable of catalyzing both (alpha)N-methylation of the SS and (epsilon)N-methylation of the LS. A homologue of the Rubisco LSMT gene (rbcMT-S) has also been identified in spinach that is closely related to Rubisco LSMT sequences from pea and tobacco. Two mRNAs are produced from rbcMT-S, and both long and short forms of the spinach cDNAs were expressed in Escherichia coli cells and shown to catalyze methylation of the alpha-amino group of the N-terminal methionine of the SS of Rubisco. Thus, the absence of lysine-14 methylation in species like spinach is apparently a consequence of a monofunctional protein methyltransferase incapable of methylating Lys-14, with activity limited to methylation of the SS.

  16. Mapping of a conformational epitope on the cashew allergen Ana o 2: a discontinuous large subunit epitope dependent upon homologous or heterologous small subunit association.

    PubMed

    Xia, Lixin; Willison, LeAnna N; Porter, Lauren; Robotham, Jason M; Teuber, Suzanne S; Sathe, Shridhar K; Roux, Kenneth H

    2010-05-01

    The 11S globulins are members of the cupin protein superfamily and represent an important class of tree nut allergens for which a number of linear epitopes have been mapped. However, specific conformational epitopes for these allergens have yet to be described. We have recently reported a cashew Ana o 2 conformational epitope defined by murine mAb 2B5 and competitively inhibited by a subset of patient IgE antibodies. The 2B5 epitope appears to reside on the large (acidic) subunit, is dependent upon small (basic) subunit association for expression, and is highly susceptible to denaturation. Here we fine map the epitope using a combination of recombinant chimeric cashew Ana o 2-soybean Gly m 6 chimeras, deletion and point mutations, molecular modeling, and electron microscopy of 2B5-Ana o 2 immune complexes. Key residues appear confined to a 24 amino acid segment near the N-terminus of the large subunit peptide, a portion of which makes direct contact with the small subunit. These data provide an explanation for both the small subunit dependence and the structurally labile nature of the epitope.

  17. A definition of the domains Archaea, Bacteria and Eucarya in terms of small subunit ribosomal RNA characteristics

    NASA Technical Reports Server (NTRS)

    Winker, S.; Woese, C. R.

    1991-01-01

    The number of small subunit rRNA sequences is now great enough that the three domains Archaea, Bacteria and Eucarya (Woese et al., 1990) can be reliably defined in terms of their sequence "signatures". Approximately 50 homologous positions (or nucleotide pairs) in the small subunit rRNA characterize and distinguish among the three. In addition, the three can be recognized by a variety of nonhomologous rRNA characters, either individual positions and/or higher-order structural features. The Crenarchaeota and the Euryarchaeota, the two archaeal kingdoms, can also be defined and distinguished by their characteristic compositions at approximately fifteen positions in the small subunit rRNA molecule.

  18. A definition of the domains Archaea, Bacteria and Eucarya in terms of small subunit ribosomal RNA characteristics

    SciTech Connect

    Winker, S.; Woese, C.R.

    1994-11-01

    The number of small subunit rRNA sequences is not great enough that the three domains Archaea, Bacteria, and Eucarya (Woese, et al., 1990) can be reliably defined in terms of their sequence ``signatures.`` Approximately 50 homologous positions (or nucleotide pairs) in the small subunit rRNA characterized and distinguish among the three. In addition, the three can be recognized by a variety of nonhomologous rRNA characters, either individual positions and/or higher-order structural features. The Crenarchaeota and the Euryarchaeota, the two archaeal kingdoms, can also be defined and distinguished by their characteristic composition at approximately fifteen positions in the small subunit rRNA molecule.

  19. Chemical probing of adenine residues within the secondary structure of rabbit /sup 18/S ribosomal RNA

    SciTech Connect

    Rairkar, A.; Rubino, H.M.; Lockard, R.E.

    1988-01-26

    The location of unpaired adenine residues within the secondary structure of rabbit /sup 18/S ribosomal RNA was determined by chemical probing. Naked /sup 18/S rRNA was first prepared by digestion of purified 40S subunits with matrix-bound proteinase K in sodium dodecyl sulfate, thereby omitting the use of nucleic acid denaturants. Adenines within naked /sup 18/S rRNA were chemically probed by using either diethyl pyrocarbonate or dimethyl sulfate, which specifically react with unpaired nucleotides. Adenine modification sites were identified by polyacrylamide sequencing gel electrophoresis either upon aniline-induced strand scission of /sup 32/P-end-labeled intact and fragmented rRNA or by primer extension using sequence-specific DNA oligomers with reverse transcriptase. The data indicate good agreement between the general pattern of adenine reactivity and the location of unpaired regions in /sup 18/S rRNA determined by comparative sequence analysis. The overall reactivity of adenine residues toward single-strand-specific chemical probes was, also, similar for both rabbit and Escherichia coli small rRNA. The number of strongly reactive adenines appearing within phylogenetically determined helical segments, however, was greater in rabbit /sup 18/S rRNA than for E. coli /sup 16/S rRNA. Some of these adenines were found clustered in specific helices. Such differences suggest a greater irregularity of many of the helical elements within mammalian /sup 18/S rRNA, as compared with prokaryotic /sup 16/S rRNA. These helical irregularities could be important for protein association and also may represent biologically relevant flexible regions of the molecule.

  20. A Small Covalent Allosteric Inhibitor of Human Cytomegalovirus DNA Polymerase Subunit Interactions.

    PubMed

    Chen, Han; Coseno, Molly; Ficarro, Scott B; Mansueto, My Sam; Komazin-Meredith, Gloria; Boissel, Sandrine; Filman, David J; Marto, Jarrod A; Hogle, James M; Coen, Donald M

    2017-02-10

    Human cytomegalovirus DNA polymerase comprises a catalytic subunit, UL54, and an accessory subunit, UL44, the interaction of which may serve as a target for the development of new antiviral drugs. Using a high-throughput screen, we identified a small molecule, (5-((dimethylamino)methylene-3-(methylthio)-6,7-dihydrobenzo[c]thiophen-4(5H)-one), that selectively inhibits the interaction of UL44 with a UL54-derived peptide in a time-dependent manner, full-length UL54, and UL44-dependent long-chain DNA synthesis. A crystal structure of the compound bound to UL44 revealed a covalent reaction with lysine residue 60 and additional noncovalent interactions that cause steric conflicts that would prevent the UL44 connector loop from interacting with UL54. Analyses of the reaction of the compound with model substrates supported a resonance-stabilized conjugation mechanism, and substitution of the lysine reduced the ability of the compound to inhibit UL44-UL54 peptide interactions. This novel covalent inhibitor of polymerase subunit interactions may serve as a starting point for new, needed drugs to treat human cytomegalovirus infections.

  1. Substrate specificity determinants of the methanogen homoaconitase enzyme: structure and function of the small subunit.

    PubMed

    Jeyakanthan, Jeyaraman; Drevland, Randy M; Gayathri, Dasara Raju; Velmurugan, Devadasan; Shinkai, Akeo; Kuramitsu, Seiki; Yokoyama, Shigeyuki; Graham, David E

    2010-03-30

    The aconitase family of hydro-lyase enzymes includes three classes of proteins that catalyze the isomerization of alpha-hydroxy acids to beta-hydroxy acids. Besides aconitase, isopropylmalate isomerase (IPMI) proteins specifically catalyze the isomerization of alpha,beta-dicarboxylates with hydrophobic gamma-chain groups, and homoaconitase (HACN) proteins catalyze the isomerization of tricarboxylates with variable chain length gamma-carboxylate groups. These enzymes' stereospecific hydro-lyase activities make them attractive catalysts to produce diastereomers from unsaturated precursors. However, sequence similarity and convergent evolution among these proteins lead to widespread misannotation and uncertainty about gene function. To find the substrate specificity determinants of homologous IPMI and HACN proteins from Methanocaldococcus jannaschii, the small-subunit HACN protein (MJ1271) was crystallized for X-ray diffraction. The structural model showed characteristic residues in a flexible loop region between alpha2 and alpha3 that distinguish HACN from IPMI and aconitase proteins. Site-directed mutagenesis of MJ1271 produced loop-region variant proteins that were reconstituted with wild-type MJ1003 large-subunit protein. The heteromers formed promiscuous hydro-lyases with reduced activity but broader substrate specificity. Both R26K and R26V variants formed relatively efficient IPMI enzymes, while the T27A variant had uniformly lower specificity constants for both IPMI and HACN substrates. The R26V T27Y variant resembles the MJ1277 IPMI small subunit in its flexible loop sequence but demonstrated the broad substrate specificity of the R26V variant. These mutations may reverse the evolution of HACN activity from an ancestral IPMI gene, demonstrating the evolutionary potential for promiscuity in hydro-lyase enzymes. Understanding these specificity determinants enables the functional reannotation of paralogous HACN and IPMI genes in numerous genome sequences. These

  2. Structural insights on the small subunit of DNA topoisomerase I from the unicellular parasite Leishmania donovani.

    PubMed

    Díaz González, Rosario; Pérez Pertejo, Yolanda; Redondo, Carmen M; Pommier, Yves; Balaña-Fouce, Rafael; Reguera, Rosa M

    2007-12-01

    Leishmania donovani, the causative organism of visceral leishmaniasis, contains a unique heterodimeric DNA topoisomerase IB (LdTop1). The catalytically active enzyme consists of a large subunit (LdTop1L), which contains the non-conserved N-terminal end and a phylogenetically conserved core domain, and of a small subunit (LdTop1S) which harbours the C-terminal region with a characteristic tyrosine residue in the active site. Heterologous co-expression of LdTop1L and LdTop1S in a topoisomerase I deficient yeast strain, reconstitutes a fully functional enzyme which can be used for structural studies. The role played by the non-conserved N-terminal extension of LdTop1S in both relaxation activity and CPT sensitivity of LdTop1 has been examined co-expressing the full-length LdTop1L with several deletions of LdTop1S lacking growing sequences of the N-terminal end. The sequential deletion study shows that the first 174 amino acids of LdTop1S are dispensable in terms of relaxation activity and DNA cleavage. It is also described that the trapping of the covalent complex between LdTop1 and DNA by CPT requires a pentapeptide between amino acid residues 175 and 179 of LdTop1S. Our results suggest the crucial role played by the N-terminal extension of the small subunit of DNA topoisomerase I.

  3. Molecular architecture of the 90S small subunit pre-ribosome

    PubMed Central

    Sun, Qi; Zhu, Xing; Qi, Jia; An, Weidong; Lan, Pengfei; Tan, Dan; Chen, Rongchang; Wang, Bing; Zheng, Sanduo; Zhang, Cheng; Chen, Xining; Zhang, Wei; Chen, Jing; Dong, Meng-Qiu; Ye, Keqiong

    2017-01-01

    Eukaryotic small ribosomal subunits are first assembled into 90S pre-ribosomes. The complete 90S is a gigantic complex with a molecular mass of approximately five megadaltons. Here, we report the nearly complete architecture of Saccharomyces cerevisiae 90S determined from three cryo-electron microscopy single particle reconstructions at 4.5 to 8.7 angstrom resolution. The majority of the density maps were modeled and assigned to specific RNA and protein components. The nascent ribosome is assembled into isolated native-like substructures that are stabilized by abundant assembly factors. The 5' external transcribed spacer and U3 snoRNA nucleate a large subcomplex that scaffolds the nascent ribosome. U3 binds four sites of pre-rRNA, including a novel site on helix 27 but not the 3' side of the central pseudoknot, and crucially organizes the 90S structure. The 90S model provides significant insight into the principle of small subunit assembly and the function of assembly factors. DOI: http://dx.doi.org/10.7554/eLife.22086.001 PMID:28244370

  4. Isolation of the catalytically competent small subunit of ribulose bisphosphate carboxylase/oxygenase from spinach under an extremely alkaline condition.

    PubMed

    Incharoensakdi, A; Takabe, T; Takabe, T; Akazawa, T

    1986-07-16

    A method for isolating the small subunit (B) of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from spinach leaf using an alkaline buffer (pH 11.2) in combination with sucrose gradient centrifugation is described. Although the yield of isolated subunit B (ca. 20%) was comparable to that previously described (ca. 25%) using the acid precipitation method [Andrews, T.J. and Lorimer, G.H. (1985) J. Biol. Chem. 260: 4632-4636], the isolated subunit B in this report suffered less denaturation (ca. 30%) as estimated from kinetic analysis of its reassembly with large subunit (A) derived from Aphanothece halophytica. Studies on the kinetic properties of the reassembled enzyme molecules suggested that spinach subunit B does not influence the affinity of the enzyme for substrate CO2. The catalytic core (A8) of spinach RuBisCO could not be isolated in the native form.

  5. Orthologs of the small RPB8 subunit of the eukaryotic RNA polymerases are conserved in hyperthermophilic Crenarchaeota and "Korarchaeota".

    PubMed

    Koonin, Eugene V; Makarova, Kira S; Elkins, James G

    2007-12-14

    Although most of the key components of the transcription apparatus, and in particular, RNA polymerase (RNAP) subunits, are conserved between archaea and eukaryotes, no archaeal homologs of the small RPB8 subunit of eukaryotic RNAP have been detected. We report that orthologs of RPB8 are encoded in all sequenced genomes of hyperthermophilic Crenarchaeota and a recently sequenced "korarchaeal" genome, but not in Euryarchaeota or the mesophilic crenarchaeon Cenarchaeum symbiosum. These findings suggest that all 12 core subunits of eukaryotic RNAPs were already present in the last common ancestor of the extant archaea.

  6. Structure-based design of small peptide inhibitors of protein kinase CK2 subunit interaction

    PubMed Central

    Laudet, Béatrice; Barette, Caroline; Dulery, Vincent; Renaudet, Olivier; Dumy, Pascal; Metz, Alexandra; Prudent, Renaud; Deshiere, Alexandre; Dideberg, Otto; Filhol, Odile; Cochet, Claude

    2007-01-01

    X-ray crystallography studies, as well as live-cell fluorescent imaging, have recently challenged the traditional view of protein kinase CK2. Unbalanced expression of catalytic and regulatory CK2 subunits has been observed in a variety of tissues and tumours. Thus the potential intersubunit flexibility suggested by these studies raises the likely prospect that the CK2 holoenzyme complex is subject to disassembly and reassembly. In the present paper, we show evidence for the reversible multimeric organization of the CK2 holoenzyme complex in vitro. We used a combination of site-directed mutagenesis, binding experiments and functional assays to show that, both in vitro and in vivo, only a small set of primary hydrophobic residues of CK2β which contacts at the centre of the CK2α/CK2β interface dominates affinity. The results indicate that a double mutation in CK2β of amino acids Tyr188 and Phe190, which are complementary and fill up a hydrophobic pocket of CK2α, is the most disruptive to CK2α binding both in vitro and in living cells. Further characterization of hotspots in a cluster of hydrophobic amino acids centred around Tyr188–Phe190 led us to the structure-based design of small-peptide inhibitors. One conformationally constrained 11-mer peptide (Pc) represents a unique CK2β-based small molecule that was particularly efficient (i) to antagonize the interaction between the CK2 subunits, (ii) to inhibit the assembly of the CK2 holoenzyme complex, and (iii) to strongly affect its substrate preference. PMID:17714077

  7. Integrative structural analysis of the UTPB complex, an early assembly factor for eukaryotic small ribosomal subunits.

    PubMed

    Zhang, Cheng; Sun, Qi; Chen, Rongchang; Chen, Xining; Lin, Jinzhong; Ye, Keqiong

    2016-09-06

    Ribosome assembly is an essential and conserved cellular process in eukaryotes that requires numerous assembly factors. The six-subunit UTPB complex is an essential component of the 90S precursor of the small ribosomal subunit. Here, we analyzed the molecular architecture of UTPB using an integrative structural biology approach. We mapped the major interactions that associate each of six UTPB proteins. Crystallographic studies showed that Utp1, Utp21, Utp12 and Utp13 are evolutionarily related and form a dimer of dimers (Utp1-Utp21, Utp12-Utp13) through their homologous helical C-terminal domains. Molecular docking with crosslinking restraints showed that the WD domains of Utp12 and Utp13 are associated, as are the WD domains of Utp1, Utp21 and Utp18. Electron microscopy images of the entire UTPB complex revealed that it predominantly adopts elongated conformations and possesses internal flexibility. We also determined crystal structures of the WD domain of Utp18 and the HAT and deviant HAT domains of Utp6. A structural model of UTPB was derived based on these data.

  8. The bacteriophage T4 gene for the small subunit of ribonucleotide reductase contains an intron.

    PubMed Central

    Sjöberg, B M; Hahne, S; Mathews, C Z; Mathews, C K; Rand, K N; Gait, M J

    1986-01-01

    The bacteriophage T4 gene nrdB codes for the small subunit of the enzyme ribonucleotide reductase. The T4 nrdB gene was localized between 136.1 kb and 137.8 kb in the T4 genetic map according to the deduced structural homology of the protein to the amino acid sequence of its bacterial counterpart, the B2 subunit of Escherichia coli. This positions the C-terminal end of the T4 nrdB gene approximately 2 kb closer to the T4 gene 63 than earlier anticipated from genetic recombinational analyses. The most surprising feature of the T4 nrdB gene is the presence of an approximately 625 bp intron which divides the structural gene into two parts. This is the second example of a prokaryotic structural gene with an intron. The first prokaryotic intron was reported in the nearby td gene, coding for the bacteriophage T4-specific thymidylate synthase enzyme. The nucleotide sequence at the exon-intron junctions of the T4 nrdB gene is similar to that of the junctions of the T4 td gene: the anticipated exon-intron boundary at the donor site ends with a TAA stop codon and there is an ATG start codon at the putative downstream intron-exon boundary of the acceptor site. In the course of this work the denA gene of T4 (endonuclease II) was also located. PMID:3530746

  9. Expression of Calcium Channel Subunit Variants in Small Mesenteric Arteries of WKY and SHR

    PubMed Central

    Fromme, Samantha

    2015-01-01

    BACKGROUND Enhanced function of dihydropyridine-sensitive Ca2+ channels (CaV) in hypertensive arterial myocytes (HAM) is well accepted. Increased protein expression of pore forming α1-subunits contributes to this effect, but cannot explain all of the differences in CaV properties in HAM. We hypothesized that differences in expression of CaV subunits and/or their splice variants also contribute. METHODS RNA, protein, and myocytes were isolated from small mesenteric arteries (SMA) of 20-week-old male WKY and SHR and analyzed by polymerase chain reaction (PCR), sequencing, immunoblotting, and patch clamp methods. RESULTS Cav1.2 α1, β2c, and α2δ1d were the dominant subunits expressed in both WKY and SHR with a smaller amount of β3a. Real-time PCR indicated that the mRNA abundance of β3a and α2δ1 but not total Cav1.2 α1 or β2c were significantly larger in SHR. Analysis of alternative splicing of Cav1.2 α1 showed no differences in abundance of mutually exclusive exons1b, 8, 21 and 32 or alternative exons33 and 45. However, inclusion of exon9* was higher and a 73 nucleotide (nt) deletion in exon15 (exon15Δ73) was lower in SHR. Immunoblot analysis showed higher protein levels of Cav1.2 α1 (1.61±0.05), β3 (1.80±0.32), and α2δ1 (1.80±0.24) but not β2 in SHR. CONCLUSIONS The lower abundance of exon15Δ73 transcripts in SHR results in a larger fraction of total Cav1.2 mRNA coding for full-length CaV protein, and the higher abundance of exon9* transcripts and CaVβ3a protein likely contribute to differences in gating and kinetics of CaV currents in SHR. Functional studies of Ca2+ currents in native SMA myocytes and HEK cells transiently transfected with CaV subunits support these conclusions. PMID:25820242

  10. Isolation and characterization of rubisco small subunit gene promoter from common wheat (Triticum aestivum L.).

    PubMed

    Mukherjee, Shalini; Stasolla, Claudio; Brûlé-Babel, Anita; Ayele, Belay T

    2015-01-01

    Choice of an appropriate promoter is critical to express target genes in intended tissues and developmental stages. However, promoters capable of directing gene expression in specific tissues and stages are not well characterized in monocot species. To identify such a promoter in wheat, this study isolated a partial sequence of the wheat small subunit of RuBisCO (TarbcS) promoter. In silico analysis revealed the presence of elements that are characteristic to rbcS promoters of other, mainly dicot, species. Transient expression of the TarbcS:GUS in immature wheat embryos and tobacco leaves but not in the wheat roots indicate the functionality of the TarbcS promoter fragment in directing the expression of target genes in green plant tissues.

  11. AtPAP2 modulates the import of the small subunit of Rubisco into chloroplasts

    PubMed Central

    Zhang, Renshan; Guan, Xiaoqian; Law, Yee-Song; Sun, Feng; Chen, Shuai; Wong, Kam Bo

    2016-01-01

    ABSTRACT Arabidopsis thaliana purple acid phosphatase 2 (AtPAP2) is the only phosphatase that is dual-targeted to both chloroplasts and mitochondria. Like Toc33/34 of the TOC and Tom 20 of the TOM, AtPAP2 is anchored to the outer membranes of chloroplasts and mitochondria via a hydrophobic C-terminal motif. AtPAP2 on the mitochondria was previously shown to recognize the presequences of several nuclear-encoded mitochondrial proteins and modulate the import of pMORF3 into the mitochondria. Here we show that AtPAP2 binds to the small subunit of Rubisco (pSSU) and that chloroplast import experiments demonstrated that pSSU was imported less efficiently into pap2 chloroplasts than into wild-type chloroplasts. We propose that AtPAP2 is an outer membrane-bound phosphatase receptor that facilitates the import of selected proteins into chloroplasts. PMID:27700374

  12. Postimport methylation of the small subunit of ribulose-1,5-bisphosphate carboxylase in chloroplasts.

    PubMed

    Grimm, R; Grimm, M; Eckerskorn, C; Pohlmeyer, K; Röhl, T; Soll, J

    1997-05-26

    Electron impact mass spectronomy analysis of the amino-terminal amino acid of the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase (Rubisco) showed that the amino-terminal methionine residue is post-translationally modified to N-methyl-methionine. Modification of the amino-terminal methionine residue was found in mature SSU proteins from the dicotyledonous plants pea and spinach as well as the monocotyledonous plants barley and corn. SSU methyltransferase is a soluble protein in the chloroplast stroma and accepts heterologously expressed non-methylated SSU as a substrate using S-adenosylmethionine as methyl-group donor. We show that this modification occurs after post-translational uptake of the precursor form of SSU into chloroplasts and processing to its mature size. This reaction represents a new step in the import and assembly pathway of Rubisco holoenzyme.

  13. Small subunit ribosomal DNA suggests that the xenophyophorean Syringammina corbicula is a foraminiferan.

    PubMed

    Pawlowski, Jan; Holzmann, Maria; Fahrni, Jose; Richardson, Susan L

    2003-01-01

    Xenophyophorea are giant deep-sea rhizopodial protists of enigmatic origins. Although species were described as Foraminifera or sponges in the early literature, the xenophyophoreans are currently classified either as a class of Rhizopoda or an independent phylum. To establish the phylogenetic position of Xenophyophorea, we analysed the small subunit (SSU) rRNA gene sequence of Syringammina corbicula Richardson, a newly described xenophyophorean species from the Cape Verde Plateau. The SSUrDNA analyses showed that S. corbicula is closely related to Rhizammina algaeformis, a tubular deep-sea foraminiferan. Both species branch within a group of monothalamous (single-chambered) Foraminifera, which include also such agglutinated genera as Toxisarcon, Rhabdammina, and Saccammina, and the organic-walled genera Gloiogullmia and Cylindrogullmia. Our results are congruent with observations of similar cytoplasmic organisation in Rhizammina and Syringammina. Thus, the Xenophyophorea appear to be a highly specialised group of deep-sea Foraminifera.

  14. Imp3p and Imp4p mediate formation of essential U3–precursor rRNA (pre-rRNA) duplexes, possibly to recruit the small subunit processome to the pre-rRNA

    PubMed Central

    Gérczei, Tímea; Correll, Carl C.

    2004-01-01

    In eukaryotes, formation of short duplexes between the U3 small nucleolar RNA (snoRNA) and the precursor rRNA (pre-rRNA) at multiple sites is a prerequisite for three endonucleolytic cleavages that initiate small subunit biogenesis by releasing the 18S rRNA precursor from the pre-rRNA. The most likely role of these RNA duplexes is to guide the U3 snoRNA and its associated proteins, designated the small subunit processome, to the target cleavage sites on the pre-rRNA. Studies by others in Saccharomyces cerevisiae have identified the proteins Mpp10p, Imp3p, and Imp4p as candidates to mediate U3–pre-rRNA interactions. We report here that Imp3p and Imp4p appear to stabilize an otherwise unstable duplex between the U3 snoRNA hinge region and complementary bases in the external transcribed spacer of the pre-rRNA. In addition, Imp4p, but not Imp3p, seems to rearrange the U3 box A stem structure to expose the site that base-pairs with the 5′ end of the 18S rRNA, thereby mediating duplex formation at a second site. By mediating formation of both essential U3–pre-rRNA duplexes, Imp3p and Imp4p may help the small subunit processome to dock onto the pre-rRNA, an event indispensable for ribosome biogenesis and hence for cell growth. PMID:15489263

  15. Functional Expression Profile of Voltage-Gated K(+) Channel Subunits in Rat Small Mesenteric Arteries.

    PubMed

    Cox, Robert H; Fromme, Samantha

    2016-06-01

    Multiple K v channel complexes contribute to total K v current in numerous cell types and usually subserve different physiological functions. Identifying the complete compliment of functional K v channel subunits in cells is a prerequisite to understanding regulatory function. It was the goal of this work to determine the complete K v subunit compliment that contribute to functional K v currents in rat small mesenteric artery (SMA) myocytes as a prelude to studying channel regulation. Using RNA prepared from freshly dispersed myocytes, high levels of K v 1.2, 1.5, and 2.1 and lower levels of K v 7.4 α-subunit expressions were demonstrated by quantitative PCR and confirmed by Western blotting. Selective inhibitors correolide (K v 1; COR), stromatoxin (K v 2.1; ScTx), and linopirdine (K v 7.4; LINO) decreased K v current at +40 mV in SMA by 46 ± 4, 48 ± 4, and 6.5 ± 2 %, respectively, and K v current in SMA was insensitive to α-dendrotoxin. Contractions of SMA segments pretreated with 100 nmol/L phenylephrine were enhanced by 27 ± 3, 30 ± 8, and 7 ± 3 % of the response to 120 mmol/L KCl by COR, ScTX, and LINO, respectively. The presence of K v 6.1, 9.3, β1.1, and β1.2 was demonstrated by RT-PCR using myocyte RNA with expressions of K vβ1.2 and K v 9.3 about tenfold higher than K vβ1.1 and K v 6.1, respectively. Selective inhibitors of K v 1.3, 3.4, 4.1, and 4.3 channels also found at the RNA and/or protein level had no significant effect on K v current or contraction. These results suggest that K v current in rat SMA myocytes are dominated equally by two major components consisting of K v 1.2-1.5-β1.2 and K v 2.1-9.3 channels along with a smaller contribution from K v 7.4 channels but differences in voltage dependence of activation allows all three to provide significant contributions to SMA function at physiological voltages.

  16. Crystallization of the Nonameric Small Terminase Subunit of Bacteriophage P22

    SciTech Connect

    A Roy; A Bhardwaj; G Cingolani

    2011-12-31

    The packaging of viral genomes into preformed empty procapsids is powered by an ATP-dependent genome-translocating motor. This molecular machine is formed by a heterodimer consisting of large terminase (L-terminase) and small terminase (S-terminase) subunits, which is assembled into a complex of unknown stoichiometry, and a dodecameric portal protein. There is considerable confusion in the literature regarding the biologically relevant oligomeric state of terminases, which, like portal proteins, form ring-like structures. The number of subunits in a hollow oligomeric protein defines the internal diameter of the central channel and the ability to fit DNA inside. Thus, knowledge of the exact stoichiometry of terminases is critical to decipher the mechanisms of terminase-dependent DNA translocation. Here, the gene encoding bacteriophage P22 S-terminase in Escherichia coli has been overexpressed and the protein purified under native conditions. In the absence of detergents and/or denaturants that may cause disassembly of the native oligomer and formation of aberrant rings, it was found that P22 S-terminase assembles into a concentration-independent nonamer of {approx}168 kDa. Nonameric S-terminase was crystallized in two different crystal forms at neutral pH. Crystal form I belonged to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 144.2, b = 144.2, c = 145.3 {angstrom}, and diffracted to 3.0 {angstrom} resolution. Crystal form II belonged to space group P2{sub 1}, with unit-cell parameters a = 76.48, b = 100.9, c = 89.95 {angstrom}, {beta} = 93.73{sup o}, and diffracted to 1.75 {angstrom} resolution. Preliminary crystallographic analysis of crystal form II confirms that the S-terminase crystals contain a nonamer in the asymmetric unit and are suitable for high-resolution structure determination.

  17. Crystallization of the Nonameric Small Terminase Subunit of bacteriophage P22

    SciTech Connect

    A Roy; A Bhardwaj; G Cingoloni

    2011-12-31

    The packaging of viral genomes into preformed empty procapsids is powered by an ATP-dependent genome-translocating motor. This molecular machine is formed by a heterodimer consisting of large terminase (L-terminase) and small terminase (S-terminase) subunits, which is assembled into a complex of unknown stoichiometry, and a dodecameric portal protein. There is considerable confusion in the literature regarding the biologically relevant oligomeric state of terminases, which, like portal proteins, form ring-like structures. The number of subunits in a hollow oligomeric protein defines the internal diameter of the central channel and the ability to fit DNA inside. Thus, knowledge of the exact stoichiometry of terminases is critical to decipher the mechanisms of terminase-dependent DNA translocation. Here, the gene encoding bacteriophage P22 S-terminase in Escherichia coli has been overexpressed and the protein purified under native conditions. In the absence of detergents and/or denaturants that may cause disassembly of the native oligomer and formation of aberrant rings, it was found that P22 S-terminase assembles into a concentration-independent nonamer of {approx}168 kDa. Nonameric S-terminase was crystallized in two different crystal forms at neutral pH. Crystal form I belonged to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 144.2, b = 144.2, c = 145.3 {angstrom}, and diffracted to 3.0 {angstrom} resolution. Crystal form II belonged to space group P2{sub 1}, with unit-cell parameters a = 76.48, b = 100.9, c = 89.95 {angstrom}, {beta} = 93.73{sup o}, and diffracted to 1.75 {angstrom} resolution. Preliminary crystallographic analysis of crystal form II confirms that the S-terminase crystals contain a nonamer in the asymmetric unit and are suitable for high-resolution structure determination.

  18. Yeast Dun1 Kinase Regulates Ribonucleotide Reductase Small Subunit Localization in Response to Iron Deficiency.

    PubMed

    Sanvisens, Nerea; Romero, Antonia M; Zhang, Caiguo; Wu, Xiaorong; An, Xiuxiang; Huang, Mingxia; Puig, Sergi

    2016-04-29

    Ribonucleotide reductase (RNR) is an essential iron-dependent enzyme that catalyzes deoxyribonucleotide synthesis in eukaryotes. Living organisms have developed multiple strategies to tightly modulate RNR function to avoid inadequate or unbalanced deoxyribonucleotide pools that cause DNA damage and genome instability. Yeast cells activate RNR in response to genotoxic stress and iron deficiency by facilitating redistribution of its small heterodimeric subunit Rnr2-Rnr4 from the nucleus to the cytoplasm, where it forms an active holoenzyme with large Rnr1 subunit. Dif1 protein inhibits RNR by promoting nuclear import of Rnr2-Rnr4. Upon DNA damage, Dif1 phosphorylation by the Dun1 checkpoint kinase and its subsequent degradation enhances RNR function. In this report, we demonstrate that Dun1 kinase triggers Rnr2-Rnr4 redistribution to the cytoplasm in response to iron deficiency. We show that Rnr2-Rnr4 relocalization by low iron requires Dun1 kinase activity and phosphorylation site Thr-380 in the Dun1 activation loop, but not the Dun1 forkhead-associated domain. By using different Dif1 mutant proteins, we uncover that Dun1 phosphorylates Dif1 Ser-104 and Thr-105 residues upon iron scarcity. We observe that the Dif1 phosphorylation pattern differs depending on the stimuli, which suggests different Dun1 activating pathways. Importantly, the Dif1-S104A/T105A mutant exhibits defects in nucleus-to-cytoplasm redistribution of Rnr2-Rnr4 by iron limitation. Taken together, these results reveal that, in response to iron starvation, Dun1 kinase phosphorylates Dif1 to stimulate Rnr2-Rnr4 relocalization to the cytoplasm and promote RNR function.

  19. Succession of Microbial Communities during Hot Composting as Detected by PCR–Single-Strand-Conformation Polymorphism-Based Genetic Profiles of Small-Subunit rRNA Genes

    PubMed Central

    Peters, Sabine; Koschinsky, Stefanie; Schwieger, Frank; Tebbe, Christoph C.

    2000-01-01

    A cultivation-independent technique for genetic profiling of PCR-amplified small-subunit rRNA genes (SSU rDNA) was chosen to characterize the diversity and succession of microbial communities during composting of an organic agricultural substrate. PCR amplifications were performed with DNA directly extracted from compost samples and with primers targeting either (i) the V4–V5 region of eubacterial 16S rRNA genes, (ii) the V3 region in the 16S rRNA genes of actinomycetes, or (iii) the V8–V9 region of fungal 18S rRNA genes. Homologous PCR products were converted to single-stranded DNA molecules by exonuclease digestion and were subsequently electrophoretically separated by their single-strand-conformation polymorphism (SSCP). Genetic profiles obtained by this technique showed a succession and increasing diversity of microbial populations with all primers. A total of 19 single products were isolated from the profiles by PCR reamplification and cloning. DNA sequencing of these molecular isolates showed similarities in the range of 92.3 to 100% to known gram-positive bacteria with a low or high G+C DNA content and to the SSU rDNA of γ-Proteobacteria. The amplified 18S rRNA gene sequences were related to the respective gene regions of Candida krusei and Candida tropicalis. Specific molecular isolates could be attributed to different composting stages. The diversity of cultivated bacteria isolated from samples taken at the end of the composting process was low. A total of 290 isolates were related to only 6 different species. Two or three of these species were also detectable in the SSCP community profiles. Our study indicates that community SSCP profiles can be highly useful for the monitoring of bacterial diversity and community successions in a biotechnologically relevant process. PMID:10698754

  20. rRNA suppressor of a eukaryotic translation initiation factor 5B/initiation factor 2 mutant reveals a binding site for translational GTPases on the small ribosomal subunit.

    PubMed

    Shin, Byung-Sik; Kim, Joo-Ran; Acker, Michael G; Maher, Kathryn N; Lorsch, Jon R; Dever, Thomas E

    2009-02-01

    The translational GTPases promote initiation, elongation, and termination of protein synthesis by interacting with the ribosome. Mutations that impair GTP hydrolysis by eukaryotic translation initiation factor 5B/initiation factor 2 (eIF5B/IF2) impair yeast cell growth due to failure to dissociate from the ribosome following subunit joining. A mutation in helix h5 of the 18S rRNA in the 40S ribosomal subunit and intragenic mutations in domain II of eIF5B suppress the toxic effects associated with expression of the eIF5B-H480I GTPase-deficient mutant in yeast by lowering the ribosome binding affinity of eIF5B. Hydroxyl radical mapping experiments reveal that the domain II suppressors interface with the body of the 40S subunit in the vicinity of helix h5. As the helix h5 mutation also impairs elongation factor function, the rRNA and eIF5B suppressor mutations provide in vivo evidence supporting a functionally important docking of domain II of the translational GTPases on the body of the small ribosomal subunit.

  1. Sequencing for complete rDNA sequences (18S, ITS1, 5.8S, ITS2, and 28S rDNA) of Demodex and phylogenetic analysis of Acari based on 18S and 28S rDNA.

    PubMed

    Zhao, Ya-E; Wu, Li-Ping; Hu, Li; Xu, Yang; Wang, Zheng-Hang; Liu, Wen-Yan

    2012-11-01

    Due to the difficulty of DNA extraction for Demodex, few studies dealt with the identification and the phyletic evolution of Demodex at molecular level. In this study, we amplified, sequenced, and analyzed a complete (Demodex folliculorum) and an almost complete (D12 missing) (Demodex brevis) ribosomal DNA (rDNA) sequence and also analyzed the primary sequences of divergent domains in small-subunit ribosomal RNA (rRNA) of 51 species and in large-subunit rRNA of 43 species from four superfamilies in Acari (Cheyletoidea, Tetranychoidea, Analgoidea, and Ixodoidea). The results revealed that 18S rDNA sequence was relatively conserved in rDNA-coding regions and was not evolving as rapidly as 28S rDNA sequence. The evolutionary rates of transcribed spacer regions were much higher than those of the coding regions. The maximum parsimony trees of 18S and 28S rDNA appeared to be almost identical, consistent with their morphological classification. Based on the fact that the resolution capability of sequence length and the divergence of the 13 segments (D1-D6, D7a, D7b, and D8-D12) of 28S rDNA were stronger than that of the nine variable regions (V1-V9) of 18S rDNA, we were able to identify Demodex (Cheyletoidea) by the indels occurring in D2, D6, and D8.

  2. Soybean ribulose bisphosphate carboxylase small subunit: Mechanisms and determinants of RNA turnover. Annual progress report

    SciTech Connect

    Meagher, R.B.

    1993-12-31

    An in vitro degradation system has been developed from petunia and soybean polysomes in order to investigate the mechanisms and determinants controlling RNA turnover in higher plants. This system faithfully degrades soybean ribulose-1,5-bisphosphate carboxylase small subunit (rbcS) mRNA into the same products observed in total RNA preparations. In previous years it was shown that the most stable products represent a nested constellation of fragments, which are shortened from their 3{prime} ends, and have intact 5{prime} ends. Exogenous rbcS RNA tagged with novel 5{prime} sequence 15 or 56 bp long were synthesized in vitro as Sp6 and T7 runoff transcripts, respectively. When added to the system they were degraded faithfully into constellation of products which were 15 or 56 bp longer than the endogenous products, respectively. Detailed kinetics on the appearance of these exogenous products confirmed degradation proceeds in an overall 3{prime} to 5{prime} direction but suggested that there are multiple pathways through which the RNA may be degraded. To further demonstrate a precursor product relationships, in vitro synthesized transcripts truncated at their 3{prime} ends were shown to degrade into the expected smaller fragments previously mapped in the 5{prime} portion of the rbcS RNA.

  3. Phylogenetic Analysis of Cryptosporidium Parasites Based on the Small-Subunit rRNA Gene Locus

    PubMed Central

    Xiao, Lihua; Escalante, Lillian; Yang, Chunfu; Sulaiman, Irshad; Escalante, Anannias A.; Montali, Richard J.; Fayer, Ronald; Lal, Altaf A.

    1999-01-01

    Biological data support the hypothesis that there are multiple species in the genus Cryptosporidium, but a recent analysis of the available genetic data suggested that there is insufficient evidence for species differentiation. In order to resolve the controversy in the taxonomy of this parasite genus, we characterized the small-subunit rRNA genes of Cryptosporidium parvum, Cryptosporidium baileyi, Cryptosporidium muris, and Cryptosporidium serpentis and performed a phylogenetic analysis of the genus Cryptosporidium. Our study revealed that the genus Cryptosporidium contains the phylogenetically distinct species C. parvum, C. muris, C. baileyi, and C. serpentis, which is consistent with the biological characteristics and host specificity data. The Cryptosporidium species formed two clades, with C. parvum and C. baileyi belonging to one clade and C. muris and C. serpentis belonging to the other clade. Within C. parvum, human genotype isolates and guinea pig isolates (known as Cryptosporidium wrairi) each differed from bovine genotype isolates by the nucleotide sequence in four regions. A C. muris isolate from cattle was also different from parasites isolated from a rock hyrax and a Bactrian camel. Minor differences were also detected between C. serpentis isolates from snakes and lizards. Based on the genetic information, a species- and strain-specific PCR-restriction fragment length polymorphism diagnostic tool was developed. PMID:10103253

  4. Revised small subunit rRNA analysis provides further evidence that Foraminifera are related to Cercozoa.

    PubMed

    Berney, Cédric; Pawlowski, Jan

    2003-01-01

    There is accumulating evidence that the general shape of the ribosomal DNA-based phylogeny of Eukaryotes is strongly biased by the long-branch attraction phenomenon, leading to an artifactual basal clustering of groups that are probably highly derived. Among these groups, Foraminifera are of particular interest, because their deep phylogenetic position in ribosomal trees contrasts with their Cambrian appearance in the fossil record. A recent actin-based phylogeny of Eukaryotes has proposed that Foraminifera might be closely related to Cercozoa and, thus, branch among the so-called crown of Eukaryotes. Here, we reanalyze the small-subunit ribosomal RNA gene (SSU rDNA) phylogeny by removing all long-branching lineages that could artifactually attract foraminiferan sequences to the base of the tree. Our analyses reveal that Foraminifera branch together with the marine testate filosean Gromia oviformis as a sister group to Cercozoa, in agreement with actin phylogeny. Our study confirms the utility of SSU rDNA as a phylogenetic marker of megaevolutionary history, provided that the artifacts due to the heterogeneity of substitution rates in ribosomal genes are circumvented.

  5. Rubisco small subunits from the unicellular green alga Chlamydomonas complement Rubisco-deficient mutants of Arabidopsis.

    PubMed

    Atkinson, Nicky; Leitão, Nuno; Orr, Douglas J; Meyer, Moritz T; Carmo-Silva, Elizabete; Griffiths, Howard; Smith, Alison M; McCormick, Alistair J

    2017-04-01

    Introducing components of algal carbon concentrating mechanisms (CCMs) into higher plant chloroplasts could increase photosynthetic productivity. A key component is the Rubisco-containing pyrenoid that is needed to minimise CO2 retro-diffusion for CCM operating efficiency. Rubisco in Arabidopsis was re-engineered to incorporate sequence elements that are thought to be essential for recruitment of Rubisco to the pyrenoid, namely the algal Rubisco small subunit (SSU, encoded by rbcS) or only the surface-exposed algal SSU α-helices. Leaves of Arabidopsis rbcs mutants expressing 'pyrenoid-competent' chimeric Arabidopsis SSUs containing the SSU α-helices from Chlamydomonas reinhardtii can form hybrid Rubisco complexes with catalytic properties similar to those of native Rubisco, suggesting that the α-helices are catalytically neutral. The growth and photosynthetic performance of complemented Arabidopsis rbcs mutants producing near wild-type levels of the hybrid Rubisco were similar to those of wild-type controls. Arabidopsis rbcs mutants expressing a Chlamydomonas SSU differed from wild-type plants with respect to Rubisco catalysis, photosynthesis and growth. This confirms a role for the SSU in influencing Rubisco catalytic properties.

  6. Small-angle x-ray scattering studies of the manganese stabilizing subunit in photosystem II.

    SciTech Connect

    Svensson, B.; Tiede, D. M.; Barry, B. A.; Univ. of Minnesota

    2002-08-29

    Small-angle X-ray scattering studies (SAXS) were used to determine the size, shape, and oligomeric composition of the manganese stabilizing protein (MSP) of photosystem II. This extrinsic protein subunit plays an important role in photosynthetic oxygen evolution. As its name implies, MSP stabilizes the tetranuclear Mn cluster of the water oxidation complex. Removal of MSP lowers activity and decreases the stability of active-site manganese. Reconstitution of MSP reverses these effects. In this study, MSP was extracted from spinach PSII membranes using CaCl{sub 2} or urea. Through the use of MALDI-TOF mass spectrometry, the molecular weight of MSP was determined to be 26.53 kDa. X-ray scattering results show that both samples display a monodisperse scattering pattern; this pattern is consistent with a homogeneous protein solution. The CaCl{sub 2} extracted and urea extracted MSP samples have radii of gyration of 25.9 {+-} 0.4 and 27.0 {+-} 0.01 {angstrom}, respectively. MSP is shown to be monomeric in solution. This was determined using a cytochrome c standard and the scattering intensity, extrapolated to zero scattering angle, which is proportional to the molecular weight. This SAXS study suggests that, in solution, MSP is a monomeric, elongated prolate ellipsoid with dimensions, 112 x 23 x 23 {angstrom}{sup 3} and an axial ratio of 4.8.

  7. A genetic link between epigenetic repressor AS1-AS2 and a putative small subunit processome in leaf polarity establishment of Arabidopsis

    PubMed Central

    Matsumura, Yoko; Ohbayashi, Iwai; Takahashi, Hiro; Kojima, Shoko; Ishibashi, Nanako; Keta, Sumie; Nakagawa, Ayami; Hayashi, Rika; Saéz-Vásquez, Julio; Echeverria, Manuel; Sugiyama, Munetaka; Nakamura, Kenzo; Machida, Chiyoko

    2016-01-01

    ABSTRACT Although the DEAD-box RNA helicase family is ubiquitous in eukaryotes, its developmental role remains unelucidated. Here, we report that cooperative action between the Arabidopsis nucleolar protein RH10, an ortholog of human DEAD-box RNA helicase DDX47, and the epigenetic repressor complex of ASYMMETRIC-LEAVES1 (AS1) and AS2 (AS1-AS2) is critical to repress abaxial (ventral) genes ETT/ARF3 and ARF4, which leads to adaxial (dorsal) development in leaf primordia at shoot apices. Double mutations of rh10-1 and as2 (or as1) synergistically up-regulated the abaxial genes, which generated abaxialized filamentous leaves with loss of the adaxial domain. DDX47 is part of the small subunit processome (SSUP) that mediates rRNA biogenesis. In rh10-1 we found various defects in SSUP-related events, such as: accumulation of 35S/33S rRNA precursors; reduction in the 18S/25S ratio; and nucleolar hypertrophy. Double mutants of as2 with mutations of genes that encode other candidate SSUP-related components such as nucleolin and putative rRNA methyltransferase exhibited similar synergistic defects caused by up-regulation of ETT/ARF3 and ARF4. These results suggest a tight link between putative SSUP and AS1-AS2 in repression of the abaxial-determining genes for cell fate decisions for adaxial development. PMID:27334696

  8. Cloning and sequencing of the genes encoding the large and small subunits of the periplasmic (NiFeSe) hydrogenase of Desulfovibrio baculatus

    SciTech Connect

    Menon, N.K.; Peck, H.D. Jr.; Le Gall, J.; Przybyla, A.E.

    1987-12-01

    The genes coding for the large and small subunits of the periplasmic hydrogenase from Desulfovibrio baculatus have been cloned and sequenced. The genes are arranged in an operon with the small subunit gene preceding the large subunit gene. The small subunit gene codes for a 32 amino acid leader sequence supporting the periplasmic localization of the protein, however no ferredoxin-like or other characteristic iron-sulfur coordination sites were observed. The periplasmic hydrogenases from D. baculatus (an NiFeSe protein) and D. vulgaris (an Fe protein) exhibit no homology suggesting that they are structurally different, unrelated entities.

  9. Small Molecule Inhibition of the TNF Family Cytokine CD40 Ligand Through a Subunit Fracture Mechanism

    SciTech Connect

    L Silvian; J Friedman; K Strauch; T Cachero; E Day; F Qian; B Cunningham; A Fung; L Sun; et al.

    2011-12-31

    BIO8898 is one of several synthetic organic molecules that have recently been reported to inhibit receptor binding and function of the constitutively trimeric tumor necrosis factor (TNF) family cytokine CD40 ligand (CD40L, aka CD154). Small molecule inhibitors of protein-protein interfaces are relatively rare, and their discovery is often very challenging. Therefore, to understand how BIO8898 achieves this feat, we characterized its mechanism of action using biochemical assays and X-ray crystallography. BIO8898 inhibited soluble CD40L binding to CD40-Ig with a potency of IC{sub 50} = 25 {mu}M and inhibited CD40L-dependent apoptosis in a cellular assay. A co-crystal structure of BIO8898 with CD40L revealed that one inhibitor molecule binds per protein trimer. Surprisingly, the compound binds not at the surface of the protein but by intercalating deeply between two subunits of the homotrimeric cytokine, disrupting a constitutive protein-protein interface and breaking the protein's 3-fold symmetry. The compound forms several hydrogen bonds with the protein, within an otherwise hydrophobic binding pocket. In addition to the translational splitting of the trimer, binding of BIO8898 was accompanied by additional local and longer-range conformational perturbations of the protein, both in the core and in a surface loop. Binding of BIO8898 is reversible, and the resulting complex is stable and does not lead to detectable dissociation of the protein trimer. Our results suggest that a set of core aromatic residues that are conserved across a subset of TNF family cytokines might represent a generic hot-spot for the induced-fit binding of trimer-disrupting small molecules.

  10. Functional determinants in transit sequences: import and partial maturation by vascular plant chloroplasts of the ribulose-1,5- bisphosphate carboxylase small subunit of Chlamydomonas

    PubMed Central

    1985-01-01

    The precursor of the ribulose-1,5-bisphosphate carboxylase small subunit and other proteins from Chlamydomonas reinhardtii are efficiently transported into chloroplasts isolated from spinach and pea. Thus, similar determinants specify precursor-chloroplast interactions in the alga and vascular plants. Removal of all or part of its transit sequence was found to block import of the algal small subunit into isolated chloroplasts. Comparison of available sequences revealed a nine amino acid segment conserved in the transit sequences of all small subunit precursors. A protease in the vascular plant chloroplasts recognized this region in the Chlamydomonas precursor and produced an intermediate form of the small subunit. We propose that processing of the small subunit precursor involves at least two proteolytic events; only one of these has been evolutionarily conserved. PMID:3965471

  11. Molecular Evolution of the Small Subunit of Ribulose Bisphosphate Carboxylase: Nucleotide Substitution and Gene Conversion

    PubMed Central

    Meagher, R. B.; Berry-Lowe, S.; Rice, K.

    1989-01-01

    The nucleotide sequences encoding the mature portion of 31 ribulose 1,5-bisphosphate carboxylase small subunit (SSU) genes from 17 genera of plants, green algae and cyanobacteria were examined. Among the 465 pairwise sequence comparisons, SSU multigene family members within the same species were more similar to each other in nonsynonymous or replacement nucleotide substitutions (RNS) than they were to SSU sequences in any other organism. The concerted evolution of independent SSU gene lineages within closely related plant species suggests that homogenization of RNS positions has occurred at least once in the life of each genus. The rate of expected RNS among mature SSU sequences was calculated to be 1.25 X 10(-9)/site/yr for the first 70 million years (MY) of divergence with a significant slowing to 0.13 X 10(-9)/site/yr for the next 1,400 MY. The data suggest that mature SSU sequences do not accumulate more than 20% differences in the RNS positions without compensatory changes in other components of this enzyme system. During the first 70 MY of divergence between species, the rate of expected synonymous or silent nucleotide substitutions (SNS) is ~6.6 X 10(-9)/site/yr. This is five times the RNS rate and is similar to the silent rate observed in animals. In striking contrast, SNS and RNS do not show this correlation among SSU gene family members within a species. A mechanism involving gene conversion within the exons followed by selection for biased gene conversion products with conservation of RNS positions and divergence of SNS positions is discussed. A SSU gene tree based on corrected RNS for 31 SSU sequences is presented and agrees well with a species tree based on morphological and cytogenetic traits for the 17 genera examined. SSU gene comparisons may be useful in predicting phylogenetic relationships and in some cases divergence times of various plant, algal and cyanobacterial species. PMID:2515110

  12. Assembly properties of dominant and recessive mutations in the small mouse neurofilament (NF-L) subunit

    PubMed Central

    1990-01-01

    We have generated a set of amino- and carboxy-terminal deletions of the NF-L neurofilament gene and determined the assembly properties of the encoded subunits after coexpression with vimentin or wild-type NF-L. NF- L molecules missing greater than 30% (31 amino acids of the head) or 90% (128 amino acids of the tail) failed to incorporate into intermediate filament networks. Carboxy-terminal deletions into the rod domain yield dominant mutants that disrupt arrays assembled from wild- type subunits, even when present at levels of approximately 2% of the wild-type subunits. Even mutants retaining 55% of the tail (61 amino acids) disrupt normal arrays when accumulated above approximately 10% of wild-type subunits. Since deletion of greater than 90% of the head domain produces "recessive" assembly incompetent subunits that do not affect wild-type filament arrays, whereas smaller deletions yield efficient network disruption, we conclude that some sequence(s) in the head domain (within residues 31-87) are required for the earliest steps in filament assembly. Insertional mutagenesis in the nonhelical spacer region within the rod domain reveals that as many as eight additional amino acids can be tolerated without disrupting assembly competence. PMID:2121744

  13. A single gene encodes two different transcripts for the ADP-glucose pyrophosphorylase small subunit from barley (Hordeum vulgare).

    PubMed Central

    Thorbjørnsen, T; Villand, P; Kleczkowski, L A; Olsen, O A

    1996-01-01

    ADP-glucose pyrophosphorylase (AGPase), a heterotetrameric enzyme composed of two small and two large subunits, catalyses the first committed step of starch synthesis in plant tissues. In an attempt to learn more about the organization and expression of the small-subunit gene of AGPase, we have studied the small-subunit transcripts as well as the structure of the gene encoding these transcripts in barley (Hordeum vulgare L. cv. Bomi). Two different transcripts (bepsF1 and blps14) were identified: bepF1 was abundantly expressed in the starchy endosperm but not in leaves, whereas blps14 was isolated from leaves but was also found to be present at a moderate level in the starchy endosperm. The sequences for the two transcripts are identical over approx. 90% of the length, with differences being confined solely to their 5' ends. In blps14, the unique 5' end is 259 nt long and encodes a putative plastid transit peptide sequence. For the 178-nt 5' end of bepsF1, on the other hand, no transit peptide sequence could be recognized. A lambda clone that hybridized to the AGPase transcripts was isolated from a barley genomic library and characterized. The restriction map has suggested a complex organization of the gene, with alternative exons encoding the different 5' ends of the two transcripts followed by nine exons coding for the common part of the transcripts. The sequence of a portion of the genomic clone, covering the alternative 5'-end exons as well as upstream regions, has verified that both transcripts are encoded by the gene. The results suggest that the small-subunit gene of barley AGPase transcribes two different mRNAs by a mechanism classified as alternative splicing. PMID:8546676

  14. An Immunological Analysis of Dystroglycan Subunits: Lessons Learned from a Small Cohort of Non-Congenital Dystrophic Patients

    PubMed Central

    Pavoni, Ernesto; Sciandra, Francesca; Tasca, Giorgio; Tittarelli, Roberta; Bozzi, Manuela; Giardina, Bruno; Ricci, Enzo; Brancaccio, Andrea

    2011-01-01

    The dystroglycan (DG) expression pattern can be altered in severe muscular dystrophies. In fact, some congenital muscular dystrophies (CMDs) and limb-girdle muscular dystrophies (LGMDs) are caused by point mutations identified in six glycosyltransferase genes which are likely to target different steps along the posttranslational “O-glycosylation route” leading to a fully decorated and functional α-DG subunit. Indeed, hypoglycosylation of α-DG is thought to represent a major pathological event, in that it could reduce the DG’s ability to bind the basement membrane components, thus leading to sarcolemmal instability and necrosis. In order to set up an efficient standard immunological protocol, taking advantage of a wide panel of antibodies, we have analyzed the two DG subunits in a small cohort of adult dystrophic patients, whom an extensive medical examination had already clinically classified as affected by LGMD (5), Miyoshi (1) or distal (1) myopathy. Immunofluorescence analysis of skeletal muscle tissue sections revealed a proper sarcolemmal localization of the DG subunits in all the patients analyzed. However, Western blot analysis of lectin enriched skeletal muscle samples revealed an abnormal glycosylation of α-DG in two patients. Our work reinforces the notion that a careful immunological and biochemical analysis of the two DG subunits should be always considered as a prerequisite for the identification of new putative cases of dystroglycanopathy. PMID:22046204

  15. A mechanism for intergenomic integration: abundance of ribulose bisphosphate carboxylase small-subunit protein influences the translation of the large-subunit mRNA.

    PubMed

    Rodermel, S; Haley, J; Jiang, C Z; Tsai, C H; Bogorad, L

    1996-04-30

    Multimeric protein complexes in chloroplasts and mitochondria are generally composed of products of both nuclear and organelle genes of the cell. A central problem of eukaryotic cell biology is to identify and understand the molecular mechanisms for integrating the production and accumulation of the products of the two separate genomes. Ribulose bisphosphate carboxylase (Rubisco) is localized in the chloroplasts of photosynthetic eukaryotic cells and is composed of small subunits (SS) and large subunits (LS) coded for by nuclear rbcS and chloroplast rbcL genes, respectively. Transgenic tobacco plants containing antisense rbcS DNA have reduced levels of rbcS mRNA, normal levels of rbcL mRNA, and coordinately reduced LS and SS proteins. Our previous experiments indicated that the rate of translation of rbcL mRNA might be reduced in some antisense plants; direct evidence is presented here. After a short-term pulse there is less labeled LS protein in the transgenic plants than in wild-type plants, indicating that LS accumulation is controlled in the mutants at the translational and/or posttranslational levels. Consistent with a primary restriction at translation, fewer rbcL mRNAs are associated with polysomes of normal size and more are free or are associated with only a few ribosomes in the antisense plants. Effects of the rbcS antisense mutation on mRNA and protein accumulation, as well as on the distribution of mRNAs on polysomes, appear to be minimal for other chloroplast and nuclear photosynthetic genes. Our results suggest that SS protein abundance specifically contributes to the regulation of LS protein accumulation at the level of rbcL translation initiation.

  16. Functional characterization of sequence motifs in the transit peptide of Arabidopsis small subunit of rubisco.

    PubMed

    Lee, Dong Wook; Lee, Sookjin; Lee, Gil-Je; Lee, Kwang Hee; Kim, Sanguk; Cheong, Gang-Won; Hwang, Inhwan

    2006-02-01

    The transit peptides of nuclear-encoded chloroplast proteins are necessary and sufficient for targeting and import of proteins into chloroplasts. However, the sequence information encoded by transit peptides is not fully understood. In this study, we investigated sequence motifs in the transit peptide of the small subunit of the Rubisco complex by examining the ability of various mutant transit peptides to target green fluorescent protein reporter proteins to chloroplasts in Arabidopsis (Arabidopsis thaliana) leaf protoplasts. We divided the transit peptide into eight blocks (T1 through T8), each consisting of eight or 10 amino acids, and generated mutants that had alanine (Ala) substitutions or deletions, of one or two T blocks in the transit peptide. In addition, we generated mutants that had the original sequence partially restored in single- or double-T-block Ala (A) substitution mutants. Analysis of chloroplast import of these mutants revealed several interesting observations. Single-T-block mutations did not noticeably affect targeting efficiency, except in T1 and T4 mutations. However, double-T mutants, T2A/T4A, T3A/T6A, T3A/T7A, T4A/T6A, and T4A/T7A, caused a 50% to 100% loss in targeting ability. T3A/T6A and T4A/T6A mutants produced only precursor proteins, whereas T2A/T4A and T4A/T7A mutants produced only a 37-kD protein. Detailed analyses revealed that sequence motifs ML in T1, LKSSA in T3, FP and RK in T4, CMQVW in T6, and KKFET in T7 play important roles in chloroplast targeting. In T1, the hydrophobicity of ML is important for targeting. LKSSA in T3 is functionally equivalent to CMQVW in T6 and KKFET in T7. Furthermore, subcellular fractionation revealed that Ala substitution in T1, T3, and T6 produced soluble precursors, whereas Ala substitution in T4 and T7 produced intermediates that were tightly associated with membranes. These results demonstrate that the transit peptide contains multiple motifs and that some of them act in concert or

  17. Rnr4p, a novel ribonucleotide reductase small-subunit protein.

    PubMed Central

    Wang, P J; Chabes, A; Casagrande, R; Tian, X C; Thelander, L; Huffaker, T C

    1997-01-01

    Ribonucleotide reductases catalyze the formation of deoxyribonucleotides by the reduction of the corresponding ribonucleotides. Eukaryotic ribonucleotide reductases are alpha2beta2 tetramers; each of the larger, alpha subunits possesses binding sites for substrate and allosteric effectors, and each of the smaller, beta subunits contains a binuclear iron complex. The iron complex interacts with a specific tyrosine residue to form a tyrosyl free radical which is essential for activity. Previous work has identified two genes in the yeast Saccharomyces cerevisiae, RNR1 and RNR3, that encode alpha subunits and one gene, RNR2, that encodes a beta subunit. Here we report the identification of a second gene from this yeast, RNR4, that encodes a protein with significant similarity to the beta-subunit proteins. The phenotype of rnr4 mutants is consistent with that expected for a defect in ribonucleotide reductase; rnr4 mutants are supersensitive to the ribonucleotide reductase inhibitor hydroxyurea and display an S-phase arrest at their restrictive temperature. rnr4 mutant extracts are deficient in ribonucleotide reductase activity, and this deficiency can be remedied by the addition of exogenous Rnr4p. As is the case for the other RNR genes, RNR4 is induced by agents that damage DNA. However, Rnr4p lacks a number of sequence elements thought to be essential for iron binding, and mutation of the critical tyrosine residue does not affect Rnr4p function. These results suggest that Rnr4p is catalytically inactive but, nonetheless, does play a role in the ribonucleotide reductase complex. PMID:9315671

  18. Structural Comparison, Substrate Specificity, and Inhibitor Binding of AGPase Small Subunit from Monocot and Dicot: Present Insight and Future Potential

    PubMed Central

    Choudhury, Manabendra D.; Modi, Mahendra K.

    2014-01-01

    ADP-glucose pyrophosphorylase (AGPase) is the first rate limiting enzyme of starch biosynthesis pathway and has been exploited as the target for greater starch yield in several plants. The structure-function analysis and substrate binding specificity of AGPase have provided enormous potential for understanding the role of specific amino acid or motifs responsible for allosteric regulation and catalytic mechanisms, which facilitate the engineering of AGPases. We report the three-dimensional structure, substrate, and inhibitor binding specificity of AGPase small subunit from different monocot and dicot crop plants. Both monocot and dicot subunits were found to exploit similar interactions with the substrate and inhibitor molecule as in the case of their closest homologue potato tuber AGPase small subunit. Comparative sequence and structural analysis followed by molecular docking and electrostatic surface potential analysis reveal that rearrangements of secondary structure elements, substrate, and inhibitor binding residues are strongly conserved and follow common folding pattern and orientation within monocot and dicot displaying a similar mode of allosteric regulation and catalytic mechanism. The results from this study along with site-directed mutagenesis complemented by molecular dynamics simulation will shed more light on increasing the starch content of crop plants to ensure the food security worldwide. PMID:25276800

  19. Structural comparison, substrate specificity, and inhibitor binding of AGPase small subunit from monocot and dicot: present insight and future potential.

    PubMed

    Sarma, Kishore; Sen, Priyabrata; Barooah, Madhumita; Choudhury, Manabendra D; Roychoudhury, Shubhadeep; Modi, Mahendra K

    2014-01-01

    ADP-glucose pyrophosphorylase (AGPase) is the first rate limiting enzyme of starch biosynthesis pathway and has been exploited as the target for greater starch yield in several plants. The structure-function analysis and substrate binding specificity of AGPase have provided enormous potential for understanding the role of specific amino acid or motifs responsible for allosteric regulation and catalytic mechanisms, which facilitate the engineering of AGPases. We report the three-dimensional structure, substrate, and inhibitor binding specificity of AGPase small subunit from different monocot and dicot crop plants. Both monocot and dicot subunits were found to exploit similar interactions with the substrate and inhibitor molecule as in the case of their closest homologue potato tuber AGPase small subunit. Comparative sequence and structural analysis followed by molecular docking and electrostatic surface potential analysis reveal that rearrangements of secondary structure elements, substrate, and inhibitor binding residues are strongly conserved and follow common folding pattern and orientation within monocot and dicot displaying a similar mode of allosteric regulation and catalytic mechanism. The results from this study along with site-directed mutagenesis complemented by molecular dynamics simulation will shed more light on increasing the starch content of crop plants to ensure the food security worldwide.

  20. Insights of interaction between small and large subunits of ADP-glucose pyrophosphorylase from bread wheat (Triticum aestivum L.).

    PubMed

    Danishuddin, Mohd; Chatrath, Ravish; Singh, Rajender

    2011-05-07

    Lack of knowledge of three dimensional structures of small and large subunits of ADP- glucose pyrophosphorylase (AGPase) in wheat has hindered efforts to understand the binding specifities of substrate and catalytic mechanism. Thus, to understand the structure activity relationship, 3D structures were built by homology modelling based on crystal structure of potato tuber ADP-glucose pyrophosphorylase. Selected models were refined by energy minimization and further validated by Procheck and Prosa-web analysis. Ramachandran plot showed that overall main chain and side chain parameters are favourable. Moreover, Z-score of the models from Prosa-web analysis gave the conformation that they are in the range of the template. Interaction analysis depicts the involvement of six amino acids in hydrogen bonding (AGP-SThr422-AGP-LMet138, AGP- SArg420-AGP-LGly47, AGP-SSer259-AGP-LSer306, AGP-SGlu241-AGP-LIle311, AGPSGln113- AGP-LGlu286 and AGP-SGln70-AGP-LLys291). Fifteen amino acids of small subunit were able to make hydrophobic contacts with seventeen amino acids of large subunit. Furthermore, decrease in the solvent accessible surface area in the amino acids involved in interaction were also reported. All the distances were formed in between 2.27 to 3.78Å. The present study focussed on heterodimeric structure of (AGPase). This predicted complex not only enhance our understanding of the interaction mechanism between these subunits (AGP-L and AGP-S) but also enable to further study to obtain better variants of this enzyme for the improvement of the plant yield.

  1. Cloning of the cDNAs for the small subunits of bovine and human DNA polymerase {delta} and chromosomal location of the human gene (POLD2)

    SciTech Connect

    Zhang, Jian; Tan, Cheng-Keat; Downey, K.M.

    1995-09-01

    cDNAs encoding the small subunit of bovine and human DNA polymerase {delta} have been cloned and sequenced. The predicted polypeptides, 50,885 and 51,289 Daltons, respectively, are 94% identical, similar to the catalytic subunits. The high degree of conservation of the polypeptides suggests an essential function for the small subunit in the heterodimeric core enzyme. Although the catalytic subunit of DNA polymerase 5 shares significant homology with those of the herpes virus family of DNA polymerases, the small subunit of mammalian DNA polymerase 6 is not homologous to the small subunit of either herpes simplex virus type 1 DNA polymerase (UL42 protein) or the Epstein-Barr virus DNA polymerase (BMRF1 protein). Searches of the protein databases failed to detect significant homology with any protein sequenced thus far. PCR analysis of DNA from a panel of human-hamster hybrid cell lines localized the gene (POLD2) for the small subunit of DNA polymerase 5 to human chromosome 7. 45 refs., 2 figs., 2 tabs.

  2. Dual functions of a small regulatory subunit in the mitochondrial calcium uniporter complex

    PubMed Central

    Tsai, Ming-Feng; Phillips, Charles B; Ranaghan, Matthew; Tsai, Chen-Wei; Wu, Yujiao; Williams, Carole; Miller, Christopher

    2016-01-01

    Mitochondrial Ca2+ uptake, a process crucial for bioenergetics and Ca2+ signaling, is catalyzed by the mitochondrial calcium uniporter. The uniporter is a multi-subunit Ca2+-activated Ca2+ channel, with the Ca2+ pore formed by the MCU protein and Ca2+-dependent activation mediated by MICU subunits. Recently, a mitochondrial inner membrane protein EMRE was identified as a uniporter subunit absolutely required for Ca2+ permeation. However, the molecular mechanism and regulatory purpose of EMRE remain largely unexplored. Here, we determine the transmembrane orientation of EMRE, and show that its known MCU-activating function is mediated by the interaction of transmembrane helices from both proteins. We also reveal a second function of EMRE: to maintain tight MICU regulation of the MCU pore, a role that requires EMRE to bind MICU1 using its conserved C-terminal polyaspartate tail. This dual functionality of EMRE ensures that all transport-competent uniporters are tightly regulated, responding appropriately to a dynamic intracellular Ca2+ landscape. DOI: http://dx.doi.org/10.7554/eLife.15545.001 PMID:27099988

  3. Conservation of the primary structure at the 3' end of 18S rRNA from eucaryotic cells.

    PubMed

    Hagenbüchle, O; Santer, M; Steitz, J A; Mans, R J

    1978-03-01

    DNA sequencing methods have been used to determine a sequence of about 20 nucleotides at the 3' termini of various 18S (small ribosomal subunit) RNA molecules. Polyadenylated rRNA was first synthesized using the enzyme ATP:polynucleotidyl transferase from mainze. Then in the presence of an oligonucleotide primer uniquely complementary to the end of each adenylated rRNA, a cDNA copy was produced using AMV reverse transcriptase. In every case, the cDNA transcript was of finite size, which we ascribe to the appearance of an oligonucleotide containing m62A near the 3' end of the 18S rRNAs. Sequences at the 3' termini of 18S rRNA molecules from the four eucaryotic species examined here (mouse, silk worm, wheat embryo and slime mold) are highly conserved. They also exhibit strong homology to the 3' end of E. coli 16S rRNA. Two important differences, however, are apparent. First, the 16S sequence CCUCC, implicated in mRNA binding by E. coli ribosomes, is absent from each eucaryotic rRNA sequence. Second, a purine-rich region which exhibits extensive complementarity to the 5' noncoding regions of many eucaryotic mRNAs appears consistently.

  4. Mitochondrial DNA Restriction Fragment Length Polymorphism (RFLP) and 18S Small-Subunit Ribosomal DNA PCR-RFLP Analyses of Acanthamoeba Isolated from Contact Lens Storage Cases of Residents in Southwestern Korea

    PubMed Central

    Kong, Hyun-Hee; Shin, Ji-Yeol; Yu, Hak-Sun; Kim, Jin; Hahn, Tae-Won; Hahn, Young-Ho; Chung, Dong-Il

    2002-01-01

    We applied ribosomal DNA PCR-restriction fragment length polymorphism (RFLP) and mitochondrial DNA (mtDNA) RFLP analyses to 43 Acanthamoeba environmental isolates (KA/LH1 to KA/LH43) from contact lens storage cases in southwestern Korea. These isolates were compared to American Type Culture Collection strains and clinical isolates (KA/E1 to KA/E12) from patients with keratitis. Seven riboprint patterns were seen. To identify the species of the isolates, a phylogenetic tree was constructed based on the comparison of riboprint patterns with reference strains. Four types accounted for 39 of the isolates belonging to the A. castellanii complex. The most predominant (48.8%) type was A. castellanii KA/LH2 type, which had identical riboprint and mtDNA RFLP patterns to those of A. castellanii Castellani, KA/E3 and KA/E8. The riboprint and mtDNA RFLP patterns of the KA/LH7 (20.9%) type were identical to those of A. castellanii Ma, a corneal isolate from the United States. The riboprint and mtDNA RFLP patterns of the KA/LH1 (18.6%) type were the same as those of A. lugdunensis L3a, KA/E2, and KA/E12. The prevalent pattern for each type of Acanthamoeba in southwestern Korea was very different from that from southeastern Korea and Seoul, Korea. It is noteworthy that 38 (88.4%) out of 43 isolates from contact lens storage cases of the residents in southwestern Korea revealed mtDNA RFLP and riboprint patterns identical to those found for clinical isolates in our area. This indicates that most isolates from contact lens storage cases in the surveyed area are potential keratopathogens. More attention should be paid to the disinfection of contact lens storage cases to prevent possible amoebic keratitis. PMID:11923331

  5. Mitochondrial DNA restriction fragment length polymorphism (RFLP) and 18S small-subunit ribosomal DNA PCR-RFLP analyses of Acanthamoeba isolated from contact lens storage cases of residents in southwestern Korea.

    PubMed

    Kong, Hyun-Hee; Shin, Ji-Yeol; Yu, Hak-Sun; Kim, Jin; Hahn, Tae-Won; Hahn, Young-Ho; Chung, Dong-Il

    2002-04-01

    We applied ribosomal DNA PCR-restriction fragment length polymorphism (RFLP) and mitochondrial DNA (mtDNA) RFLP analyses to 43 Acanthamoeba environmental isolates (KA/LH1 to KA/LH43) from contact lens storage cases in southwestern Korea. These isolates were compared to American Type Culture Collection strains and clinical isolates (KA/E1 to KA/E12) from patients with keratitis. Seven riboprint patterns were seen. To identify the species of the isolates, a phylogenetic tree was constructed based on the comparison of riboprint patterns with reference strains. Four types accounted for 39 of the isolates belonging to the A. castellanii complex. The most predominant (48.8%) type was A. castellanii KA/LH2 type, which had identical riboprint and mtDNA RFLP patterns to those of A. castellanii Castellani, KA/E3 and KA/E8. The riboprint and mtDNA RFLP patterns of the KA/LH7 (20.9%) type were identical to those of A. castellanii Ma, a corneal isolate from the United States. The riboprint and mtDNA RFLP patterns of the KA/LH1 (18.6%) type were the same as those of A. lugdunensis L3a, KA/E2, and KA/E12. The prevalent pattern for each type of Acanthamoeba in southwestern Korea was very different from that from southeastern Korea and Seoul, Korea. It is noteworthy that 38 (88.4%) out of 43 isolates from contact lens storage cases of the residents in southwestern Korea revealed mtDNA RFLP and riboprint patterns identical to those found for clinical isolates in our area. This indicates that most isolates from contact lens storage cases in the surveyed area are potential keratopathogens. More attention should be paid to the disinfection of contact lens storage cases to prevent possible amoebic keratitis.

  6. Purification and characterization of large and small subunits of ribulose 1,5-bisphosphate carboxylase expressed separately in Escherichia coli.

    PubMed

    Smrcka, A V; Ramage, R T; Bohnert, H J; Jensen, R G

    1991-04-01

    Procedures were developed for 95 and 80% purification to homogeneity of the large subunit (L) and small subunit (S) of ribulose 1,5-bisphosphate carboxylase/oxygenase (L8S8) from Synechococcus PCC 6301, each expressed separately in Escherichia coli. Purified L had a low specific activity in the absence of S (0.075 mumol CO2 fixed/mg holoenzyme/min). Following elution on a Pharmacia Superose 6 or 12 gel filtration column, 50% of the purified L appeared as the octamer, L8. The rest was in equilibrium with lower polymeric species and/or was retained on the column. Large and small subunits assembled rapidly into the L8S8 holoenzyme that had high specific activities, 6.2 and 3.1 mumol CO2 fixed/mg holoenzyme/min for the homologous Synechococcus L8S8 and the hybrid Synechococcus L-pea S L8S8, respectively. The CO2 dependence for carbamylation of L8 was compared to that of L8S8 as a function of pH and CO2 concentration. The pH dependence indicated an apparent pKa for L8 of 8.28 and for L8S8 of 8.15, suggesting that S may influence the pKa of the lysine involved in carbamylation. The Kact for CO2 at pH 8.4 were similar for L8 (13.5 microM) and L8S8 (15.5 microM). L8 bound 2-[14C]carboxy-D-arabinitol 1,5-bisphosphate (CABP) tightly so that most of the bound [14C]CABP survived gel filtration. A major amount of the L8-[14C]CABP complex appeared as larger polymeric aggregates when eluted in the presence of E. coli protein.

  7. The small subunit 1 of the Arabidopsis isopropylmalate isomerase is required for normal growth and development and the early stages of glucosinolate formation.

    PubMed

    Imhof, Janet; Huber, Florian; Reichelt, Michael; Gershenzon, Jonathan; Wiegreffe, Christoph; Lächler, Kurt; Binder, Stefan

    2014-01-01

    In Arabidopsis thaliana the evolutionary and functional relationship between Leu biosynthesis and the Met chain elongation pathway, the first part of glucosinolate formation, is well documented. Nevertheless the exact functions of some pathway components are still unclear. Isopropylmalate isomerase (IPMI), an enzyme usually involved in Leu biosynthesis, is a heterodimer consisting of a large and a small subunit. While the large protein is encoded by a single gene (isopropylmalate isomerase large subunit1), three genes encode small subunits (isopropylmalate isomerase small subunit1 to 3). We have now analyzed small subunit 1 (isopropylmalate isomerase small subunit1) employing artificial microRNA for a targeted knockdown of the encoding gene. Strong reduction of corresponding mRNA levels to less than 5% of wild-type levels resulted in a severe phenotype with stunted growth, narrow pale leaf blades with green vasculature and abnormal adaxial-abaxial patterning as well as anomalous flower morphology. Supplementation of the knockdown plants with leucine could only partially compensate for the morphological and developmental abnormalities. Detailed metabolite profiling of the knockdown plants revealed changes in the steady state levels of isopropylmalate and glucosinolates as well as their intermediates demonstrating a function of IPMI SSU1 in both leucine biosynthesis and the first cycle of Met chain elongation. Surprisingly the levels of free leucine slightly increased suggesting an imbalanced distribution of leucine within cells and/or within plant tissues.

  8. Cloning of a yeast gene coding for the glutamate synthase small subunit (GUS2) by complementation of Saccharomyces cerevisiae and Escherichia coli glutamate auxotrophs.

    PubMed

    González, A; Membrillo-Hernández, J; Olivera, H; Aranda, C; Macino, G; Ballario, P

    1992-02-01

    A Saccharomyces cerevisiae glutamate auxotroph, lacking NADP-glutamate dehydrogenase (NADP-GDH) and glutamate synthase (GOGAT) activities, was complemented with a yeast genomic library. Clones were obtained which still lacked NADP-GDH but showed GOGAT activity. Northern analysis revealed that the DNA fragment present in the complementing plasmids coded for a 1.5kb mRNA. Since the only GOGAT enzyme so far purified from S. cerevisiae is made up of a small and a large subunit, the size of the mRNA suggested that the cloned DNA fragment could code for the GOGAT small subunit. Plasmids were purified and used to transform Escherichia coli glutamate auxotrophs. Transformants were only recovered when the recipient strain was an E. coli GDH-less mutant lacking the small GOGAT subunit. These data show that we have cloned the structural gene coding for the yeast small subunit (GUS2). Evidence is also presented indicating that the GOGAT enzyme which is synthesized in the E. coli transformants is a hybrid comprising the large E. coli subunit and the small S. cerevisiae subunit.

  9. Late-assembly of human ribosomal protein S20 in the cytoplasm is essential for the functioning of the small subunit ribosome

    SciTech Connect

    Tai, Lin-Ru; Chou, Chang-Wei; Wu, Jing-Ying; Kirby, Ralph; Lin, Alan

    2013-11-15

    Using immuno-fluorescent probing and Western blotting analysis, we reveal the exclusive cytoplasm nature of the small subunit ribosomal protein S20. To illustrate the importance of the cellular compartmentation of S20 to the function of small subunit 40S, we created a nuclear resident S20{sub NLS} mutant gene and examined polysome profile of cells that had been transfected with the S20{sub NLS} gene. As a result, we observed the formation of recombinant 40S carried S20{sub NLS} but this recombinant 40S was never found in the polysome, suggesting such a recombinant 40S was translation incompetent. Moreover, by the tactic of the energy depletion and restoration, we were able to restrain the nuclear-resided S20{sub NLS} in the cytoplasm. Yet, along a progressive energy restoration, we observed the presence of recombinant 40S subunits carrying the S20{sub NLS} in the polysome. This proves that S20 needs to be cytoplasmic in order to make a functional 40S subunit. Furthermore, it also implies that the assembly order of ribosomal protein in eukaryote is orderly regulated. - Highlights: • The step of S20 assembled on 40S is happened in the cytoplasm. • A small subunit assembled with a nuclear S20{sub NLS} is translational incompetence. • Using energy depletion and recovery to manipulate the cellular compartment of S20{sub NLS}. • Cytoplasm-retained S20{sub NLS} is crucial for creating a functional small subunit.

  10. Isolation and 18S ribosomal DNA gene sequences of Marteilioides chungmuensis (Paramyxea), an ovarian parasite of the Pacific oyster Crassostrea gigas.

    PubMed

    Itoh, Naoki; Oda, Tadashi; Yoshinaga, Tomoyoshi; Ogawa, Kazuo

    2003-03-31

    To develop sensitive detection techniques with the aim of elucidating the life cycle of Marteilioides chungmuensis, an intracellular paramyxean infecting the ovary of the Pacific oyster Crassostrea gigas, we isolated the parasite at the sporont stage from infected oysters using a freeze-thaw procedure at -20 degrees C and differential centrifugations in discontinuous sucrose and Percoll gradients. DNA was extracted from the isolated sporonts, and a PCR amplicon of 18S small subunit ribosomal RNA gene DNA was partially sequenced. In situ hybridization using 3 parasite-specific probes designed from the obtained sequence successfully detected parasite cells in infected oysters, and confirmed that the sequenced DNA was derived from M. chungmuensis.

  11. Distribution of Mosquitoes in the South East of Argentina and First Report on the Analysis Based on 18S rDNA and COI Sequences

    PubMed Central

    Díaz-Nieto, Leonardo M.; Maciá, Arnaldo; Parisi, Gustavo; Farina, Juan L.; Vidal-Domínguez, María E.; Perotti, M. Alejandra; Berón, Corina M.

    2013-01-01

    Although Mar del Plata is the most important city on the Atlantic coast of Argentina, mosquitoes inhabiting such area are almost uncharacterized. To increase our knowledge in their distribution, we sampled specimens of natural populations. After the morphological identification based on taxonomic keys, sequences of DNA from small ribosomal subunit (18S rDNA) and cytochrome c oxidase I (COI) genes were obtained from native species and the phylogenetic analysis of these sequences were done. Fourteen species from the genera Uranotaenia, Culex, Ochlerotatus and Psorophora were found and identified. Our 18S rDNA and COI-based analysis indicates the relationships among groups at the supra-species level in concordance with mosquito taxonomy. The introduction and spread of vectors and diseases carried by them are not known in Mar del Plata, but some of the species found in this study were reported as pathogen vectors. PMID:24098700

  12. Group-specific small-subunit rRNA hybridization probes to characterize filamentous foaming in activated sludge systems.

    PubMed Central

    de los Reyes, F L; Ritter, W; Raskin, L

    1997-01-01

    Foaming in activated sludge systems is characterized by the formation of a thick, chocolate brown-colored scum that floats on the surface of aeration basins and secondary clarifiers. These viscous foams have been associated with the presence of filamentous mycolic acid-containing actinomycetes. To aid in evaluating the microbial representation in foam, we developed and characterized group-, genus-, and species-specific oligonucleotide probes targeting the small subunit rRNA of the Mycobacterium complex, Gordona spp., and Gordona (Nocardia) amarae, respectively. The use of a universal base analog, 5-nitroindole, in oligonucleotide probe design was evaluated by comparing the characteristics of two different versions of the Mycobacterium complex probe. The temperature of dissociation of each probe was determined. Probe specificity studies with a diverse collection of 67 target and nontarget rRNAs demonstrated the specificity of the probes to the target groups. Whole-cell hybridizations with fluorescein- and rhodamine-labeled probes were performed with pure cultures of various members of the Mycobacterium complex as well as with environmental samples from a full-scale activated sludge plant which experienced foaming. Quantitative membrane hybridizations with activated sludge and anaerobic digester foam showed that 15.0 to 18.3% of the total small-subunit rRNAs could be attributed to members of the Mycobacterium complex, of which a vast majority consisted of Gordona rRNA. Several G. amarae strains made up only a very small percentage of the Gordona strains present. We demonstrated that group-specific rRNA probes are useful tools for the in situ monitoring and identification of filamentous bacteria in activated sludge systems. PMID:9055425

  13. Combined large and small subunit ribosomal RNA phylogenies support a basal position of the acoelomorph flatworms.

    PubMed Central

    Telford, Maximilian J; Lockyer, Anne E; Cartwright-Finch, Chloë; Littlewood, D Timothy J

    2003-01-01

    The phylogenetic position of the phylum Platyhelminthes has been re-evaluated in the past decade by analysis of diverse molecular datasets. The consensus is that the Rhabditophora + Catenulida, which includes most of the flatworm taxa, are not primitively simple basal bilaterians but are related to coelomate phyla such as molluscs. The status of two other groups of acoelomate worms, Acoela and Nemertodermatida, is less clear. Although many characteristics unite these two groups, initial molecular phylogenetic studies placed the Nemertodermatida within the Rhabditophora, but placed the Acoela at the base of the Bilateria, distant from other flatworms. This contradiction resulted in scepticism about the basal position of acoels and led to calls for further data. We have sequenced large subunit ribosomal RNA genes from 13 rhabditophorans + catenulids, three acoels and one nemertodermatid, tripling the available data. Our analyses strongly support a basal position of both acoels and nemertodermatids. Alternative hypotheses are significantly less well supported by the data. We conclude that the Nemertodermatida and Acoela are basal bilaterians and, owing to their unique body plan and embryogenesis, should be recognized as a separate phylum, the Acoelomorpha. PMID:12803898

  14. Evaluating hypotheses of basal animal phylogeny using complete sequences of large and small subunit rRNA

    PubMed Central

    Medina, Mónica; Collins, Allen G.; Silberman, Jeffrey D.; Sogin, Mitchell L.

    2001-01-01

    We studied the evolutionary relationships among basal metazoan lineages by using complete large subunit (LSU) and small subunit (SSU) ribosomal RNA sequences for 23 taxa. After identifying competing hypotheses, we performed maximum likelihood searches for trees conforming to each hypothesis. Kishino–Hasegawa tests were used to determine whether the data (LSU, SSU, and combined) reject any of the competing hypotheses. We also conducted unconstrained tree searches, compared the resulting topologies, and calculated bootstrap indices. Shimodaira–Hasegawa tests were applied to determine whether the data reject any of the topologies resulting from the constrained and unconstrained tree searches. LSU, SSU, and the combined data strongly contradict two assertions pertaining to sponge phylogeny. Hexactinellid sponges are not likely to be the basal lineage of a monophyletic Porifera or the sister group to all other animals. Instead, Hexactinellida and Demospongia form a well-supported clade of siliceous sponges, Silicea. It remains unclear, on the basis of these data alone, whether the calcarean sponges are more closely related to Silicea or to nonsponge animals. The SSU and combined data reject the hypothesis that Bilateria is more closely related to Ctenophora than it is to Cnidaria, whereas LSU data alone do not refute either hypothesis. LSU and SSU data agree in supporting the monophyly of Bilateria, Cnidaria, Ctenophora, and Metazoa. LSU sequence data reveal phylogenetic structure in a data set with limited taxon sampling. Continued accumulation of LSU sequences should increase our understanding of animal phylogeny. PMID:11504944

  15. Evaluating hypotheses of basal animal phylogeny using complete sequences of large and small subunit rRNA

    SciTech Connect

    Medina, Monica; Collins, Allen G.; Silberman, Jeffrey; Sogin, Mitchell L.

    2001-06-21

    We studied the evolutionary relationships among basal metazoan lineages by using complete large subunit (LSU) and small subunit (SSU) ribosomal RNA sequences for 23 taxa. After identifying competing hypotheses, we performed maximum likelihood searches for trees conforming to each hypothesis. Kishino-Hasegawa tests were used to determine whether the data (LSU, SSU, and combined) reject any of the competing hypotheses. We also conducted unconstrained tree searches, compared the resulting topologies, and calculated bootstrap indices. Shimodaira-Hasegawa tests were applied to determine whether the data reject any of the topologies resulting from the constrained and unconstrained tree searches. LSU, SSU, and the combined data strongly contradict two assertions pertaining to sponge phylogeny. Hexactinellid sponges are not likely to be the basal lineage of amonophyletic Porifera or the sister group to all other animals. Instead, Hexactinellida and Demospongia form a well-supported clade of siliceous sponges, Silicea. It remains unclear, on the basis of these data alone, whether the calcarean sponges are more closely related to Silicea or to nonsponge animals. The SSU and combined data reject the hypothesis that Bilateria is more closely related to Ctenophora than it is to Cnidaria, whereas LSU data alone do not refute either hypothesis. LSU and SSU data agree in supporting the monophyly of Bilateria, Cnidaria, Ctenophora, and Metazoa. LSU sequence data reveal phylogenetic structure in a data set with limited taxon sampling. Continued accumulation of LSU sequences should increase our understanding of animal phylogeny.

  16. Preparation of polyclonal antibodies of Rubisco large and small subunits and their application in the functional analysis of the genes.

    PubMed

    Ma, Peng-Da; Lu, Tian-Cheng; Zhou, Xiao-Fu; Zhu, Xiao-Juan; Wang, Xing-Zhi

    2004-09-01

    Spinach Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) large (rbcL) and small (rbcS) subunits were separated by SDS-PAGE, and protein amount and purity were determined by Bradford assay. Polyclonal antibodies against rbcL and rbcS subunit were generated in female BALB/c mice and had no cross-reaction with each other. A total of 81 microg antigens were used and 0.3 ml anti-sera with titer of 1:5000 were yielded. The antibodies were also applicable to study rbcL and rbcS in tobacco plant Nicotiana benthamiana. Potato virus X vector pGR107 induced silencing of rbcS gene by Agrobacterium in Nicotiana benthamiana was performed. The expression level of rbcL and rbcS was lower in rbcS silenced plants than that in control plants as detected by the corresponding antibodies. This implied that the expression of rbcL was regulated by rbcS.

  17. Phosphorylation of chloroplast ribulose bisphosphate carboxylase/oxygenase small subunit by an envelope-bound protein kinase in situ.

    PubMed

    Soll, J; Buchanan, B B

    1983-06-10

    A new protein kinase of the cAMP independent type was found to be bound to the outer envelope membrane of spinach chloroplasts. While stimulated by Mg2+ and inhibited by ADP, the enzyme showed no response to conventional protein substrates and was essentially independent of pH in the physiological (pH 7 to 8) range. The new protein kinase phosphorylated the mature form of the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase and, to a lesser extent, an unidentified 24-kDa polypeptide, both of which were bound to the outer envelope membrane. The results suggest that phosphorylation of cytoplasmically synthesized protein constituents of chloroplasts is involved in their transport through the chloroplast envelope membrane barrier.

  18. A preliminary phylogeny of the scale insects (Hemiptera: Sternorrhyncha: Coccoidea) based on nuclear small-subunit ribosomal DNA.

    PubMed

    Cook, Lyn G; Gullan, Penny J; Trueman, Holly E

    2002-10-01

    Scale insects (Hemiptera: Sternorrhyncha: Coccoidea) are a speciose and morphologically specialized group of plant-feeding bugs in which evolutionary relationships and thus higher classification are controversial. Sequences derived from nuclear small-subunit ribosomal DNA were used to generate a preliminary molecular phylogeny for the Coccoidea based on 39 species representing 14 putative families. Monophyly of the archaeococcoids (comprising Ortheziidae, Margarodidae sensu lato, and Phenacoleachia) was equivocal, whereas monophyly of the neococcoids was supported. Putoidae, represented by Puto yuccae, was found to be outside the remainder of the neococcoid clade. These data are consistent with a single origin (in the ancestor of the neococcoid clade) of a chromosome system involving paternal genome elimination in males. Pseudococcidae (mealybugs) appear to be sister to the rest of the neococcoids and there are indications that Coccidae (soft scales) and Kerriidae (lac scales) are sister taxa. The Eriococcidae (felt scales) was not recovered as a monophyletic group and the eriococcid genus Eriococcus sensu lato was polyphyletic.

  19. Molecular analysis of lungworms from European bison (Bison bonasus) on the basis of small subunit ribosomal RNA gene (SSU).

    PubMed

    Pyziel, Anna M

    2014-03-01

    Dictyocaulosis (Nematoda: Trichostrongyloidea) is a widespread parasitosis of the European bison (Bison bonasus) inhabiting Bialowieza Primeval Forest. Bearing in mind the current coexistence of bison with wild cervids, and with domestic ruminants in the 19th and 20th century, the need arose for molecular identification of lungworm species. Molecular analysis was done on adult lungworms that were obtained from the respiratory track of four free-roaming bison euthanized as a part of the population health control program. As the result of the study four identical small subunit-ribosomal RNA gene sequences from the lungworms were obtained and deposited in GenBank as sequence, 1708 bp long (GenBank KC771250). Comparative analysis of the SSU rRNA sequences revealed the European bison to be a host for the bovine lungworm Dictyocaulus viviparus.

  20. Structure of a human pre-40S particle points to a role for RACK1 in the final steps of 18S rRNA processing

    PubMed Central

    Larburu, Natacha; Montellese, Christian; O'Donohue, Marie-Françoise; Kutay, Ulrike; Gleizes, Pierre-Emmanuel; Plisson-Chastang, Célia

    2016-01-01

    Synthesis of ribosomal subunits in eukaryotes is a complex and tightly regulated process that has been mostly characterized in yeast. The discovery of a growing number of diseases linked to defects in ribosome biogenesis calls for a deeper understanding of these mechanisms and of the specificities of human ribosome maturation. We present the 19 Å resolution cryo-EM reconstruction of a cytoplasmic precursor to the human small ribosomal subunit, purified by using the tagged ribosome biogenesis factor LTV1 as bait. Compared to yeast pre-40S particles, this first three-dimensional structure of a human 40S subunit precursor shows noticeable differences with respect to the position of ribosome biogenesis factors and uncovers the early deposition of the ribosomal protein RACK1 during subunit maturation. Consistently, RACK1 is required for efficient processing of the 18S rRNA 3′-end, which might be related to its role in translation initiation. This first structural analysis of a human pre-ribosomal particle sets the grounds for high-resolution studies of conformational transitions accompanying ribosomal subunit maturation. PMID:27530427

  1. 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.

  2. Small Subunits of Serine Palmitoyltransferase (ssSPTs) and Their Physiological Roles

    DTIC Science & Technology

    2014-02-12

    blast homology searches of Tsc3p against the human genome showed no candidate homologs. However, recently two functional orthologs of Tsc3p, the small...ssSPTs in Schizosaccharomyces pombe   INTRODUCTION: Schizosaccharomyces pombe forms a major clade of the ‘Ascomycete fungi ’ but it is believed...homology search against the C. elegans genome database, using human ssSPTa as the query sequence. Blast analysis yielded one questionable homolog

  3. Secondary structure and molecular evolution of the mitochondrial small subunit ribosomal RNA in Agaricales (Euagarics clade, Homobasidiomycota).

    PubMed

    Barroso, Gérard; Sirand-Pugnet, Pascal; Mouhamadou, Bello; Labarère, Jacques

    2003-10-01

    The complete sequences and secondary structures of the mitochondrial small subunit (SSU) ribosomal RNAs of both mostly cultivated mushrooms Agaricus bisporus (1930 nt) and Lentinula edodes (2164 nt) were achieved. These secondary structures and that of Schizophyllum commune (1872 nt) were compared to that previously established for Agrocybe aegerita. The four structures are near the model established for Archae, Bacteria, plastids, and mitochondria; particularly the helices 23 and 37, described as specific to bacteria, are present. Within the four Agaricales (Homobasidiomycota), the SSU-rRNA "core" is conserved in size (966 to 1009 nt) with the exception of an unusual extension of 40 nt in the H17 helix of S. commune. The four core sequences possess 76% of conserved positions and a cluster of C in their 3' end, which could constitute a signal involved in the RNA maturation process. Among the nine putative variable domains, three (V3, V5, V7) do not show significant length variations and possess similar percentages of conserved positions (69%) than the core. The other six variable domains show important length variations, due to independent large size inserted/deleted sequences, and higher rates of nucleotide substitutions than the core (only 31% of conserved positions between the four species). Interestingly, the inserted/deleted sequences are located in few preferential sites (hot spots for insertion/deletion) where they seem to arise or disappear haphazardly during evolution. These sites are located on the surface of the tertiary structure of the 30S ribosomal subunit, at the beginning of hairpin loops; the insertions lead to a lengthening of existing hairpins or to branching loops bearing up to five additional helices.

  4. Testing three pipelines for 18S rDNA-based metabarcoding of soil faunal diversity.

    PubMed

    Yang, ChenXue; Ji, YingQiu; Wang, XiaoYang; Yang, ChunYang; Yu, Douglas W

    2013-01-01

    A number of basic and applied questions in ecology and environmental management require the characterization of soil and leaf litter faunal diversity. Recent advances in high-throughput sequencing of barcode-gene amplicons ('metabarcoding') have made it possible to survey biodiversity in a robust and efficient way. However, one obstacle to the widespread adoption of this technique is the need to choose amongst many candidates for bioinformatic processing of the raw sequencing data. We compare three candidate pipelines for the processing of 18S small subunit rDNA metabarcode data from solid substrates: (i) USEARCH/CROP, (ii) Denoiser/UCLUST, and (iii) OCTUPUS. The three pipelines produced reassuringly similar and highly correlated assessments of community composition that are dominated by taxa known to characterize the sampled environments. However, OCTUPUS appears to inflate phylogenetic diversity, because of higher sequence noise. We therefore recommend either the USEARCH/CROP or Denoiser/UCLUST pipelines, both of which can be run within the QIIME (Quantitative Insights Into Microbial Ecology) environment.

  5. 18S ribosomal RNA gene sequences of Cochliopodium (Himatismenida) and the phylogeny of Amoebozoa.

    PubMed

    Kudryavtsev, Alexander; Bernhard, Detlef; Schlegel, Martin; Chao, Ema E Y; Cavalier-Smith, Thomas

    2005-08-01

    Cochliopodium is a very distinctive genus of discoid amoebae covered by a dorsal tectum of carbohydrate microscales. Its phylogenetic position is unclear, since although sharing many features with naked "gymnamoebae", the tectum sets it apart. We sequenced 18S ribosomal RNA genes from three Cochliopodium species (minus, spiniferum and Cochliopodium sp., a new species resembling C. minutum). Phylogenetic analysis shows Cochliopodium as robustly holophyletic and within Amoebozoa, in full accord with morphological data. Cochliopodium is always one of the basal branches within Amoebozoa but its precise position is unstable. In Bayesian analysis it is sister to holophyletic Glycostylida, but distance trees mostly place it between Dermamoeba and a possibly artifactual long-branch cluster including Thecamoeba. These positions are poorly supported and basal amoebozoan branching ill-resolved, making it unclear whether Discosea (Glycostylida, Himatismenida, Dermamoebida) is holophyletic; however, Thecamoeba seems not specifically related to Dermamoeba. We also sequenced the small-subunit rRNA gene of Vannella persistens, which constantly grouped with other Vannella species, and two Hartmannella strains. Our trees suggest that Vexilliferidae, Variosea and Hartmannella are polyphyletic, confirming the existence of two very distinct Hartmannella clades: that comprising H. cantabrigiensis and another divergent species is sister to Glaeseria, whilst Hartmannella vermiformis branches more deeply.

  6. 18S rDNA phylogeny of lamproderma and allied genera (Stemonitales, Myxomycetes, Amoebozoa).

    PubMed

    Fiore-Donno, Anna Maria; Kamono, Akiko; Meyer, Marianne; Schnittler, Martin; Fukui, Manabu; Cavalier-Smith, Thomas

    2012-01-01

    The phylogenetic position of the slime-mould genus Lamproderma (Myxomycetes, Amoebozoa) challenges traditional taxonomy: although it displays the typical characters of the order Stemonitales, it appears to be sister to Physarales. This study provides a small subunit (18S or SSU) ribosomal RNA gene-based phylogeny of Lamproderma and its allies, with new sequences from 49 specimens in 12 genera. We found that the order Stemonitales and Lamproderma were both ancestral to Physarales and that Lamproderma constitutes several clades intermingled with species of Diacheopsis, Colloderma and Elaeomyxa. We suggest that these genera may have evolved from Lamproderma by multiple losses of fruiting body stalks and that many taxonomic revisions are needed. We found such high genetic diversity within three Lamproderma species that they probably consist of clusters of sibling species. We discuss the contrasts between genetic and morphological divergence and implications for the morphospecies concept, highlighting the phylogenetically most reliable morphological characters and pointing to others that have been overestimated. In addition, we showed that the first part (~600 bases) of the SSU rDNA gene is a valuable tool for phylogeny in Myxomycetes, since it displayed sufficient variability to distinguish closely related taxa and never failed to cluster together specimens considered of the same species.

  7. 18S rDNA Phylogeny of Lamproderma and Allied Genera (Stemonitales, Myxomycetes, Amoebozoa)

    PubMed Central

    Fiore-Donno, Anna Maria; Kamono, Akiko; Meyer, Marianne; Schnittler, Martin; Fukui, Manabu; Cavalier-Smith, Thomas

    2012-01-01

    The phylogenetic position of the slime-mould genus Lamproderma (Myxomycetes, Amoebozoa) challenges traditional taxonomy: although it displays the typical characters of the order Stemonitales, it appears to be sister to Physarales. This study provides a small subunit (18S or SSU) ribosomal RNA gene-based phylogeny of Lamproderma and its allies, with new sequences from 49 specimens in 12 genera. We found that the order Stemonitales and Lamproderma were both ancestral to Physarales and that Lamproderma constitutes several clades intermingled with species of Diacheopsis, Colloderma and Elaeomyxa. We suggest that these genera may have evolved from Lamproderma by multiple losses of fruiting body stalks and that many taxonomic revisions are needed. We found such high genetic diversity within three Lamproderma species that they probably consist of clusters of sibling species. We discuss the contrasts between genetic and morphological divergence and implications for the morphospecies concept, highlighting the phylogenetically most reliable morphological characters and pointing to others that have been overestimated. In addition, we showed that the first part (∼600 bases) of the SSU rDNA gene is a valuable tool for phylogeny in Myxomycetes, since it displayed sufficient variability to distinguish closely related taxa and never failed to cluster together specimens considered of the same species. PMID:22530009

  8. Secondary structure of rabbit 18S ribosomal RNA determined from biochemical and phylogenetic data

    SciTech Connect

    Rairkar, A.; Rubino, H.; Lockard, R.E.

    1986-05-01

    To understand the functional role of 18S rRNA in the eukaryotic 40S subunit, its higher order structure must first be determined. Native deproteinized 18S rRNA was isolated from purified rabbit 40S subunits, fractionated on SDS-sucrose density gradients and concentrated using centricon-30 microconcentrators. The structure of native 18S rRNA was probed chemically with both diethylpyrocarbonate (DEPC) and dimethyl sulfate (DMS) which react with unpaired adenosine and guanosine residues, respectively. After /sup 32/P-end-labeling of intact and fragmented RNA, the modified nucleotides were identified by polyacrylamide sequencing gel electrophoresis upon aniline induced strand scission. On the basis of both the biochemical and phylogenetic data, a secondary structure model is proposed which includes the two major G + C rich insertion elements. A comparison of the structure data with previously published phylogenetic models suggests an instability of certain predicted helices. These unstable helices may normally be stabilized by ribosomal proteins and could represent the flexible elements involved in biologically significant conformational switches within 40S subunit.

  9. Global eukaryote phylogeny: Combined small- and large-subunit ribosomal DNA trees support monophyly of Rhizaria, Retaria and Excavata.

    PubMed

    Moreira, David; von der Heyden, Sophie; Bass, David; López-García, Purificación; Chao, Ema; Cavalier-Smith, Thomas

    2007-07-01

    Resolution of the phylogenetic relationships among the major eukaryotic groups is one of the most important problems in evolutionary biology that is still only partially solved. This task was initially addressed using a single marker, the small-subunit ribosomal DNA (SSU rDNA), although in recent years it has been shown that it does not contain enough phylogenetic information to robustly resolve global eukaryotic phylogeny. This has prompted the use of multi-gene analyses, especially in the form of long concatenations of numerous conserved protein sequences. However, this approach is severely limited by the small number of taxa for which such a large number of protein sequences is available today. We have explored the alternative approach of using only two markers but a large taxonomic sampling, by analysing a combination of SSU and large-subunit (LSU) rDNA sequences. This strategy allows also the incorporation of sequences from non-cultivated protists, e.g., Radiozoa (=radiolaria minus Phaeodarea). We provide the first LSU rRNA sequences for Heliozoa, Apusozoa (both Apusomonadida and Ancyromonadida), Cercozoa and Radiozoa. Our Bayesian and maximum likelihood analyses for 91 eukaryotic combined SSU+LSU sequences yielded much stronger support than hitherto for the supergroup Rhizaria (Cercozoa plus Radiozoa plus Foraminifera) and several well-recognised groups and also for other problematic clades, such as the Retaria (Radiozoa plus Foraminifera) and, with more moderate support, the Excavata. Within opisthokonts, the combined tree strongly confirms that the filose amoebae Nuclearia are sisters to Fungi whereas other Choanozoa are sisters to animals. The position of some bikont taxa, notably Heliozoa and Apusozoa, remains unresolved. However, our combined trees suggest a more deeply diverging position for Ancyromonas, and perhaps also Apusomonas, than for other bikonts, suggesting that apusozoan zooflagellates may be central for understanding the early evolution of

  10. E2F1 promote the aggressiveness of human colorectal cancer by activating the ribonucleotide reductase small subunit M2

    SciTech Connect

    Fang, Zejun; Gong, Chaoju; Liu, Hong; Zhang, Xiaomin; Mei, Lingming; Song, Mintao; Qiu, Lanlan; Luo, Shuchai; Zhu, Zhihua; Zhang, Ronghui; Gu, Hongqian; Chen, Xiang

    2015-08-21

    As the ribonucleotide reductase small subunit, the high expression of ribonucleotide reductase small subunit M2 (RRM2) induces cancer and contributes to tumor growth and invasion. In several colorectal cancer (CRC) cell lines, we found that the expression levels of RRM2 were closely related to the transcription factor E2F1. Mechanistic studies were conducted to determine the molecular basis. Ectopic overexpression of E2F1 promoted RRM2 transactivation while knockdown of E2F1 reduced the levels of RRM2 mRNA and protein. To further investigate the roles of RRM2 which was activated by E2F1 in CRC, CCK-8 assay and EdU incorporation assay were performed. Overexpression of E2F1 promoted cell proliferation in CRC cells, which was blocked by RRM2 knockdown attenuation. In the migration and invasion tests, overexpression of E2F1 enhanced the migration and invasion of CRC cells which was abrogated by silencing RRM2. Besides, overexpression of RRM2 reversed the effects of E2F1 knockdown partially in CRC cells. Examination of clinical CRC specimens demonstrated that both RRM2 and E2F1 were elevated in most cancer tissues compared to the paired normal tissues. Further analysis showed that the protein expression levels of E2F1 and RRM2 were parallel with each other and positively correlated with lymph node metastasis (LNM), TNM stage and distant metastasis. Consistently, the patients with low E2F1 and RRM2 levels have a better prognosis than those with high levels. Therefore, we suggest that E2F1 can promote CRC proliferation, migration, invasion and metastasis by regulating RRM2 transactivation. Understanding the role of E2F1 in activating RRM2 transcription will help to explain the relationship between E2F1 and RRM2 in CRC and provide a novel predictive marker for diagnosis and prognosis of the disease. - Highlights: • E2F1 promotes RRM2 transactivation in CRC cells. • E2F1 promotes the proliferation of CRC cells by activating RRM2. • E2F1 promotes the migration and

  11. Using the small subunit of nuclear ribosomal DNA to reveal the phylogenetic position of the plerocercoid larvae of Spirometra tapeworms.

    PubMed

    Zhang, Xi; Duan, Jiang Yang; Wang, Zhong Quan; Jiang, Peng; Liu, Ruo Dan; Cui, Jing

    2017-04-01

    Although medically important, the systematics of Spirometra and the taxonomic position of S. erinaceieuropaei remain unclear. In this study, the 18S rDNA gene of S. erinaceieuropaei sparganum from naturally infected frogs caught in 14 geographical locations of China was sequenced. In addition, all available 18S sequences of the family Diphyllobothriidae in the Genbank database were included to reconstruct the phylogeny of diphyllobothriid tapeworms. The secondary structure model of the 18S rDNA was also predicated to further explore the sequence variation. Phylogenetic analyses were performed using maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI) methods. The intraspecific divergences of 18S rDNA in Chinese sparganum isolates ranged from 0.0 to 0.4%. Regions of V2, V4 and V7 were the most variable regions in the secondary structure of 18S rDNA. With the exception of genera Duthiersia and Probothriocephalus, other genera (i.e., Adenocephalus, Diphyllobothrium, Diplogonoporus, Duthiersia, Schistocephalus and Spirometra) selected in the Diphyllobothriidae shared similar topologies of V2, V4 and V7 structures. The topology of generated phylogenetic trees revealed close relationships among Adenocephalus, Digramma, Diphyllobothrium, Diplogonoporus, Ligula, Sparganum and Spirometra. The exact phylogenetic position of Spirometra species should be further analyzed with more sampling and more useful molecular markers.

  12. Evaluation of nearest-neighbor methods for detection of chimeric small-subunit rRNA sequences.

    PubMed Central

    Robison-Cox, J F; Bateson, M M; Ward, D M

    1995-01-01

    Detection of chimeric artifacts formed when PCR is used to retrieve naturally occurring small-subunit (SSU) rRNA sequences may rely on demonstrating that different sequence domains have different phylogenetic affiliations. We evaluated the CHECK_CHIMERA method of the Ribosomal Database Project and another method which we developed, both based on determining nearest neighbors of different sequence domains, for their ability to discern artificially generated SSU rRNA chimeras from authentic Ribosomal Database Project sequences. The reliability of both methods decreases when the parental sequences which contribute to chimera formation are more than 82 to 84% similar. Detection is also complicated by the occurrence of authentic SSU rRNA sequences that behave like chimeras. We developed a naive statistical test based on CHECK_CHIMERA output and used it to evaluate previously reported SSU rRNA chimeras. Application of this test also suggests that chimeras might be formed by retrieving SSU rRNAs as cDNA. The amount of uncertainty associated with nearest-neighbor analyses indicates that such tests alone are insufficient and that better methods are needed. PMID:7538272

  13. The rubisco small subunit is involved in tobamovirus movement and Tm-2²-mediated extreme resistance.

    PubMed

    Zhao, Jinping; Liu, Qi; Zhang, Haili; Jia, Qi; Hong, Yiguo; Liu, Yule

    2013-01-01

    The multifunctional movement protein (MP) of Tomato mosaic tobamovirus (ToMV) is involved in viral cell-to-cell movement, symptom development, and resistance gene recognition. However, it remains to be elucidated how ToMV MP plays such diverse roles in plants. Here, we show that ToMV MP interacts with the Rubisco small subunit (RbCS) of Nicotiana benthamiana in vitro and in vivo. In susceptible N. benthamiana plants, silencing of NbRbCS enabled ToMV to induce necrosis in inoculated leaves, thus enhancing virus local infectivity. However, the development of systemic viral symptoms was delayed. In transgenic N. benthamiana plants harboring Tobacco mosaic virus resistance-2² (Tm-2²), which mediates extreme resistance to ToMV, silencing of NbRbCS compromised Tm-2²-dependent resistance. ToMV was able to establish efficient local infection but was not able to move systemically. These findings suggest that NbRbCS plays a vital role in tobamovirus movement and plant antiviral defenses.

  14. Phylogenetic relationships between Vorticella convallaria and other species inferred from small subunit rRNA gene sequences.

    PubMed

    Itabashi, Takeshi; Mikami, Kazuyuki; Fang, Jie; Asai, Hiroshi

    2002-08-01

    Vorticellid ciliates generally dwell in freshwater. In nature, the species have up until now been identified by comparison with previous descriptions. It is difficult to identify between species of the genus Vorticella, because the morphological markers of vorticellid ciliates described in reports are limited and variable. Unfortunately, culturing them has only succeeded with certain species such as Vorticella convallaria, but many others have been impossible to culture. To find out whether the sequence of a small subunit rRNA gene was an appropriate marker to identify vorticellid ciliates, the gene was aligned and compared. Finding a new convenient method will contribute to research on vorticellid ciliates. In strains of V. convallaria, classified morphologically, some varieties of the SSrRNA gene sequences were recognized, but there were large variations within the same species. According to the phylogenetic tree, these strains are closely related. However, the difference was not as big as between Vorticella and Carchesium. In addition, Carchesium constructed a distinct clade from the genus Vorticella and Epistylis. These results show the possibility that the SSrRNA gene is one of the important markers to identify species of Vorticella. This study is first to approach and clarify the complicated taxa in the genus Vorticella.

  15. Quantitative Analysis of Small-Subunit rRNA Genes in Mixed Microbial Populations via 5′-Nuclease Assays

    PubMed Central

    Suzuki, Marcelino T.; Taylor, Lance T.; DeLong, Edward F.

    2000-01-01

    Few techniques are currently available for quantifying specific prokaryotic taxa in environmental samples. Quantification of specific genotypes has relied mainly on oligonucleotide hybridization to extracted rRNA or intact rRNA in whole cells. However, low abundance and cellular rRNA content limit the application of these techniques in aquatic environments. In this study, we applied a newly developed quantitative PCR assay (5′-nuclease assay, also known as TaqMan) to quantify specific small-subunit (SSU) rRNA genes (rDNAs) from uncultivated planktonic prokaryotes in Monterey Bay. Primer and probe combinations for quantification of SSU rDNAs at the domain and group levels were developed and tested for specificity and quantitative reliability. We examined the spatial and temporal variations of SSU rDNAs from Synechococcus plus Prochlorococcus and marine Archaea and compared the results of the quantitative PCR assays to those obtained by alternative methods. The 5′-nuclease assays reliably quantified rDNAs over at least 4 orders of magnitude and accurately measured the proportions of genes in artificial mixtures. The spatial and temporal distributions of planktonic microbial groups measured by the 5′-nuclease assays were similar to the distributions estimated by quantitative oligonucleotide probe hybridization, whole-cell hybridization assays, and flow cytometry. PMID:11055900

  16. Rfc5, a small subunit of replication factor C complex, couples DNA replication and mitosis in budding yeast.

    PubMed Central

    Sugimoto, K; Shimomura, T; Hashimoto, K; Araki, H; Sugino, A; Matsumoto, K

    1996-01-01

    The inhibition of DNA synthesis prevents mitotic entry through the action of the S phase checkpoint. In the yeast Saccharomyces cerevisiae, an essential protein kinase, Spk1/Mec2/Rad53/Sad1, controls the coupling of S phase to mitosis. In an attempt to identify genes that genetically interact with Spk1, we have isolated a temperature-sensitive mutation, rfc5-1, that can be suppressed by overexpression of SPK1. The RFC5 gene encodes a small subunit of replication factor C complex. At the restrictive temperature, rfc5-1 mutant cells entered mitosis with unevenly separated or fragmented chromosomes, resulting in loss of viability. Thus, the rfc5 mutation defective for DNA replication is also impaired in the S phase checkpoint. Overexpression of POL30, which encodes the proliferating cell nuclear antigen, suppressed the replication defect of the rfc5 mutant but not its checkpoint defect. Taken together, these results suggested that replication factor C has a direct role in sensing the state of DNA replication and transmitting the signal to the checkpoint machinery. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8692942

  17. Phylogenetics of Bonamia parasites based on small subunit and internal transcribed spacer region ribosomal DNA sequence data.

    PubMed

    Hill, Kristina M; Stokes, Nancy A; Webb, Stephen C; Hine, P Mike; Kroeck, Marina A; Moore, James D; Morley, Margaret S; Reece, Kimberly S; Burreson, Eugene M; Carnegie, Ryan B

    2014-07-24

    The genus Bonamia (Haplosporidia) includes economically significant oyster parasites. Described species were thought to have fairly circumscribed host and geographic ranges: B. ostreae infecting Ostrea edulis in Europe and North America, B. exitiosa infecting O. chilensis in New Zealand, and B. roughleyi infecting Saccostrea glomerata in Australia. The discovery of B. exitiosa-like parasites in new locations and the observation of a novel species, B. perspora, in non-commercial O. stentina altered this perception and prompted our wider evaluation of the global diversity of Bonamia parasites. Samples of 13 oyster species from 21 locations were screened for Bonamia spp. by PCR, and small subunit and internal transcribed spacer regions of Bonamia sp. ribosomal DNA were sequenced from PCR-positive individuals. Infections were confirmed histologically. Phylogenetic analyses using parsimony and Bayesian methods revealed one species, B. exitiosa, to be widely distributed, infecting 7 oyster species from Australia, New Zealand, Argentina, eastern and western USA, and Tunisia. More limited host and geographic distributions of B. ostreae and B. perspora were confirmed, but nothing genetically identifiable as B. roughleyi was found in Australia or elsewhere. Newly discovered diversity included a Bonamia sp. in Dendostrea sandvicensis from Hawaii, USA, that is basal to the other Bonamia species and a Bonamia sp. in O. edulis from Tomales Bay, California, USA, that is closely related to both B. exitiosa and the previously observed Bonamia sp. from O. chilensis in Chile.

  18. Evaluation of nearest-neighbor methods for detection of chimeric small-subunit rRNA sequences

    NASA Technical Reports Server (NTRS)

    Robison-Cox, J. F.; Bateson, M. M.; Ward, D. M.

    1995-01-01

    Detection of chimeric artifacts formed when PCR is used to retrieve naturally occurring small-subunit (SSU) rRNA sequences may rely on demonstrating that different sequence domains have different phylogenetic affiliations. We evaluated the CHECK_CHIMERA method of the Ribosomal Database Project and another method which we developed, both based on determining nearest neighbors of different sequence domains, for their ability to discern artificially generated SSU rRNA chimeras from authentic Ribosomal Database Project sequences. The reliability of both methods decreases when the parental sequences which contribute to chimera formation are more than 82 to 84% similar. Detection is also complicated by the occurrence of authentic SSU rRNA sequences that behave like chimeras. We developed a naive statistical test based on CHECK_CHIMERA output and used it to evaluate previously reported SSU rRNA chimeras. Application of this test also suggests that chimeras might be formed by retrieving SSU rRNAs as cDNA. The amount of uncertainty associated with nearest-neighbor analyses indicates that such tests alone are insufficient and that better methods are needed.

  19. A molecular phylogeny of the marine red algae (Rhodophyta) based on the nuclear small-subunit rRNA gene.

    PubMed Central

    Ragan, M A; Bird, C J; Rice, E L; Gutell, R R; Murphy, C A; Singh, R K

    1994-01-01

    A phylogeny of marine Rhodophyta has been inferred by a number of methods from nucleotide sequences of nuclear genes encoding small subunit rRNA from 39 species in 15 orders. Sequence divergences are relatively large, especially among bangiophytes and even among congeners in this group. Subclass Bangiophycidae appears polyphyletic, encompassing at least three lineages, with Porphyridiales distributed between two of these. Subclass Florideophycidae is monophyletic, with Hildenbrandiales, Corallinales, Ahnfeltiales, and a close association of Nemaliales, Acrochaetiales, and Palmariales forming the four deepest branches. Cermiales may represent a convergence of vegetative and reproductive morphologies, as family Ceramiaceae is at best weakly related to the rest of the order, and one of its members appears to be allied to Gelidiales. Except for Gigartinales, for which more data are required, the other florideophyte orders appear distinct and taxonomically justified. A good correlation was observed with taxonomy based on pit-plug ultrastructure. Tests under maximum-likelihood and parsimony of alternative phylogenies based on structure and chemistry refuted suggestions that Acrochaetiales is the most primitive florideophyte order and that Gelidiales and Hildenbrandiales are sister groups. PMID:8041780

  20. Small angle X-ray scattering of wheat seed-storage proteins: alpha-, gamma- and omega-gliadins and the high molecular weight (HMW) subunits of glutenin.

    PubMed

    Thomson, N H; Miles, M J; Popineau, Y; Harries, J; Shewry, P; Tatham, A S

    1999-03-19

    Small angle X-ray scattering in solution was performed on seed-storage proteins from wheat. Three different groups of gliadins (alpha-, gamma- and omega-) and a high molecular weight (HMW) subunit of glutenin (1Bx20) were studied to determine molecular size parameters. All the gliadins could be modelled as prolate ellipsoids with extended conformations. The HMW subunit existed as a highly extended rod-like particle in solution with a length of about 69 nm and a diameter of about 6.4 nm. Specific aggregation effects were observed which may reflect mechanisms of self-assembly that contribute to the unique viscoelastic properties of wheat dough.

  1. Phylogeny of gregarines (Apicomplexa) as inferred from small-subunit rDNA and beta-tubulin.

    PubMed

    Leander, Brian S; Clopton, Richard E; Keeling, Patrick J

    2003-01-01

    Gregarines are thought to be deep-branching apicomplexans. Accordingly, a robust inference of gregarine phylogeny is crucial to any interpretation of apicomplexan evolution, but molecular sequences from gregarines are restricted to a small number of small-subunit (SSU) rDNA sequences from derived taxa. This work examines the usefulness of SSU rDNA and beta-tubulin sequences for inferring gregarine phylogeny. SSU rRNA genes from Lecudina (Mingazzini) sp., Monocystis agilis Stein, Leidyana migrator Clopton and Gregarina polymorpha Dufour, as well as the beta-tubulin gene from Leidyana migrator, were sequenced. The results of phylogenetic analyses of alveolate taxa using both genes were consistent with an early origin of gregarines and the putative 'sister' relationship between gregarines and Cryptosporidium, but neither phylogeny was strongly supported. In addition, two SSU rDNA sequences from unidentified marine eukaryotes were found to branch among the gregarines: one was a sequence derived from the haemolymph parasite of the giant clam, Tridacna crocea, and the other was a sequence misattributed to the foraminiferan Ammonium beccarii. In all of our analyses, the SSU rDNA sequence from Colpodella sp. clustered weakly with the apicomplexans, which is consistent with ultrastructural data. Altogether, the exact position of gregarines with respect to Cryptosporidium and other apicomplexans remains to be confirmed, but the congruence of SSU rDNA and beta-tubulin trees with one another and with morphological data does suggest that further sampling of molecular data will eventually put gregarine diversity into a phylogenetic context.

  2. Added resolution among ordinal level relationships of tapeworms (Platyhelminthes: Cestoda) with complete small and large subunit nuclear ribosomal RNA genes.

    PubMed

    Waeschenbach, Andrea; Webster, Bonnie L; Bray, Rodney A; Littlewood, D T J

    2007-10-01

    The addition of large subunit ribosomal DNA (lsrDNA) to small subunit ribosomal DNA (ssrDNA) has been shown to add resolution to phylogenies at various taxonomic levels for a diversity of phyla. We added nearly complete lsrDNA (4057-4593bp) sequences to ssrDNA (1940-2228bp) for 26 ingroup and 3 outgroup taxa in an attempt to provide an improved ordinal phylogeny for the Cestoda. Ten lsrDNA and seven ssrDNA sequences were generated from new taxa and 13 existing partial lsrDNA sequences were sequenced to completion. The majority of phylogenetic signal in the combined analysis came from lsrDNA (69.6% of parsimonious informative sites, as opposed to 30.4% obtained from ssrDNA), resulting in almost identical topologies for lsrDNA and lsr+ssrDNA (pairwise symmetric distance=6) in model-based analyses. Topology testing found trees based on partial lsrDNA (domains D1-D3)+ssrDNA and complete lsr+ssrDNA to differ significantly; the addition of lsrDNA domains D4-D12 had a significant effect on topology. Overall nodal support was greatest in the combined analysis and weakest for ssrDNA only. Our molecular phylogenies differed significantly from those based on morphology alone. Acetabulate lineages form a monophyletic group, with the Tetraphyllidea being paraphyletic. Support for the combined data was high for the following topology: (Litobothriidea (Lecanicephalidea (Rhinebothrium/Rhodobothrium (Clistobothrium (Pachybothrium(Acanthobothrium Proteocephalidea) (Mesocestoididae, Nippotaeniidea, Cyclophyllidea, Tetrabothriidea)))))); all genus names refer to tetraphyllidean lineages. Although the interrelationships among the four most derived taxa remain uncertain, overall ambiguity of the acetabulate interrelationships was reduced. The Pseudophyllidea were recovered as polyphyletic, with support for a sister-group relationship between Diphyllobothriidae and Haplobothriidea. The monophyly of the Trypanorhyncha was recovered for the first time based on molecular data. The positions

  3. Molecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis.

    PubMed

    Godon, J J; Zumstein, E; Dabert, P; Habouzit, F; Moletta, R

    1997-07-01

    The bacterial community structure of a fluidized-bed reactor fed by vinasses (wine distillation waste) was analyzed. After PCR amplification, four small-subunit (SSU) rDNA clone libraries of Bacteria, Archaea, Procarya, and Eucarya populations were established. The community structure was determined by operational taxonomic unit (OTU) phylogenetic analyses of 579 partial rDNA sequences (about 500 bp long). A total of 146 OTUs were found, comprising 133, 6, and 7 from the Bacteria, Archaea, and Eucarya domains, respectively. A total of 117 bacterial OTU were affiliated with major phyla: low-G+C gram-positive bacteria, Cytophaga-Flexibacter-Bacteroides, Proteobacteria, high-G+C gram-positive bacteria, and Spirochaetes, where the clone distribution was 34, 26, 17, 6, and 4%, respectively. The other 16 bacterial OTUs represent 13% of the clones. They were either affiliated with narrow phyla such as Planctomyces-Chlamydia, green nonsulfur bacteria, or Synergistes, or deeply branched on the phylogenetic tree. A large number of bacterial OTUs are not closely related to any other hitherto determined sequences. The most frequent bacterial OTUs represents less than 5% of the total bacterial SSU rDNA sequences. However, the 20 more frequent bacterial OTUs describe at least 50% of these sequences. Three of the six Archaea OTUs correspond to 95% of the Archaea population and are very similar to already known methanogenic species: Methanosarcina barkeri, Methanosarcina frisius, and Methanobacterium formicicum. In contrast, the three other Archaea OTUs are unusual and are related to thermophilic microorganisms such as Crenarchaea or Thermoplasma spp. Five percent of the sequences analyzed were chimeras and were removed from the analysis.

  4. Multiple Group I Introns in the Small-Subunit rDNA of Botryosphaeria dothidea: Implication for Intraspecific Genetic Diversity

    PubMed Central

    Xu, Chao; Wang, Chunsheng; Sun, Xinyao; Zhang, Rong; Gleason, Mark L.; Eiji, Tanaka; Sun, Guangyu

    2013-01-01

    Botryosphaeria dothidea is a widespread and economically important pathogen on various fruit trees, and it often causes die-back and canker on limbs and fruit rot. In characterizing intraspecies genetic variation within this fungus, group I introns, rich in rDNA of fungi, may provide a productive region for exploration. In this research, we analysed complete small subunit (SSU) ribosomal DNA (rDNA) sequences of 37 B. dothidea strains, and found four insertions, designated Bdo.S943, Bdo.S1199-A, Bdo.S1199-B and Bdo.S1506, at three positions. Sequence analysis and structure prediction revealed that both Bdo.S943 and Bdo.S1506 belonged to subgroup IC1 of group I introns, whereas Bdo.S1199-A and Bdo.S1199-B corresponded to group IE introns. Moreover, Bdo.S1199-A was found to host an open reading frame (ORF) for encoding the homing endonuclease (HE), whereas Bdo.S1199-B, an evolutionary descendant of Bdo.S1199-A, included a degenerate HE. The above four introns were novel, and were the first group I introns observed and characterized in this species. Differential distribution of these introns revealed that all strains could be separated into four genotypes. Genotype III (no intron) and genotype IV (Bdo.S1199-B) were each found in only one strain, whereas genotype I (Bdo.S1199-A) and genotype II (Bdo.S943 and Bdo.S1506) occurred in 95% of the strains. There is a correlation between B. dothidea genotypes and hosts or geographic locations. Thus, these newly discovered group I introns can help to advance understanding of genetic differentiation within B. dothidea. PMID:23844098

  5. Multiple group I introns in the small-subunit rDNA of Botryosphaeria dothidea: implication for intraspecific genetic diversity.

    PubMed

    Xu, Chao; Wang, Chunsheng; Sun, Xinyao; Zhang, Rong; Gleason, Mark L; Eiji, Tanaka; Sun, Guangyu

    2013-01-01

    Botryosphaeria dothidea is a widespread and economically important pathogen on various fruit trees, and it often causes die-back and canker on limbs and fruit rot. In characterizing intraspecies genetic variation within this fungus, group I introns, rich in rDNA of fungi, may provide a productive region for exploration. In this research, we analysed complete small subunit (SSU) ribosomal DNA (rDNA) sequences of 37 B. dothidea strains, and found four insertions, designated Bdo.S943, Bdo.S1199-A, Bdo.S1199-B and Bdo.S1506, at three positions. Sequence analysis and structure prediction revealed that both Bdo.S943 and Bdo.S1506 belonged to subgroup IC1 of group I introns, whereas Bdo.S1199-A and Bdo.S1199-B corresponded to group IE introns. Moreover, Bdo.S1199-A was found to host an open reading frame (ORF) for encoding the homing endonuclease (HE), whereas Bdo.S1199-B, an evolutionary descendant of Bdo.S1199-A, included a degenerate HE. The above four introns were novel, and were the first group I introns observed and characterized in this species. Differential distribution of these introns revealed that all strains could be separated into four genotypes. Genotype III (no intron) and genotype IV (Bdo.S1199-B) were each found in only one strain, whereas genotype I (Bdo.S1199-A) and genotype II (Bdo.S943 and Bdo.S1506) occurred in 95% of the strains. There is a correlation between B. dothidea genotypes and hosts or geographic locations. Thus, these newly discovered group I introns can help to advance understanding of genetic differentiation within B. dothidea.

  6. Molecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis.

    PubMed Central

    Godon, J J; Zumstein, E; Dabert, P; Habouzit, F; Moletta, R

    1997-01-01

    The bacterial community structure of a fluidized-bed reactor fed by vinasses (wine distillation waste) was analyzed. After PCR amplification, four small-subunit (SSU) rDNA clone libraries of Bacteria, Archaea, Procarya, and Eucarya populations were established. The community structure was determined by operational taxonomic unit (OTU) phylogenetic analyses of 579 partial rDNA sequences (about 500 bp long). A total of 146 OTUs were found, comprising 133, 6, and 7 from the Bacteria, Archaea, and Eucarya domains, respectively. A total of 117 bacterial OTU were affiliated with major phyla: low-G+C gram-positive bacteria, Cytophaga-Flexibacter-Bacteroides, Proteobacteria, high-G+C gram-positive bacteria, and Spirochaetes, where the clone distribution was 34, 26, 17, 6, and 4%, respectively. The other 16 bacterial OTUs represent 13% of the clones. They were either affiliated with narrow phyla such as Planctomyces-Chlamydia, green nonsulfur bacteria, or Synergistes, or deeply branched on the phylogenetic tree. A large number of bacterial OTUs are not closely related to any other hitherto determined sequences. The most frequent bacterial OTUs represents less than 5% of the total bacterial SSU rDNA sequences. However, the 20 more frequent bacterial OTUs describe at least 50% of these sequences. Three of the six Archaea OTUs correspond to 95% of the Archaea population and are very similar to already known methanogenic species: Methanosarcina barkeri, Methanosarcina frisius, and Methanobacterium formicicum. In contrast, the three other Archaea OTUs are unusual and are related to thermophilic microorganisms such as Crenarchaea or Thermoplasma spp. Five percent of the sequences analyzed were chimeras and were removed from the analysis. PMID:9212428

  7. NRF-1, and AP-1 regulate the promoter of the human calpain small subunit 1 (CAPNS1) gene.

    PubMed

    Asangani, Irfan A; Rasheed, Suhail A K; Leupold, Jörg H; Post, Stefan; Allgayer, Heike

    2008-02-29

    Ubiquitously expressed micro- and m-calpain are cysteine proteases with broad functions in cell spreading, migration, proliferation, apoptosis, and in tumor invasion. They are heterodimers, with a distinct large 80-kDa catalytic, and a common small 28-kDa regulatory subunit (Capn4/CAPNS1). CAPNS1 is required to maintain stability and activity of both calpains. Despite its biological importance, the transcriptional regulation of this gene has not been studied, and the CAPNS1 promoter has not yet been characterized. In this study, we identified the main transcriptional start site, and cloned and characterized the ~2.0 kb upstream region of the CAPNS1 gene. Deletion analysis identified the core promoter located within region -187/+174. Site-directed mutagenesis, EMSA- and supershift analysis identified Sp1-, NRF-1-, and AP-1-binding elements within the CAPNS1 core promoter. Binding of NRF-1, Sp1 and AP-1 to the natural core promoter was confirmed by chromatin immunoprecipitation (ChIP). Site-directed mutagenesis at the NRF-1 site in HeLa and MCF7 cells substantially reduced core promoter activity by 70%, whereas mutation of the AP-1-binding and Sp1-binding site reduced promoter activity by 50% and 30%, respectively. Double mutation of the NRF-1 and the AP-1 site reduced promoter activity by 90%. In Drosophila SL2 cells, ectopic expression of NRF-1 led to a significant induction of CAPNS1 promoter activity. Furthermore, an siRNA against NRF-1 substantially reduced promoter activity in HeLa cells, which was paralleled by a significant downregulation of CAPNS1 mRNA. These results reveal that especially NRF-1, along with AP-1 and, to a minor extent, an Sp1 site, is essential for human CAPNS1 promoter activity and gene expression.

  8. Unexpected high digestion rate of cooked starch by the Ct-Maltase-Glucoamylase small intestine mucosal alpha-glucosidase subunit

    Technology Transfer Automated Retrieval System (TEKTRAN)

    For starch digestion to glucose, two luminal alpha-amylases and four gut mucosal alpha-glucosidase subunits are employed. The aim of this research was to investigate, for the first time, direct digestion capability of individual mucosal alpha-glucosidases on cooked (gelatinized) starch. Gelatinized ...

  9. 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.

  10. The fission yeast Cdc1 protein, a homologue of the small subunit of DNA polymerase delta, binds to Pol3 and Cdc27.

    PubMed Central

    MacNeill, S A; Moreno, S; Reynolds, N; Nurse, P; Fantes, P A

    1996-01-01

    cdc1+ is required for cell cycle progression in Schizosaccharomyces pombe. Cells carrying temperature-sensitive cdc1 mutants undergo cell cycle arrest when shifted to the restrictive temperature, becoming highly elongated. Here we describe the cloning and sequencing of cdc1+, which is shown to encode a 462 residue protein that displays significant sequence similarity to the small subunit of mammalian DNA polymerase delta. cdc1+ interacts genetically with pol3+, which encodes the large subunit of DNA polymerase delta in fission yeast, and the Cdc1 protein binds to Pol3 in vitro, strongly suggesting that Cdc1 is likely to be the small subunit of Pol delta. In addition, we show that cdc1+ overexpression is sufficient to rescue cells carrying temperature-sensitive cdc27 alleles and that the Cdc1 and Cdc27 proteins interact in vivo and in vitro. Deletion of either cdc1+ or cdc27+ results in cell cycle arrest with the arrested cells having a single nucleus with 2C DNA content. No evidence was obtained for a cut phenotype, indicating that neither cdc1+ nor cdc27+ is required for checkpoint function. cdc1 mutant cells are supersensitive to the DNA synthesis inhibitor hydroxyurea and to the DNA damaging agent MMS, display increased frequency of mini-chromosome loss and have an extended S phase. Images PMID:8887553

  11. Metabolism of 18S rRNA in rat liver cells in different functional states of protein-synthesizing apparatus

    SciTech Connect

    Chirkov, G.P.; Druzhinina, M.K.; Todorov, I.N.

    1986-04-10

    The ratio of the absolute radioactivities of 28S and 18S RNAs in the fractions of membrane-bound and free polysomes and the fraction of free rat liver ribosomes was studied under conditions of inhibition of translation by cycloheximide, insulin, and cAMP. It was found that insulin and cAMP, in contrast to cycloheximide, do not induce selective degradation of 18S rRNA. The results are discussed from the standpoint of the possible role of the phosphorylation of protein S6 in the degradation of the 40S ribosomal subunit.

  12. Nop9 is a PUF-like protein that prevents premature cleavage to correctly process pre-18S rRNA

    PubMed Central

    Zhang, Jun; McCann, Kathleen L.; Qiu, Chen; Gonzalez, Lauren E.; Baserga, Susan J.; Hall, Traci M. Tanaka

    2016-01-01

    Numerous factors direct eukaryotic ribosome biogenesis, and defects in a single ribosome assembly factor may be lethal or produce tissue-specific human ribosomopathies. Pre-ribosomal RNAs (pre-rRNAs) must be processed stepwise and at the correct subcellular locations to produce the mature rRNAs. Nop9 is a conserved small ribosomal subunit biogenesis factor, essential in yeast. Here we report a 2.1-Å crystal structure of Nop9 and a small-angle X-ray-scattering model of a Nop9:RNA complex that reveals a ‘C'-shaped fold formed from 11 Pumilio repeats. We show that Nop9 recognizes sequence and structural features of the 20S pre-rRNA near the cleavage site of the nuclease, Nob1. We further demonstrate that Nop9 inhibits Nob1 cleavage, the final processing step to produce mature small ribosomal subunit 18S rRNA. Together, our results suggest that Nop9 is critical for timely cleavage of the 20S pre-rRNA. Moreover, the Nop9 structure exemplifies a new class of Pumilio repeat proteins. PMID:27725644

  13. Nop9 is a PUF-like protein that prevents premature cleavage to correctly process pre-18S rRNA

    SciTech Connect

    Zhang, Jun; McCann, Kathleen L.; Qiu, Chen; Gonzalez, Lauren E.; Baserga, Susan J.; Hall, Traci M. Tanaka

    2016-10-11

    Numerous factors direct eukaryotic ribosome biogenesis, and defects in a single ribosome assembly factor may be lethal or produce tissue-specific human ribosomopathies. Pre-ribosomal RNAs (pre-rRNAs) must be processed stepwise and at the correct subcellular locations to produce the mature rRNAs. Nop9 is a conserved small ribosomal subunit biogenesis factor, essential in yeast. Here we report a 2.1-Å crystal structure of Nop9 and a small-angle X-ray-scattering model of a Nop9:RNA complex that reveals a ‘C’-shaped fold formed from 11 Pumilio repeats. We show that Nop9 recognizes sequence and structural features of the 20S pre-rRNA near the cleavage site of the nuclease, Nob1. We further demonstrate that Nop9 inhibits Nob1 cleavage, the final processing step to produce mature small ribosomal subunit 18S rRNA. Together, our results suggest that Nop9 is critical for timely cleavage of the 20S pre-rRNA. Moreover, the Nop9 structure exemplifies a new class of Pumilio repeat proteins.

  14. Natural-abundance stable carbon isotopes of small-subunit ribosomal RNA (SSU rRNA) from Guaymas Basin (Mexico)

    NASA Astrophysics Data System (ADS)

    MacGregor, B. J.; Mendlovitz, H.; Albert, D.; Teske, A. P.

    2012-12-01

    Small-subunit ribosomal RNA (SSU rRNA) is a phylogenetically informative molecule found in all species. Because it is poorly preserved in most environments, it is a useful marker for active microbial populations. We are using the natural-abundance stable carbon isotopic composition of specific microbial groups to help identify the carbon substrates contributing to microbial biomass in a variety of marine environments. At Guaymas Basin, hydrothermal fluids interact with abundant sedimentary organic carbon to produce natural gas and petroleum. Where this reaches the sediment surface, it can support dense patches of seafloor life, including Beggiatoa mats. We report here on the stable carbon isotopic composition of SSU rRNA from a Beggiatoa mat transect, a cold background site, a warm site with high oil concentration, and a second Beggiatoa mat. The central part of the transect mat overlay the steepest temperature gradient, and was visually dominated by orange Beggiatoa. This was fringed by white Beggiatoa mat and bare, but still warm, sediment. Methane concentrations were saturating beneath the orange and white mats and at the oily site, lower beneath bare sediment, and below detection at the background site. Our initial hypotheses were that rRNA isotopic composition would be strongly influenced by methane supply, and that archaeal rRNA might be lighter than bacterial due to contributions from methanogens and anaerobic methane oxidizers. We used biotin-labeled oligonucleotides to capture Bacterial and Archaeal SSU rRNA for isotopic determination. Background-site rRNA was isotopically heaviest, and bacterial RNA from below 2 cm at the oily site was lightest, consistent with control by methane. Within the transect mat, however, the pattern was more complicated; at some sediment depths, rRNA from the mat periphery was isotopically lightest. Part of this may be due to the spatially and temporally variable paths followed by hydrothermal fluid, which can include horizontal

  15. Molecular genetic analysis of the heterodimeric splicing factor U2AF: the RS domain on either the large or small Drosophila subunit is dispensable in vivo

    PubMed Central

    Rudner, David Z.; Breger, Kevin S.; Rio, Donald C.

    1998-01-01

    The pre-mRNA splicing factor U2AF (U2 snRNP auxiliary factor) has an essential role in 3′ splice site selection. U2AF binds the intron pyrimidine tract between the branchpoint and the 3′ splice site and recruits U2 snRNP to the branch site at an early step in spliceosome assembly. Human U2AF is a heterodimer composed of large (hU2AF65) and small (hU2AF35) subunits. Both subunits contain a domain enriched in arginine–serine dipeptide repeats termed an RS domain. The two U2AF RS domains have been assigned essential and independent roles in spliceosome assembly in vitro—the hU2AF65 RS domain is required to target U2 snRNP to the branch site and the hU2AF35 RS domain is necessary for protein–protein interactions with constitutive and alternative splicing factors. We have investigated the functional requirements for the RS domains on the Drosophila U2AF homolog in vivo. In sharp contrast to its essential role in U2 snRNP recruitment in vitro, the RS domain on the Drosophila large subunit homolog (dU2AF50) was completely dispensable in vivo. Prompted by this unexpected result, we analyzed the RS domain on the Drosophila small subunit homolog (dU2AF38). Despite its requirement for enhancer-dependent splicing activity in vitro, the dU2AF38 RS domain was also inessential in vivo. Finally, we have tested whether the Drosophila U2AF heterodimer requires any RS domain. Flies mutant for both the small and large subunits could not be rescued by dU2AF50ΔRS and dU2AF38ΔRS transgenes. Therefore, in contrast to the separate roles assigned to the U2AF RS domains in vitro, our genetic data suggest that they may have redundant functions in vivo. PMID:9531538

  16. A small novel A-kinase anchoring protein (AKAP) that localizes specifically protein kinase A-regulatory subunit I (PKA-RI) to the plasma membrane.

    PubMed

    Burgers, Pepijn P; Ma, Yuliang; Margarucci, Luigi; Mackey, Mason; van der Heyden, Marcel A G; Ellisman, Mark; Scholten, Arjen; Taylor, Susan S; Heck, Albert J R

    2012-12-21

    Protein kinase A-anchoring proteins (AKAPs) provide spatio-temporal specificity for the omnipotent cAMP-dependent protein kinase (PKA) via high affinity interactions with PKA regulatory subunits (PKA-RI, RII). Many PKA-RII-AKAP complexes are heavily tethered to cellular substructures, whereas PKA-RI-AKAP complexes have remained largely undiscovered. Here, using a cAMP affinity-based chemical proteomics strategy in human heart and platelets, we uncovered a novel, ubiquitously expressed AKAP, termed small membrane (sm)AKAP due to its specific localization at the plasma membrane via potential myristoylation/palmitoylation anchors. In vitro binding studies revealed specificity of smAKAP for PKA-RI (K(d) = 7 nM) over PKA-RII (K(d) = 53 nM) subunits, co-expression of smAKAP with the four PKA R subunits revealed an even more exclusive specificity of smAKAP for PKA-RIα/β in the cellular context. Applying the singlet oxygen-generating electron microscopy probe miniSOG indicated that smAKAP is tethered to the plasma membrane and is particularly dense at cell-cell junctions and within filopodia. Our preliminary functional characterization of smAKAP provides evidence that, like PKA-RII, PKA-RI can be tightly tethered by a novel repertoire of AKAPs, providing a new perspective on spatio-temporal control of cAMP signaling.

  17. Exploring the potential of small RNA subunit and ITS sequences for resolving phylogenetic relationships within the phylum Ctenophora.

    PubMed

    Simion, Paul; Bekkouche, Nicolas; Jager, Muriel; Quéinnec, Eric; Manuel, Michaël

    2015-04-01

    Ctenophores are a phylum of non-bilaterian marine (mostly planktonic) animals, characterised by several unique synapomorphies (e.g., comb rows, apical organ). Relationships between and within the nine recognised ctenophore orders are far from understood, notably due to a paucity of phylogenetically informative anatomical characters. Previous attempts to address ctenophore phylogeny using molecular data (18S rRNA) led to poorly resolved trees but demonstrated the paraphyly of the order Cydippida. Here we compiled an updated 18S rRNA data set, notably including a few newly sequenced species representing previously unsampled families (Lampeidae, Euryhamphaeidae), and we constructed an additional more rapidly evolving ITS1 + 5.8S rRNA + ITS2 alignment. These data sets were analysed separately and in combination under a probabilistic framework, using different methods (maximum likelihood, Bayesian inference) and models (e.g., doublet model to accommodate secondary structure; data partitioning). An important lesson from our exploration of these datasets is that the fast-evolving internal transcribed spacer (ITS) regions are useful markers for reconstructing high-level relationships within ctenophores. Our results confirm the paraphyly of the order Cydippida (and thus a "cydippid-like" ctenophore common ancestor) and suggest that the family Mertensiidae could be the sister group of all other ctenophores. The family Lampeidae (also part of the former "Cydippida") is probably the sister group of the order Platyctenida (benthic ctenophores). The order Beroida might not be monophyletic, due to the position of Beroe abyssicola outside of a clade grouping the other Beroe species and members of the "Cydippida" family Haeckeliidae. Many relationships (e.g. between Pleurobrachiidae, Beroida, Cestida, Lobata, Thalassocalycida) remain unresolved. Future progress in understanding ctenophore phylogeny will come from the use of additional rapidly evolving markers and improvement of

  18. Ribosomal 18S rRNA base pairs with mRNA during eukaryotic translation initiation

    PubMed Central

    Martin, Franck; Ménétret, Jean-François; Simonetti, Angelita; Myasnikov, Alexander G.; Vicens, Quentin; Prongidi-Fix, Lydia; Natchiar, S. Kundhavai; Klaholz, Bruno P.; Eriani, Gilbert

    2016-01-01

    Eukaryotic mRNAs often contain a Kozak sequence that helps tether the ribosome to the AUG start codon. The mRNA of histone H4 (h4) does not undergo classical ribosome scanning but has evolved a specific tethering mechanism. The cryo-EM structure of the rabbit ribosome complex with mouse h4 shows that the mRNA forms a folded, repressive structure at the mRNA entry site on the 40S subunit next to the tip of helix 16 of 18S ribosomal RNA (rRNA). Toe-printing and mutational assays reveal that an interaction exists between a purine-rich sequence in h4 mRNA and a complementary UUUC sequence of helix h16. Together the present data establish that the h4 mRNA harbours a sequence complementary to an 18S rRNA sequence which tethers the mRNA to the ribosome to promote proper start codon positioning, complementing the interactions of the 40S subunit with the Kozak sequence that flanks the AUG start codon. PMID:27554013

  19. Rubisco oligomers composed of linked small and large subunits assemble in tobacco plastids and have higher affinities for CO2 and O2.

    PubMed

    Whitney, Spencer Michael; Kane, Heather Jean; Houtz, Robert L; Sharwood, Robert Edward

    2009-04-01

    Manipulation of Rubisco within higher plants is complicated by the different genomic locations of the large (L; rbcL) and small (S; RbcS) subunit genes. Although rbcL can be accurately modified by plastome transformation, directed genetic manipulation of the multiple nuclear-encoded RbcS genes is more challenging. Here we demonstrate the viability of linking the S and L subunits of tobacco (Nicotiana tabacum) Rubisco using a flexible 40-amino acid tether. By replacing the rbcL in tobacco plastids with an artificial gene coding for a S40L fusion peptide, we found that the fusions readily assemble into catalytic (S40L)8 and (S40L)16 oligomers that are devoid of unlinked S subunits. While there was little or no change in CO2/O2 specificity or carboxylation rate of the Rubisco oligomers, their Kms for CO2 and O2 were reduced 10% to 20% and 45%, respectively. In young maturing leaves of the plastome transformants (called ANtS40L), the S40L-Rubisco levels were approximately 20% that of wild-type controls despite turnover of the S40L-Rubisco oligomers being only slightly enhanced relative to wild type. The reduced Rubisco content in ANtS40L leaves is partly attributed to problems with folding and assembly of the S40L peptides in tobacco plastids that relegate approximately 30% to 50% of the S40L pool to the insoluble protein fraction. Leaf CO2-assimilation rates in ANtS40L at varying pCO2 corresponded with the kinetics and reduced content of the Rubisco oligomers. This fusion strategy provides a novel platform to begin simultaneously engineering Rubisco L and S subunits in tobacco plastids.

  20. 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.

  1. Identification of Theileria parva and Theileria sp. (buffalo) 18S rRNA gene sequence variants in the African Buffalo (Syncerus caffer) in southern Africa.

    PubMed

    Chaisi, Mamohale E; Sibeko, Kgomotso P; Collins, Nicola E; Potgieter, Fred T; Oosthuizen, Marinda C

    2011-12-15

    Theileria parva is the causative agent of Corridor disease in cattle in South Africa. The African buffalo (Syncerus caffer) is the reservoir host, and, as these animals are important for eco-tourism in South Africa, it is compulsory to test and certify them disease free prior to translocation. A T. parva-specific real-time polymerase chain reaction (PCR) test based on the small subunit ribosomal RNA (18S rRNA) gene is one of the tests used for the diagnosis of the parasite in buffalo and cattle in South Africa. However, because of the high similarity between the 18S rRNA gene sequences of T. parva and Theileria sp. (buffalo), the latter is also amplified by the real-time PCR primers, although it is not detected by the T. parva-specific hybridization probes. Preliminary sequencing studies have revealed a small number of sequence differences within the 18S rRNA gene in both species but the extent of this sequence variation is unknown. The aim of the current study was to sequence the 18S rRNA genes of T. parva and Theileria sp. (buffalo), and to determine whether all identified genotypes can be correctly detected by the real-time PCR assay. The reverse line blot (RLB) hybridization assay was used to identify T. parva and Theileria sp. (buffalo) positive samples from buffalo blood samples originating from the Kruger National Park, Hluhluwe-iMfolozi Park, the Greater Limpopo Transfrontier Park, and a private game ranch in the Hoedspruit area. T. parva and Theileria sp. (buffalo) were identified in 42% and 28%, respectively, of 252 samples, mainly as mixed infections. The full-length 18S rRNA gene of selected samples was amplified, cloned and sequenced. From a total of 20 sequences obtained, 10 grouped with previously published T. parva sequences from GenBank while 10 sequences grouped with a previously published Theileria sp. (buffalo) sequence. All these formed a monophyletic group with known pathogenic Theileria species. Our phylogenetic analyses confirm the

  2. Retrovirus Restriction by TRIM5 Proteins Requires Recognition of Only a Small Fraction of Viral Capsid Subunits

    PubMed Central

    Shi, Jiong; Friedman, David B.

    2013-01-01

    The host restriction factors TRIM5α and TRIMCyp potently inhibit retrovirus infection by binding to the incoming retrovirus capsid. TRIM5 proteins are dimeric, and their association with the viral capsid appears to be enhanced by avidity effects owing to formation of higher-order oligomeric complexes. We examined the stoichiometric requirement for TRIM5 functional recognition by quantifying the efficiencies of restriction of HIV-1 and murine leukemia virus (MLV) particles containing various proportions of restriction-sensitive and -insensitive CA subunits. Both TRIMCyp and TRIM5α inhibited infection of retrovirus particles containing as little as 25% of the restriction-sensitive CA protein. Accordingly, we also observed efficient binding of TRIMCyp in vitro to capsid assemblies containing as little as one-fourth wild-type CA protein. Paradoxically, the ability of HIV-1 particles to abrogate TRIMCyp restriction in trans was more strongly dependent on the fraction of wild-type CA than was restriction of infection. Collectively, our results indicate that TRIM5 restriction factors bind to retroviral capsids in a highly cooperative manner and suggest that TRIM5 can engage a capsid lattice containing a minimum of three or fewer recognizable subunits per hexamer. Our study supports a model in which localized binding of TRIM5 to the viral capsid nucleates rapid polymerization of a TRIM5 lattice on the capsid surface. PMID:23785198

  3. Phylogenetic analysis and the evolution of the 18S rRNA gene typing system of Acanthamoeba.

    PubMed

    Fuerst, Paul A; Booton, Gregory C; Crary, Monica

    2015-01-01

    Species of Acanthamoeba were first described using morphological characters including cyst structure and cytology of nuclear division. More than 20 nominal species were proposed using these methods. Morphology, especially cyst shape and size, has proven to be plastic and dependent upon culture conditions. The DNA sequence of the nuclear small-subunit (18S) rRNA, the Rns gene, has become the most widely accepted method for rapid diagnosis and classification of Acanthamoeba. The Byers-Fuerst lab first proposed an Rns typing system in 1996. Subsequent refinements, with an increasing DNA database and analysis of diagnostic fragments within the gene, have become widely accepted by the Acanthamoeba research community. The development of the typing system, including its current state of implementation is illustrated by three cases: (i) the division between sequence types T13 and T16; (ii) the diversity within sequence supertype T2/T6, and (iii) verification of a new sequence type, designated T20. Molecular studies make clear the disconnection between phylogenetic relatedness and species names, as applied for the genus Acanthamoeba. Future reconciliation of genetic types with species names must become a priority, but the possible shortcomings of the use of a single gene when reconstructing the evolutionary history of the acanthamoebidae must also be resolved.

  4. Comparison of potential diatom 'barcode' genes (the 18S rRNA gene and ITS, COI, rbcL) and their effectiveness in discriminating and determining species taxonomy in the Bacillariophyta.

    PubMed

    Guo, Liliang; Sui, Zhenghong; Zhang, Shu; Ren, Yuanyuan; Liu, Yuan

    2015-04-01

    Diatoms form an enormous group of photoautotrophic micro-eukaryotes and play a crucial role in marine ecology. In this study, we evaluated typical genes to determine whether they were effective at different levels of diatom clustering analysis to assess the potential of these regions for barcoding taxa. Our test genes included nuclear rRNA genes (the nuclear small-subunit rRNA gene and the 5.8S rRNA gene+ITS-2), a mitochondrial gene (cytochrome c-oxidase subunit 1, COI), a chloroplast gene [ribulose-1,5-biphosphate carboxylase/oxygenase large subunit (rbcL)] and the universal plastid amplicon (UPA). Calculated genetic divergence was highest for the internal transcribed spacer (ITS; 5.8S+ITS-2) (p-distance of 1.569, 85.84% parsimony-informative sites) and COI (6.084, 82.14%), followed by the 18S rRNA gene (0.139, 57.69%), rbcL (0.120, 42.01%) and UPA (0.050, 14.97%), which indicated that ITS and COI were highly divergent compared with the other tested genes, and that their nucleotide compositions were variable within the whole group of diatoms. Bayesian inference (BI) analysis showed that the phylogenetic trees generated from each gene clustered diatoms at different phylogenetic levels. The 18S rRNA gene was better than the other genes in clustering higher diatom taxa, and both the 18S rRNA gene and rbcL performed well in clustering some lower taxa. The COI region was able to barcode species of some genera within the Bacillariophyceae. ITS was a potential marker for DNA based-taxonomy and DNA barcoding of Thalassiosirales, while species of Cyclotella, Skeletonema and Stephanodiscus gathered in separate clades, and were paraphyletic with those of Thalassiosira. Finally, UPA was too conserved to serve as a diatom barcode.

  5. Structural and functional studies of Bud23-Trm112 reveal 18S rRNA N7-G1575 methylation occurs on late 40S precursor ribosomes.

    PubMed

    Létoquart, Juliette; Huvelle, Emmeline; Wacheul, Ludivine; Bourgeois, Gabrielle; Zorbas, Christiane; Graille, Marc; Heurgué-Hamard, Valérie; Lafontaine, Denis L J

    2014-12-23

    The eukaryotic small ribosomal subunit carries only four ribosomal (r) RNA methylated bases, all close to important functional sites. N(7)-methylguanosine (m(7)G) introduced at position 1575 on 18S rRNA by Bud23-Trm112 is at a ridge forming a steric block between P- and E-site tRNAs. Here we report atomic resolution structures of Bud23-Trm112 in the apo and S-adenosyl-L-methionine (SAM)-bound forms. Bud23 and Trm112 interact through formation of a β-zipper involving main-chain atoms, burying an important hydrophobic surface and stabilizing the complex. The structures revealed that the coactivator Trm112 undergoes an induced fit to accommodate its methyltransferase (MTase) partner. We report important structural similarity between the active sites of Bud23 and Coffea canephora xanthosine MTase, leading us to propose and validate experimentally a model for G1575 coordination. We identify Bud23 residues important for Bud23-Trm112 complex formation and recruitment to pre-ribosomes. We report that though Bud23-Trm112 binds precursor ribosomes at an early nucleolar stage, m(7)G methylation occurs at a late step of small subunit biogenesis, implying specifically delayed catalytic activation. Finally, we show that Bud23-Trm112 interacts directly with the box C/D snoRNA U3-associated DEAH RNA helicase Dhr1 supposedly involved in central pseudoknot formation; this suggests that Bud23-Trm112 might also contribute to controlling formation of this irreversible and dramatic structural reorganization essential to overall folding of small subunit rRNA. Our study contributes important new elements to our understanding of key molecular aspects of human ribosomopathy syndromes associated with WBSCR22 (human Bud23) malfunction.

  6. The utility of diversity profiling using Illumina 18S rRNA gene amplicon deep sequencing to detect and discriminate Toxoplasma gondii among the cyst-forming coccidia.

    PubMed

    Cooper, Madalyn K; Phalen, David N; Donahoe, Shannon L; Rose, Karrie; Šlapeta, Jan

    2016-01-30

    Next-generation sequencing (NGS) has the capacity to screen a single DNA sample and detect pathogen DNA from thousands of host DNA sequence reads, making it a versatile and informative tool for investigation of pathogens in diseased animals. The technique is effective and labor saving in the initial identification of pathogens, and will complement conventional diagnostic tests to associate the candidate pathogen with a disease process. In this report, we investigated the utility of the diversity profiling NGS approach using Illumina small subunit ribosomal RNA (18S rRNA) gene amplicon deep sequencing to detect Toxoplasma gondii in previously confirmed cases of toxoplasmosis. We then tested the diagnostic approach with species-specific PCR genotyping, histopathology and immunohistochemistry of toxoplasmosis in a Risso's dolphin (Grampus griseus) to systematically characterise the disease and associate causality. We show that the Euk7A/Euk570R primer set targeting the V1-V3 hypervariable region of the 18S rRNA gene can be used as a species-specific assay for cyst-forming coccidia and discriminate T. gondii. Overall, the approach is cost-effective and improves diagnostic decision support by narrowing the differential diagnosis list with more certainty than was previously possible. Furthermore, it supplements the limitations of cryptic protozoan morphology and surpasses the need for species-specific PCR primer combinations.

  7. Nematode 18S rRNA gene is a reliable tool for environmental biosafety assessment of transgenic banana in confined field trials.

    PubMed

    Nakacwa, R; Kiggundu, A; Talwana, H; Namaganda, J; Lilley, C; Tushemereirwe, W; Atkinson, H

    2013-10-01

    Information on relatedness in nematodes is commonly obtained by DNA sequencing of the ribosomal internal transcribed spacer region. However, the level of diversity at this locus is often insufficient for reliable species differentiation. Recent findings suggest that the sequences of a fragment of the small subunit nuclear ribosomal DNA (18S rRNA or SSU), identify genera of soil nematodes and can also distinguish between species in some cases. A database of soil nematode genera in a Ugandan soil was developed using 18S rRNA sequences of individual nematodes from a GM banana confined field trial site at the National Agricultural Research Laboratories, Kawanda in Uganda. The trial was planted to evaluate transgenic bananas for resistance to black Sigatoka disease. Search for relatedness of the sequences gained with entries in a public genomic database identified a range of 20 different genera and sometimes distinguished species. Molecular markers were designed from the sequence information to underpin nematode faunal analysis. This approach provides bio-indicators for disturbance of the soil environment and the condition of the soil food web. It is being developed to support environmental biosafety analysis by detecting any perturbance by transgenic banana or other GM crops on the soil environment.

  8. Analysis of U3 snoRNA and small subunit processome components in the parasitic protist Entamoeba histolytica.

    PubMed

    Srivastava, Ankita; Ahamad, Jamaluddin; Ray, Ashwini Kumar; Kaur, Devinder; Bhattacharya, Alok; Bhattacharya, Sudha

    2014-02-01

    In the early branching parasitic protist Entamoeba histolytica, pre-rRNA synthesis continues when cells are subjected to growth stress, but processing slows down and unprocessed pre-rRNA accumulates. To gain insight into the regulatory mechanisms leading to accumulation, it is necessary to define the pre-rRNA processing machinery in E. histolytica. We searched the E. histolytica genome sequence for homologs of the SSU processome, which contains the U3snoRNA, and 72 proteins in yeast. We could identify 57 of the proteins with high confidence. Of the rest, 6 were absent in human, and 4 were non-essential in yeast. The remaining 5 were absent in other parasite genomes as well. Analysis of U3snoRNA showed that the E. histolytica U3snoRNA adopted the same conserved secondary structure as seen in yeast and human. The predicted structure was verified by chemical modification followed by primer extension (SHAPE). Further we showed that the predicted interactions of Eh_U3snoRNA boxes A and A' with pre-18S rRNA were highly conserved both in position and sequence. The predicted interactions of 5'-hinge and 3'-hinge sequences of Eh_U3 snoRNA with the 5'-ETS sequences were conserved in position but not in sequence. Transcription of selected genes of SSU processome was tested by northern analysis, and transcripts of predicted sizes were obtained. During serum starvation, when unprocessed pre-RNA accumulated, the transcript levels of some of these genes declined. This is the first report on pre-rRNA processing machinery in E. histolytica, and shows that the components are well conserved with respect to yeast and human.

  9. The Small Subunit of Snapdragon Geranyl Diphosphate Synthase Modifies the Chain Length Specificity of Tobacco Geranylgeranyl Diphosphate Synthase in Planta[W

    PubMed Central

    Orlova, Irina; Nagegowda, Dinesh A.; Kish, Christine M.; Gutensohn, Michael; Maeda, Hiroshi; Varbanova, Marina; Fridman, Eyal; Yamaguchi, Shinjiro; Hanada, Atsushi; Kamiya, Yuji; Krichevsky, Alexander; Citovsky, Vitaly; Pichersky, Eran; Dudareva, Natalia

    2009-01-01

    Geranyl diphosphate (GPP), the precursor of many monoterpene end products, is synthesized in plastids by a condensation of dimethylallyl diphosphate and isopentenyl diphosphate (IPP) in a reaction catalyzed by homodimeric or heterodimeric GPP synthase (GPPS). In the heterodimeric enzymes, a noncatalytic small subunit (GPPS.SSU) determines the product specificity of the catalytic large subunit, which may be either an active geranylgeranyl diphosphate synthase (GGPPS) or an inactive GGPPS-like protein. Here, we show that expression of snapdragon (Antirrhinum majus) GPPS.SSU in tobacco (Nicotiana tabacum) plants increased the total GPPS activity and monoterpene emission from leaves and flowers, indicating that the introduced catalytically inactive GPPS.SSU found endogenous large subunit partner(s) and formed an active snapdragon/tobacco GPPS in planta. Bimolecular fluorescence complementation and in vitro enzyme analysis of individual and hybrid proteins revealed that two of four GGPPS-like candidates from tobacco EST databases encode bona fide GGPPS that can interact with snapdragon GPPS.SSU and form a functional GPPS enzyme in plastids. The formation of chimeric GPPS in transgenic plants also resulted in leaf chlorosis, increased light sensitivity, and dwarfism due to decreased levels of chlorophylls, carotenoids, and gibberellins. In addition, these transgenic plants had reduced levels of sesquiterpene emission, suggesting that the export of isoprenoid intermediates from the plastids into the cytosol was decreased. These results provide genetic evidence that GPPS.SSU modifies the chain length specificity of phylogenetically distant GGPPS and can modulate IPP flux distribution between GPP and GGPP synthesis in planta. PMID:20028839

  10. Two F-18s in Autonomous Formation Flight

    NASA Technical Reports Server (NTRS)

    2001-01-01

    This 32 second video clip shows two F-18s in NASA's Autonomous Formation Flight (AFF) program. The aircraft use smoke contrails to gather data on wingtip vortices. Flight research attempts to utilize the energy in the vortices for more efficient flight.

  11. Cloning and characterization of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) cDNA from green microalga Ankistrodesmus convolutus.

    PubMed

    Thanh, Tran; Chi, Vu Thi Quynh; Abdullah, Mohd Puad; Omar, Hishamuddin; Noroozi, Mostafa; Napis, Suhaimi

    2011-11-01

    An initial study on gene cloning and characterization of unicellular green microalga Ankistrodesmus convolutus was carried out to isolate and characterize the full-length cDNA of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) as a first step towards elucidating the structure of A. convolutus RbcS gene. The full-length of A. convolutus RbcS cDNA (AcRbcS) contained 28 bp of 5' untranslated region (UTR), 225 bp of 3' non-coding region, and an open reading frame of 165 amino acids consisting of a chloroplast transit peptide with 24 amino acids and a mature protein of 141 amino acids. The amino acid sequence has high identity to those of other green algae RbcS genes. The AcRbcS contained a few conserved domains including protein kinase C phosphorylation site, tyrosine kinase phosphorylation site and N-myristoylation sites. The AcRbcS was successfully expressed in Escherichia coli and a ~21 kDa of anticipated protein band was observed on SDS-PAGE. From the phylogenetic analysis of RbcS protein sequences, it was found that the RbcS of A. convolutus has closer genetic relationship with green microalgae species compared to those of green seaweed and green macroalgae species. Southern hybridization analysis revealed that the AcRbcS is a member of a small multigene family comprising of two to six members in A. convolutus genome. Under different illumination conditions, RT-PCR analysis showed that AcRbcS transcription was reduced in the dark, and drastically recovered in the light condition. Results presented in this paper established a good foundation for further study on the photosynthetic process of A. convolutus and other green algae species where little information is known on Rubisco small subunit.

  12. Phylogeny of organisms investigated by the base-pair changes in the stem regions of small and large ribosomal subunit RNAs.

    PubMed

    Otsuka, J; Terai, G; Nakano, T

    1999-02-01

    In order to obtain the evolutionary distance data that are as purely additive as possible, we have developed a novel method for evaluating the evolutionary distances from the base-pair changes in stem regions of ribosomal RNAs (rRNAs). The application of this method to small-subunit (SSU) and large-subunit (LSU) rRNAs provides the distance data, with which both the unweighted pair group method of analysis and the neighbor-joining method give almost the same tree topology of most organisms except for some Protoctista, thermophilic bacteria, parasitic organisms, and endosymbionts. Although the evolutionary distances calculated with LSU rRNAs are somewhat longer than those with SSU rRNAs, the difference, probably due to a slight difference in functional constraint, is substantially decreased when the distances are converted into the divergence times of organisms by the measure of the time scale estimated in each type of rRNAs. The divergence times of main branches agree fairly well with the geological record of organisms, at least after the appearance of oxygen-releasing photosynthesis, although the divergence times of Eukaryota, Archaebacteria, and Eubacteria are somewhat overestimated in comparison with the geological record of Earth formation. This result is explained by considering that the mutation rate is determined by the accumulation of misrepairs for DNA damage caused by radiation and that the effect of radiation had been stronger before the oxygen molecules became abundant in the atmosphere of the Earth.

  13. Discrimination between Gyrodactylus salaris, G. derjavini and G. truttae (Platyhelminthes: Monogenea) using restriction fragment length polymorphisms and an oligonucleotide probe within the small subunit ribosomal RNA gene.

    PubMed

    Cunningham, C O; McGillivray, D M; MacKenzie, K; Melvin, W T

    1995-07-01

    The small subunit ribosomal RNA (srRNA) gene was amplified from Gyrodactylus salaris using the polymerase chain reaction (PCR), cloned, and the complete gene sequence of 1966 bp determined. The V4 region of the srRNA gene was identified and amplified from single specimens of G. salaris, G. derjavini and G. truttae. Comparison of the V4 sequences from these three species revealed sequence differences from which restriction fragment length polymorphisms (RFLPs) were predicted and an oligonucleotide probe (GsV4) specific to G. salaris designed. Digestion of the amplified V4 region of the srRNA gene with Hae III and either Alw I, BstY I, Dde I or Mbo I provided a means of discriminating between G. salaris, G. derjavini and G. truttae. The GsV4 probe was used to detect the srRNA gene from G. salaris in Southern and dot blots of the amplified V4 region.

  14. Cryptosporidium is more closely related to the gregarines than to coccidia as shown by phylogenetic analysis of apicomplexan parasites inferred using small-subunit ribosomal RNA gene sequences.

    PubMed

    Carreno, R A; Martin, D S; Barta, J R

    1999-11-01

    The phylogenetic placement of gregarine parasites (Apicomplexa: Gregarinasina) within the Apicomplexa was derived by comparison of small-subunit ribosomal RNA gene sequences. Gregarine sequences were obtained from Gregarina niphandrodes Clopton, Percival, and Janovy, 1991, and Monocystis agilis Stein, 1848 (Eugregarinorida Léger 1900), as well as from Ophriocystis elektroscirrha McLaughlin and Myers, 1970 (Neogregarinorida Grassé 1953). The sequences were aligned with several other gregarine and apicomplexan sequences from GenBank and the resulting data matrix analyzed by parsimony and maximum-likelihood methods. The gregarines form a monophyletic clade that is a sister group to Cryptosporidium spp. The gregarine/ Cryptosporidium clade is separate from the other major apicomplexan clade containing the coccidia, adeleids, piroplasms, and haemosporinids. The trees indicate that the genus Cryptosporidium has a closer phylogenetic affinity with the gregarines than with the coccidia. These results do not support the present classification of the Cryptosporidiidae in the suborder Eimerioirina Léger, 1911.

  15. The reduction in small ribosomal subunit abundance in ethanol-stressed cells of Bacillus subtilis is mediated by a SigB-dependent antisense RNA.

    PubMed

    Mars, Ruben A T; Mendonça, Karoline; Denham, Emma L; van Dijl, Jan Maarten

    2015-10-01

    One of the best-characterized general stress responses in bacteria is the σB-mediated stress response of the Gram-positive soil bacterium Bacillus subtilis. The σB regulon contains approximately 200 protein-encoding genes and 136 putative regulatory RNAs. One of these σB-dependent RNAs, named S1136-S1134, was recently mapped as being transcribed from the S1136 promoter on the opposite strand of the essential rpsD gene, which encodes the ribosomal primary-binding protein S4. Accordingly, S1136-S1134 transcription results in an rpsD-overlapping antisense RNA (asRNA). Upon exposure of B. subtilis to ethanol, the S1136 promoter was found to be induced, while rpsD transcription was downregulated. By quantitative PCR, we show that the activation of transcription from the S1136 promoter is directly responsible for the downregulation of rpsD upon ethanol exposure. We also show that this downregulation of rpsD leads to a reduced level of the small (30S) ribosomal subunit upon ethanol stress. The activation of the S1136 promoter thus represents the first example of antisense transcription-mediated regulation in the general stress response of B. subtilis and implicates the reduction of ribosomal protein abundance as a new aspect in the σB-dependent stress response. We propose that the observed reduction in the level of the small ribosomal subunit, which contains the ribosome-decoding center, may protect B. subtilis cells against misreading and spurious translation of possibly toxic aberrant peptides under conditions of ethanol stress.

  16. Rice Stripe Tenuivirus Nonstructural Protein 3 Hijacks the 26S Proteasome of the Small Brown Planthopper via Direct Interaction with Regulatory Particle Non-ATPase Subunit 3

    PubMed Central

    Xu, Yi; Wu, Jianxiang; Fu, Shuai; Li, Chenyang; Zhu, Zeng-Rong

    2015-01-01

    ABSTRACT The ubiquitin/26S proteasome system plays a vital role in regulating host defenses against pathogens. Previous studies have highlighted different roles for the ubiquitin/26S proteasome in defense during virus infection in both mammals and plants, but their role in the vectors that transmit those viruses is still unclear. In this study, we determined that the 26S proteasome is present in the small brown planthopper (SBPH) (Laodelphgax striatellus) and has components similar to those in plants and mammals. There was an increase in the accumulation of Rice stripe virus (RSV) in the transmitting vector SBPH after disrupting the 26S proteasome, indicating that the SBPH 26S proteasome plays a role in defense against RSV infection by regulating RSV accumulation. Yeast two-hybrid analysis determined that a subunit of the 26S proteasome, named RPN3, could interact with RSV NS3. Transient overexpression of RPN3 had no effect on the RNA silencing suppressor activity of RSV NS3. However, NS3 could inhibit the ability of SBPH rpn3 to complement an rpn3 mutation in yeast. Our findings also indicate that the direct interaction between RPN3 and NS3 was responsible for inhibiting the complementation ability of RPN3. In vivo, we found an accumulation of ubiquitinated protein in SBPH tissues where the RSV titer was high, and silencing of rpn3 resulted in malfunction of the SBPH proteasome-mediated proteolysis. Consequently, viruliferous SBPH in which RPN3 was repressed transmitted the virus more effectively as a result of higher accumulation of RSV. Our results suggest that the RSV NS3 protein is able to hijack the 26S proteasome in SBPH via a direct interaction with the RPN3 subunit to attenuate the host defense response. IMPORTANCE We show, for the first time, that the 26S proteasome components are present in the small brown planthopper and play a role in defense against its vectored plant virus (RSV). In turn, RSV encodes a protein that subverts the SBPH 26S proteasome

  17. The Pentatricopeptide Repeat Protein EMB2654 Is Essential for Trans-Splicing of a Chloroplast Small Ribosomal Subunit Transcript1[OPEN

    PubMed Central

    Sanglard, Lilian Vincis Pereira; Bussell, John D.; Howell, Katharine A.

    2017-01-01

    We report the partial complementation and subsequent comparative molecular analysis of two nonviable mutants impaired in chloroplast translation, one (emb2394) lacking the RPL6 protein, and the other (emb2654) carrying a mutation in a gene encoding a P-class pentatricopeptide repeat protein. We show that EMB2654 is required for the trans-splicing of the plastid rps12 transcript and that therefore the emb2654 mutant lacks Rps12 protein and fails to assemble the small subunit of the plastid ribosome, explaining the loss of plastid translation and consequent embryo-lethal phenotype. Predictions of the EMB2654 binding site match a small RNA “footprint” located on the 5′ half of the trans-spliced intron that is almost absent in the partially complemented mutant. EMB2654 binds sequence specifically to this target sequence in vitro. Altered patterns in nuclease-protected small RNA fragments in emb2654 show that EMB2654 binding must be an early step in, or prior to, the formation of a large protein-RNA complex covering the free ends of the two rps12 intron halves. PMID:28011633

  18. PCR Primers for Metazoan Nuclear 18S and 28S Ribosomal DNA Sequences

    PubMed Central

    Machida, Ryuji J.; Knowlton, Nancy

    2012-01-01

    Background Metagenetic analyses, which amplify and sequence target marker DNA regions from environmental samples, are increasingly employed to assess the biodiversity of communities of small organisms. Using this approach, our understanding of microbial diversity has expanded greatly. In contrast, only a few studies using this approach to characterize metazoan diversity have been reported, despite the fact that many metazoan species are small and difficult to identify or are undescribed. One of the reasons for this discrepancy is the availability of universal primers for the target taxa. In microbial studies, analysis of the 16S ribosomal DNA is standard. In contrast, the best gene for metazoan metagenetics is less clear. In the present study, we have designed primers that amplify the nuclear 18S and 28S ribosomal DNA sequences of most metazoan species with the goal of providing effective approaches for metagenetic analyses of metazoan diversity in environmental samples, with a particular emphasis on marine biodiversity. Methodology/Principal Findings Conserved regions suitable for designing PCR primers were identified using 14,503 and 1,072 metazoan sequences of the nuclear 18S and 28S rDNA regions, respectively. The sequence similarity of both these newly designed and the previously reported primers to the target regions of these primers were compared for each phylum to determine the expected amplification efficacy. The nucleotide diversity of the flanking regions of the primers was also estimated for genera or higher taxonomic groups of 11 phyla to determine the variable regions within the genes. Conclusions/Significance The identified nuclear ribosomal DNA primers (five primer pairs for 18S and eleven for 28S) and the results of the nucleotide diversity analyses provide options for primer combinations for metazoan metagenetic analyses. Additionally, advantages and disadvantages of not only the 18S and 28S ribosomal DNA, but also other marker regions as targets

  19. The heterotrimeric G protein α subunit acts upstream of the small GTPase Rac in disease resistance of rice

    PubMed Central

    Suharsono, Utut; Fujisawa, Yukiko; Kawasaki, Tsutomu; Iwasaki, Yukimoto; Satoh, Hikaru; Shimamoto, Ko

    2002-01-01

    We used rice dwarf1 (d1) mutants lacking a single-copy Gα gene and addressed Gα's role in disease resistance. d1 mutants exhibited a highly reduced hypersensitive response to infection by an avirulent race of rice blast. Activation of PR gene expression in the leaves of the mutants infected with rice blast was delayed for 24 h relative to the wild type. H2O2 production and PR gene expression induced by sphingolipid elicitors (SE) were strongly suppressed in d1 cell cultures. Expression of the constitutively active OsRac1, a small GTPase Rac of rice, in d1 mutants restored SE-dependent defense signaling and resistance to rice blast. Gα mRNA was induced by an avirulent race of rice blast and SE application on the leaf. These results indicated the role of Gα in R gene-mediated disease resistance of rice. We have proposed a model for the defense signaling of rice in which the heterotrimeric G protein functions upstream of the small GTPase OsRac1 in the early steps of signaling. PMID:12237405

  20. Targeted Disruption of the Gene Encoding the Murine Small Subunit of Carboxypeptidase N (CPN1) Causes Susceptibility to C5a Anaphylatoxin-Mediated Shock1

    PubMed Central

    Mueller-Ortiz, Stacey L.; Wang, Dachun; Morales, John E.; Li, Li; Chang, Jui-Yoa; Wetsel, Rick A.

    2015-01-01

    Carboxypeptidase N (CPN) is a plasma zinc metalloprotease, which consists of two enzymatically active small subunits (CPN1) and two large subunits (CPN2) that protect the protein from degradation. Historically, CPN has been implicated as a major regulator of inflammation by its enzymatic cleavage of functionally important arginine and lysine amino acids from potent phlogistic molecules, such as the complement anaphylatoxins C3a and C5a. Because of no known complete CPN deficiencies, the biological impact of CPN in vivo has been difficult to evaluate. Here, we report the generation of a mouse with complete CPN deficiency by targeted disruption of the CPN1 gene. CPN1−/− mice were hypersensitive to lethal anaphylactic shock due to acute complement activation by cobra venom factor. This hypersensitivity was completely resolved in CPN1−/−/C5aR−/− but not in CPN1−/−/C3aR−/− mice. Moreover, CPN1−/− mice given C5a i.v., but not C3a, experienced 100% mortality. This C5a-induced mortality was reduced to 20% when CPN1−/− mice were treated with an antihistamine before C5a challenge. These studies describe for the first time a complete deficiency of CPN and demonstrate 1) that CPN plays a requisite role in regulating the lethal effects of anaphylatoxin-mediated shock, 2) that these lethal effects are mediated predominantly by C5a-induced histamine release, and 3) that C3a does not contribute significantly to shock following acute complement activation. PMID:19414808

  1. Small-Molecule Fusion Inhibitors Bind the pH-Sensing Stable Signal Peptide-GP2 Subunit Interface of the Lassa Virus Envelope Glycoprotein

    PubMed Central

    Shankar, Sundaresh; Whitby, Landon R.; Casquilho-Gray, Hedi E.; York, Joanne; Boger, Dale L.

    2016-01-01

    ABSTRACT Arenavirus species are responsible for severe life-threatening hemorrhagic fevers in western Africa and South America. Without effective antiviral therapies or vaccines, these viruses pose serious public health and biodefense concerns. Chemically distinct small-molecule inhibitors of arenavirus entry have recently been identified and shown to act on the arenavirus envelope glycoprotein (GPC) to prevent membrane fusion. In the tripartite GPC complex, pH-dependent membrane fusion is triggered through a poorly understood interaction between the stable signal peptide (SSP) and the transmembrane fusion subunit GP2, and our genetic studies have suggested that these small-molecule inhibitors act at this interface to antagonize fusion activation. Here, we have designed and synthesized photoaffinity derivatives of the 4-acyl-1,6-dialkylpiperazin-2-one class of fusion inhibitors and demonstrate specific labeling of both the SSP and GP2 subunits in a native-like Lassa virus (LASV) GPC trimer expressed in insect cells. Photoaddition is competed by the parental inhibitor and other chemically distinct compounds active against LASV, but not those specific to New World arenaviruses. These studies provide direct physical evidence that these inhibitors bind at the SSP-GP2 interface. We also find that GPC containing the uncleaved GP1-GP2 precursor is not susceptible to photo-cross-linking, suggesting that proteolytic maturation is accompanied by conformational changes at this site. Detailed mapping of residues modified by the photoaffinity adducts may provide insight to guide the further development of these promising lead compounds as potential therapeutic agents to treat Lassa hemorrhagic fever. IMPORTANCE Hemorrhagic fever arenaviruses cause lethal infections in humans and, in the absence of licensed vaccines or specific antiviral therapies, are recognized to pose significant threats to public health and biodefense. Lead small-molecule inhibitors that target the

  2. Molecular Identification of Ptychodera flava (Hemichordata: Enteropneusta): Reconsideration in Light of Nucleotide Polymorphism in the 18S Ribosomal RNA Gene.

    PubMed

    Urata, Makoto

    2015-06-01

    Seven nuclear and mitochondrial DNA markers were examined in 12 specimens of Ptychodera flava, a model acorn worm used in molecular biology, collected in Japan from three local populations with different modes of living. A comparison of intraspecific results did not show genetically isolated populations despite the species' enclave habitats and asexual reproduction. Moreover, both the nuclear 18S ribosomal RNA gene and mitochondrial 16S ribosomal RNA gene sequences were identical to those from Moorea in French Polynesia, nearly 10,000 kilometers away from Japan. I also provide the first definitive information regarding polymorphisms in 18S ribosomal RNA gene, the external transcribed spacer (ETS), internal transcribed spacers (ITS), and mitochondrial cytochrome c oxidase subunit 1 (mtCO1) sequence in hemichordates using newly designed primer sets, and I show both high larval vagility and certain criteria for the molecular identification of this species.

  3. Cystoisospora spp. from dogs in China and phylogenetic analysis of its 18S and ITS1 gene.

    PubMed

    He, Pengfei; Li, Jianhua; Gong, Pengtao; Huang, Jingui; Zhang, Xichen

    2012-11-23

    Cystoisospora spp. oocysts isolated from dog feces in Changchun, China were morphologically similar to those of Cystoisospora ohioensis and Cystoisospora sp. 1-MM recently isolated from dogs in Japanese. Sequencing results of the 18S subunit RNA gene from isolates in the present study were compared to other Cystoisospora spp. and the results suggested that Cystoisospora spp. from dogs in Changchun was homologous to C. ohioensis and Cystoisospora sp. 1-MM. Phylogenetic analysis of the 18S rRNA sequences showed that the Cystoisospora sp. ChangChun 1 and Cystoisospora sp. ChangChun 2 were nested in a clade with other Cystoisospora spp., including C. ohioensis, Cystoisospora belli, Cystoisospora suis, Isospora sp. Harbin/01/08 and C. orlovi,. Cystoisospora sp. ChangChun 2 was confirmed as C. ohioensis, and the other isolate was in a separate clade but the genetic relationship was relatively close to C. suis after analysis of the ITS-1gene.

  4. 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.

  5. Determination of the relative expression levels of rubisco small subunit genes in Arabidopsis by rapid amplification of cDNA ends.

    PubMed

    Yoon, M; Putterill, J J; Ross, G S; Laing, W A

    2001-04-15

    Multigene families are common in higher organisms. However, due to the close similarities between members, it is often difficult to assess the individual contribution of each gene to the overall expression of the family. In Arabidopsis thaliana, there are four genes encoding the small subunits (SSU) of ribulose-1.5-bisphosphate carboxylase oxygenase (rubisco) whose nucleotide sequences are up to 98.4% identical. In order to overcome the technical limitations associated with gene-specific probes (or primers) commonly used in existing methods, we developed a new gene expression assay based on the RACE (rapid amplification of cDNA ends) technique with a single pair of primers. With this RACE gene expression assay, we were able to determine the relative transcript levels between four Arabidopsis SSU genes. We found that the relative SSU gene expression differed significantly between plants grown at different temperatures. Our observation raises the possibility that an adaptation of rubisco to the environment may be achieved through the specific synthesis of the SSU proteins, which is determined by the relative expression levels between the SSU genes.

  6. Molecular evolution inferred from small subunit rRNA sequences: what does it tell us about phylogenetic relationships and taxonomy of the parabasalids?

    NASA Technical Reports Server (NTRS)

    Viscogliosi, E.; Edgcomb, V. P.; Gerbod, D.; Noel, C.; Delgado-Viscogliosi, P.; Sogin, M. L. (Principal Investigator)

    1999-01-01

    The Parabasala are a primitive group of protists divided into two classes: the trichomonads and the hypermastigids. Until recently, phylogeny and taxonomy of parabasalids were mainly based on the comparative analysis of morphological characters primarily linked to the development of their cytoskeleton. Recent use of molecular markers, such as small subunit (SSU) rRNA has led to now insights into the systematics of the Parabasala and other groups of prolists. An updated phylogeny based on SSU rRNA is provided and compared to that inferred from ultrastructural data. The SSU rRNA phylogeny contradicts the dogma equating simple characters with pumitive characters. Hypermastigids, possessing a hyperdeveloped cytoskeleton, exhibit the most basal emergence in the parabasalid lineage. Other observations emerge from the SSU rRNA analysis, such as the secondary loss of some cytoskeleton structures in all representatives of the Monocercomonadidae, the existence of secondarily free living taxa (reversibility of parasitism) and the evidence against the co-evolution of the endobiotic parabasalids and their animal hosts. According to phylogenies based on SSU rRNA, all the trichomonad families are not monophyletic groups, putting into question the validity of current taxonomic assignments. The precise branching order of some taxa remains unclear, but this issue can possibly be addressed by the molecular analysis of additional parabasalids. The goal of such additional analyses would be to propose, in a near future, a revision of the taxonomy of this group of protists that takes into account both molecular and morphological data.

  7. Reconsideration of the phylogenetic positions of five peritrich genera, Vorticella, Pseudovorticella, Zoothamnopsis, Zoothamnium, and Epicarchesium (Ciliophora, Peritrichia, Sessilida), based on small subunit rRNA gene sequences.

    PubMed

    Li, Lifang; Song, Weibo; Warren, Alan; Shin, Mann Kyoon; Chen, Zigui; Ji, Daode; Sun, Ping

    2008-01-01

    In order to re-evaluate the systematics of sessilid peritrich ciliates, small subunit (SSU) rRNA gene sequences were determined for 12 species belonging to five genera: Vorticella, Pseudovorticella, Epicarchesium, Zoothamnium, and Zoothamnopsis. Phylogenetic trees were deduced using Bayesian inference, maximum parsimony, and maximum likelihood methods. The phylogenetic analyses suggest that (1) sessilids which have stalks with continuous myonemes that contract in a zig-zag fashion form a separate clade from those which have stalks that contract independently and in a spiral fashion, supporting the separation of the family Zoothamniidae from the family Vorticellidae and (2) Epicarchesium and Pseudovorticella, both of which have reticulate silverline systems, are more closely related to each other than to other vorticellids, suggesting that differences in the silverline system (i.e. transverse vs. reticulate) may be the result of genuine evolutionary divergence among sessilid peritrichs. However, the newly sequenced Zoothamnopsis sinica, which has a reticulate silverline pattern, nests within the unresolved Zoothamnium species that have transverse silverline patterns. Thus, there were at least two evolutions of the reticulate silverline pattern character state from a plesiomorphic transverse state in the peritrichid ciliates. The molecular work demonstrates the genus Zoothamnium to be paraphyletic in relation to morphological studies, and suggests that Astylozoon, Opisthonecta, and Vorticella microstoma possibly share a SSU rRNA secondary structure in the helix E10-1 region.

  8. CREB1 directly activates the transcription of ribonucleotide reductase small subunit M2 and promotes the aggressiveness of human colorectal cancer

    PubMed Central

    Fang, Zejun; Lin, Aifen; Chen, Jiaoe; Zhang, Xiaomin; Liu, Hong; Li, Hongzhang; Hu, Yanyan; Zhang, Xia; Zhang, Jiangang; Qiu, Lanlan; Mei, Lingming; Shao, Jimin; Chen, Xiang

    2016-01-01

    As the small subunit of Ribonucleotide reductase (RR), RRM2 displays a very important role in various critical cellular processes such as cell proliferation, DNA repair, and senescence, etc. Importantly, RRM2 functions like a tumor driver in most types of cancer but little is known about the regulatory mechanism of RRM2 in cancer development. In this study, we found that the cAMP responsive element binding protein 1 (CREB1) acted as a transcription factor of RRM2 gene in human colorectal cancer (CRC). CREB1 directly bound to the promoter of RRM2 gene and induced its transcriptional activation. Knockdown of CREB1 decreased the expression of RRM2 at both mRNA and protein levels. Moreover, knockdown of RRM2 attenuated CREB1-induced aggressive phenotypes of CRC cells in vitro and in vivo. Analysis of the data from TCGA database and clinical CRC specimens with immunohistochemical staining also demonstrated a strong correlation between the co-expression of CREB1 and RRM2. Decreased disease survivals were observed in CRC patients with high expression levels of CREB1 or RRM2. Our results indicate CREB1 as a critical transcription factor of RRM2 which promotes tumor aggressiveness, and imply a significant correlation between CREB1 and RRM2 in CRC specimens. These may provide the possibility that CREB1 and RRM2 could be used as biomarkers or targets for CRC diagnosis and treatment. PMID:27801665

  9. A potential role for RNA turnover in the light regulation of plant gene expression: ribulose-1,5-bisphosphate carboxylase small subunit in soybean.

    PubMed Central

    Shirley, B W; Meagher, R B

    1990-01-01

    Post-transcriptional regulation of the genes encoding the small subunit (rbcS) of ribulose-1,5-bisphosphate carboxylase was examined in soybean seedlings. Substantial discrepancies were detected between relative in vitro transcription rates and steady-state RNA levels in light- and dark-grown seedling leaves, indicating that rbcS RNA may be degraded more rapidly in light than in darkness. Additional data imply that the turnover mechanism is rapidly induced by light, maintained for some time in darkness, and that it may be negatively controlled by far-red light. The proposed RNA turnover system does not affect all RNAs equally since a soybean actin gene showed equivalent in vitro transcription rates and RNA levels in light and darkness. Soybean rbcS genes may be subject to a novel mode of control in which light-induced expression is accompanied by an increased rate of RNA degradation. Models for the specific regulation of rbcS RNA stability in response to light are presented. Images PMID:2356127

  10. Development and Application of Small-Subunit rRNA Probes for Assessment of Selected Thiobacillus Species and Members of the Genus Acidiphilium

    PubMed Central

    Peccia, Jordan; Marchand, Eric A.; Silverstein, Joann; Hernandez, Mark

    2000-01-01

    Culture-dependent studies have implicated sulfur-oxidizing bacteria as the causative agents of acid mine drainage and concrete corrosion in sewers. Thiobacillus species are considered the major representatives of the acid-producing bacteria in these environments. Small-subunit rRNA genes from all of the Thiobacillus and Acidiphilium species catalogued by the Ribosomal Database Project were identified and used to design oligonucleotide DNA probes. Two oligonucleotide probes were synthesized to complement variable regions of 16S rRNA in the following acidophilic bacteria: Thiobacillus ferrooxidans and T. thiooxidans (probe Thio820) and members of the genus Acidiphilium (probe Acdp821). Using 32P radiolabels, probe specificity was characterized by hybridization dissociation temperature (Td) with membrane-immobilized RNA extracted from a suite of 21 strains representing three groups of bacteria. Fluorochrome-conjugated probes were evaluated for use with fluorescent in situ hybridization (FISH) at the experimentally determined Tds. FISH was used to identify and enumerate bacteria in laboratory reactors and environmental samples. Probing of laboratory reactors inoculated with a mixed culture of acidophilic bacteria validated the ability of the oligonucleotide probes to track specific cell numbers with time. Additionally, probing of sediments from an active acid mine drainage site in Colorado demonstrated the ability to identify numbers of active bacteria in natural environments that contain high concentrations of metals, associated precipitates, and other mineral debris. PMID:10877807

  11. Development and application of small-subunit rRNA probes for assessment of selected Thiobacillus species and members of the genus Acidiphilium.

    PubMed

    Peccia, J; Marchand, E A; Silverstein, J; Hernandez, M

    2000-07-01

    Culture-dependent studies have implicated sulfur-oxidizing bacteria as the causative agents of acid mine drainage and concrete corrosion in sewers. Thiobacillus species are considered the major representatives of the acid-producing bacteria in these environments. Small-subunit rRNA genes from all of the Thiobacillus and Acidiphilium species catalogued by the Ribosomal Database Project were identified and used to design oligonucleotide DNA probes. Two oligonucleotide probes were synthesized to complement variable regions of 16S rRNA in the following acidophilic bacteria: Thiobacillus ferrooxidans and T. thiooxidans (probe Thio820) and members of the genus Acidiphilium (probe Acdp821). Using (32)P radiolabels, probe specificity was characterized by hybridization dissociation temperature (T(d)) with membrane-immobilized RNA extracted from a suite of 21 strains representing three groups of bacteria. Fluorochrome-conjugated probes were evaluated for use with fluorescent in situ hybridization (FISH) at the experimentally determined T(d)s. FISH was used to identify and enumerate bacteria in laboratory reactors and environmental samples. Probing of laboratory reactors inoculated with a mixed culture of acidophilic bacteria validated the ability of the oligonucleotide probes to track specific cell numbers with time. Additionally, probing of sediments from an active acid mine drainage site in Colorado demonstrated the ability to identify numbers of active bacteria in natural environments that contain high concentrations of metals, associated precipitates, and other mineral debris.

  12. Genetic diversity of microbial eukaryotes in anoxic sediment around fumaroles on a submarine caldera floor based on the small-subunit rDNA phylogeny.

    PubMed

    Takishita, Kiyotaka; Miyake, Hiroshi; Kawato, Masaru; Maruyama, Tadashi

    2005-06-01

    Recent culture-independent molecular analyses have shown the diversity and ecological importance of microbial eukaryotes (protists) in various marine environments. In the present study we directly extracted DNA from anoxic sediment near active fumaroles on a submarine caldera floor at a depth of 200 m and constructed genetic libraries of PCR-amplified eukaryotic small-subunit (SSU) rDNA. By sequencing cloned SSU rDNA of the libraries and their phylogenetic analyses, it was shown that most sequences have affiliations with known major lineages of eukaryotes (Cercozoa, Alveolata, stramenopiles and Opisthokonta). In particular, some sequences were closely related to those of representatives of eukaryotic parasites, such as Phagomyxa and Cryothecomonas of Cercozoa, Pirsonia of stramenopiles and Ichthyosporea of Opisthokonta, although it is not clear whether the organisms occur in free-living or parasitic forms. In addition, other sequences did not seem to be related to any described eukaryotic lineages suggesting the existence of novel eukaryotes at a high-taxonomic level in the sediment. The community composition of microbial eukaryotes in the sediment we surveyed was different overall from those of other anoxic marine environments previously investigated.

  13. Orosomucoid Proteins Interact with the Small Subunit of Serine Palmitoyltransferase and Contribute to Sphingolipid Homeostasis and Stress Responses in Arabidopsis[OPEN

    PubMed Central

    Li, Jian; Yin, Jian; Rong, Chan; Li, Kai-En; Wu, Jian-Xin; Huang, Li-Qun; Zeng, Hong-Yun; Sahu, Sunil Kumar; Yao, Nan

    2016-01-01

    Serine palmitoyltransferase (SPT), a pyridoxyl-5′-phosphate-dependent enzyme, catalyzes the first and rate-limiting step in sphingolipid biosynthesis. In humans and yeast, orosomucoid proteins (ORMs) negatively regulate SPT and thus play an important role in maintaining sphingolipid levels. Despite the importance of sphingoid intermediates as bioactive molecules, the regulation of sphingolipid biosynthesis through SPT is not well understood in plants. Here, we identified and characterized the Arabidopsis thaliana ORMs, ORM1 and ORM2. Loss of function of both ORM1 and ORM2 (orm1 amiR-ORM2) stimulated de novo sphingolipid biosynthesis, leading to strong sphingolipid accumulation, especially of long-chain bases and ceramides. Yeast two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays confirmed that ORM1 and ORM2 physically interact with the small subunit of SPT (ssSPT), indicating that ORMs inhibit ssSPT function. We found that orm1 amiR-ORM2 plants exhibited an early-senescence phenotype accompanied by H2O2 production at the cell wall and in mitochondria, active vesicular trafficking, and formation of cell wall appositions. Strikingly, the orm1 amiR-ORM2 plants showed increased expression of genes related to endoplasmic reticulum stress and defenses and also had enhanced resistance to oxidative stress and pathogen infection. Taken together, our findings indicate that ORMs interact with SPT to regulate sphingolipid homeostasis and play a pivotal role in environmental stress tolerance in plants. PMID:27923879

  14. Genetic diversity among Babesia rossi detected in naturally infected dogs in Abeokuta, Nigeria, based on 18S rRNA gene sequences.

    PubMed

    Takeet, Michael I; Oyewusi, Adeoye J; Abakpa, Simon A V; Daramola, Olukayode O; Peters, Sunday O

    2017-03-01

    Adequate knowledge of the genetic diversity among Babesia species infecting dogs is necessary for a better understanding of the epidemiology and control of canine babesiosis. Hence, this study determined the genetic diversity among the Babesia rossi detected in dogs presented for routine examination in Veterinary Hospitals in Abeokuta, Nigeria. Blood were randomly collected from 209 dogs. Field-stained thin smears were made and DNA extracted from the blood. Partial region of the 18S small subunit ribosomal RNA (rRNA) gene was amplified, sequenced and analysed. Babesia species was detected in 16 (7.7%) of the dogs by microscopy. Electrophoresed PCR products from 39 (18.66%) dogs revealed band size of 450 bp and 2 (0.95%) dogs had band size of 430 bp. The sequences obtained from 450 bp amplicon displayed homology of 99.74% (387/388) with partial sequences of 18S rRNA gene of Babesia rossi in the GeneBank. Of the two sequences that had 430 bp amplicon, one was identified as T. annulata and second as T. ovis. A significantly (p<0.05) higher prevalence of B. rossi was detected by PCR compared to microscopy. The mean PCV of Babesia infected dogs was significantly (p<0.05) lower than non-infected dogs. Phylogenetic analysis revealed minimal diversity among B. rossi with the exception of one sequence that was greatly divergent from the others. This study suggests that more than one genotype of B. rossi may be in circulation among the dog population in the study area and this may have potential implication on clinical outcome of canine babesiosis.

  15. 2,2,2-Trifluoroethanol changes the transition kinetics and subunit interactions in the small bacterial mechanosensitive channel MscS.

    PubMed

    Akitake, Bradley; Spelbrink, Robin E J; Anishkin, Andriy; Killian, J Antoinette; de Kruijff, Ben; Sukharev, Sergei

    2007-04-15

    2,2,2-Trifluoroethanol (TFE), a low-dielectric solvent, has recently been used as a promising tool to probe the strength of intersubunit interactions in membrane proteins. An analysis of inner membrane proteins of Escherichia coli has identified several SDS-resistant protein complexes that separate into subunits upon exposure to TFE. One of these was the homo-heptameric stretch-activated mechanosensitive channel of small conductance (MscS), a ubiquitous component of the bacterial turgor-regulation system. Here we show that a substantial fraction of MscS retains its oligomeric state in cold lithium-dodecyl-sulfate gel electrophoresis. Exposure of MscS complexes to 10-15 vol % TFE in native membranes or nonionic detergent micelles before lithium-dodecyl-sulfate electrophoresis results in a complete dissociation into monomers, suggesting that at these concentrations TFE by itself disrupts or critically compromises intersubunit interactions. Patch-clamp analysis of giant E. coli spheroplasts expressing MscS shows that exposure to TFE in lower concentrations (0.5-5.0 vol %) causes leftward shifts of the dose-response curves when applied extracellularly, and rightward shifts when added from the cytoplasmic side. In the latter case, TFE increases the rate of tension-dependent inactivation and lengthens the process of recovery to the resting state. MscS responses to pressure ramps of different speeds indicate that in the presence of TFE most channels reside in the resting state and only at tensions near the activation threshold does TFE dramatically speed up inactivation. The effect of TFE is reversible as normal channel activity returns 15-30 min after a TFE washout. We interpret the observed midpoint shifts in terms of asymmetric partitioning of TFE into the membrane and distortion of the bilayer lateral pressure profile. We also relate the increased rate of inactivation and subunit separation with the capacity of TFE to perturb buried interhelical contacts in proteins

  16. Using Small Subunit Ribosomal RNA to Follow Dark Incorporation of 14C-bicarbonate by Bacteria and Archaea in Sandy Sediment

    NASA Astrophysics Data System (ADS)

    MacGregor, B. J.; Musat, N.; Kuypers, M. M.

    2007-12-01

    Small subunit ribosomal RNA (SSU rRNA) and the genes encoding it have become the basis of modern microbial phylogeny, and of numerous methods for characterizing the composition of bacterial, archaeal, and even eukaryotic communities as they occur in nature. A limitation of this approach has been that phylogeny alone is not a reliable guide to physiology, particularly for groups with no close relatives in culture. We have been developing ways of using the SSU rRNA molecule itself to identify and (eventually) quantify the carbon sources incorporated by particular phylogenetic groups. This can be done by taking advantage of natural variations in carbon isotopic composition among growth substrates, or by following incorporation of 13C- or 14C-labeled compounds. 14C has the advantage that natural background levels are negligible. In the present study, our goal is to identify species responsible for non-photosynthetic CO2 incorporation in sandy sediments of the German Wadden Sea. Sediment cores collected from the Janssand sand flats were percolated with 14C-bicarbonate at in situ temperature for 36-38h in the dark, total RNA isolated, and domain-specific oligonucleotide probes used to capture bacterial and archaeal SSU rRNA. Total and/or captured RNA was separated by denaturing polyacrylamide gel electrophoresis, and 14C detected by phosphor imager, autoradiography, or beta imager. Detection was fastest and most sensitive with the beta imager. Both Bacteria and Archaea had incorporated label, suggesting both groups may harbor non-photosynthetic autotrophs. The next step will be to use more specific capture probes. We are currently working to separate the captured domain-specific SSU rRNA on non-denaturing gels, with detection by the high-resolution mode of the beta imager, so that individual species incorporating label can be identified by RT-PCR and sequencing of labeled bands.

  17. Soybean ribulose bisphosphate carboxylase small subunit; mechanisms and determinants of RNA turnover: Annual progress report for the period June 1, 1987 through May 31, 1988

    SciTech Connect

    Meagher, R.

    1988-01-01

    SRS1 and SRS4 are two closely related soybean ribulose -1,5-bisphosphate carboxylase small subunit (SSU) genes. The promoters from both SRS1 and SRS4 can be fused to a neomycin phosphotransferase gene and these fusions produce similar levels of kanamycin resistance in transgenic petunia plants. The expression of SRS1 and SRS4 has been shown to be controlled at the level of transcription, and this transcriptional control is phytochrome mediated. Together these genes account for 2-3% of the total transcription in light-grown soybean seedlings or expanding soybean leaves. Recent experiments analyzing transcription rates of SRS1 and SRS4, steady state levels of their total and poly A+ RNA and frequency of their cDNAs in a soybean RNA library have led us to hypothesize that the expression of these two genes may also be controlled at the level of RNA turnover. Despite the 30-50 fold difference in transcription of these genes in seedlings grown in light, the steady state levels of RNA are only 4-8 fold higher in the light. When plants are shifted from darkness to light, accumulation of RNA lags far behind the striking transcriptional induction. In plants shifted from light to darkness, SRS1 transcription takes 24 hours to drop to dark-grown levels, and the steady state RNA levels take 72 hours to decay to dark-grown levels. On the other hand light-grown plants treated with far-red light shut down SRS1 transcription immediately, and the steady state levels of SSU RNA also drop rapidly. We have evidence suggesting striking differential turnover of the RNA products of SRS1 and SRS4, the SRS1 RNA being perhaps 5-10 times more stable than the SRS4 RNA.

  18. Ribosome biogenesis factor Tsr3 is the aminocarboxypropyl transferase responsible for 18S rRNA hypermodification in yeast and humans

    PubMed Central

    Meyer, Britta; Wurm, Jan Philip; Sharma, Sunny; Immer, Carina; Pogoryelov, Denys; Kötter, Peter; Lafontaine, Denis L. J.; Wöhnert, Jens; Entian, Karl-Dieter

    2016-01-01

    The chemically most complex modification in eukaryotic rRNA is the conserved hypermodified nucleotide N1-methyl-N3-aminocarboxypropyl-pseudouridine (m1acp3Ψ) located next to the P-site tRNA on the small subunit 18S rRNA. While S-adenosylmethionine was identified as the source of the aminocarboxypropyl (acp) group more than 40 years ago the enzyme catalyzing the acp transfer remained elusive. Here we identify the cytoplasmic ribosome biogenesis protein Tsr3 as the responsible enzyme in yeast and human cells. In functionally impaired Tsr3-mutants, a reduced level of acp modification directly correlates with increased 20S pre-rRNA accumulation. The crystal structure of archaeal Tsr3 homologs revealed the same fold as in SPOUT-class RNA-methyltransferases but a distinct SAM binding mode. This unique SAM binding mode explains why Tsr3 transfers the acp and not the methyl group of SAM to its substrate. Structurally, Tsr3 therefore represents a novel class of acp transferase enzymes. PMID:27084949

  19. Small molecule integrin antagonists that bind to the beta2 subunit I-like domain and activate signals in one direction and block them in the other.

    PubMed

    Shimaoka, Motomu; Salas, Azucena; Yang, Wei; Weitz-Schmidt, Gabriele; Springer, Timothy A

    2003-09-01

    Leukocyte integrins contain an inserted (I) domain in their alpha subunits and an I-like domain in their beta(2) subunit, which directly bind ligand and regulate ligand binding, respectively. We describe a novel mechanistic class of integrin inhibitors that bind to the metal ion-dependent adhesion site of the beta(2) I-like domain and prevent its interaction with and activation of the alpha(L) I domain. The inhibitors do not bind to the alpha(L) I domain but stabilize alpha/beta subunit association and can show selectivity for alpha(L)beta(2) compared to alpha(M)beta(2). The inhibitors reveal a crucial intersection for relaying conformational signals within integrin extracellular domains. While blocking signals in one direction to the I domain, the antagonists induce the active conformation of the I-like domain and stalk domains, and thus transmit conformational signals in the other direction toward the transmembrane domains.

  20. Rate accelerations in nuclear 18S rDNA of mycoheterotrophic and parasitic angiosperms.

    PubMed

    Lemaire, Benny; Huysmans, Suzy; Smets, Erik; Merckx, Vincent

    2011-09-01

    Rate variation in genes from all three genomes has been observed frequently in plant lineages with a parasitic and mycoheterotrophic mode of life. While the loss of photosynthetic ability leads to a relaxation of evolutionary constraints in genes involved in the photosynthetic apparatus, it remains to be determined how prevalent increased substitution rates are in nuclear DNA of non-photosynthetic angiosperms. In this study we infer rates of molecular evolution of 18S rDNA of all parasitic and mycoheterotorphic plant families (except Lauraceae and Polygalaceae) using relative rate tests. In several holoparasitic and mycoheterotrophic plant lineages extremely high substitution rates are observed compared to other photosynthetic angiosperms. The position and frequency of these substitutions have been identified to understand the mutation dynamics of 18S rRNA in achlorophyllous plants. Despite the presence of significantly elevated substitution rates, very few mutations occur in major functional and structural regions of the small ribosomal molecule, providing evidence that the efficiency of the translational apparatus in non-photosynthetic plants has not been affected.

  1. Karyotypes, heterochromatin, and physical mapping of 18S-26S rDNA in Cactaceae.

    PubMed

    Las Peñas, M L; Urdampilleta, J D; Bernardello, G; Forni-Martins, E R

    2009-01-01

    Karyotype analyses in members of the four Cactaceae subfamilies were performed. Numbers and karyotype formula obtained were: Pereskioideae = Pereskiaaculeata(2n = 22; 10 m + 1 sm), Maihuenioideae = Maihuenia patagonica (2n = 22, 9 m + 2 sm; 2n = 44, 18 m + 4 sm), Opuntioideae = Cumulopuntia recurvata(2n = 44; 20 m + 2 sm), Cactoideae = Acanthocalycium spiniflorum (2n = 22; 10 m + 1 sm),Echinopsis tubiflora (2n = 22; 10 m + 1 sm), Trichocereus candicans (2n = 22, 22 m). Chromosomes were small, the average chromosome length was 2.3 mum. Diploid species and the tetraploid C. recurvata had one terminal satellite, whereas the remaining tetraploid species showed four satellited chromosomes. Karyotypes were symmetrical. No CMA(-)/DAPI(+) bands were detected, but CMA(+)/DAPI(-) bands associated with NOR were always found. Pericentromeric heterochromatin was found in C. recurvata, A. spiniflorum, and the tetraploid cytotype of M. patagonica. The locations of the 18S-26S rDNA sites in all species coincided with CMA(+)/DAPI(-) bands; the same occurred with the sizes and numbers of signals for each species. This technique was applied for the first time in metaphase chromosomes in cacti. NOR-bearing pair no.1 may be homeologous in all species examined. In Cactaceae, the 18S-26S loci seem to be highly conserved.

  2. The ATPase hCINAP regulates 18S rRNA processing and is essential for embryogenesis and tumour growth

    PubMed Central

    Bai, Dongmei; Zhang, Jinfang; Li, Tingting; Hang, Runlai; Liu, Yong; Tian, Yonglu; Huang, Dadu; Qu, Linglong; Cao, Xiaofeng; Ji, Jiafu; Zheng, Xiaofeng

    2016-01-01

    Dysfunctions in ribosome biogenesis cause developmental defects and increased cancer susceptibility; however, the connection between ribosome assembly and tumorigenesis remains unestablished. Here we show that hCINAP (also named AK6) is required for human 18S rRNA processing and 40S subunit assembly. Homozygous CINAP−/− mice show embryonic lethality. The heterozygotes are viable and show defects in 18S rRNA processing, whereas no delayed cell growth is observed. However, during rapid growth, CINAP haploinsufficiency impairs protein synthesis. Consistently, hCINAP depletion in fast-growing cancer cells inhibits ribosome assembly and abolishes tumorigenesis. These data demonstrate that hCINAP reduction is a specific rate-limiting controller during rapid growth. Notably, hCINAP is highly expressed in cancers and correlated with a worse prognosis. Genome-wide polysome profiling shows that hCINAP selectively modulates cancer-associated translatome to promote malignancy. Our results connect the role of hCINAP in ribosome assembly with tumorigenesis. Modulation of hCINAP expression may be a promising target for cancer therapy. PMID:27477389

  3. In Azotobacter vinelandii hydrogenase, substitution of serine for the cysteine residues at positions 62, 65, 294, and 297 in the small (HoxK) subunit affects H2 oxidation [corrected

    PubMed Central

    Sayavedra-Soto, L A; Arp, D J

    1993-01-01

    The essential role of the small (HoxK) subunit of hydrogenase of Azotobacter vinelandii in H2 oxidation was established. This was achieved by modification of the two Cys-X2-Cys amino acid motifs at the N and C termini of the HoxK subunit (Cys-62, -65, -294, and -297). The Cys codons were individually mutated to Ser codons. Modifications in these two motifs resulted in loss of hydrogenase activity. At the N terminus, the mutations of the codons for the motif Cys-62-Thr-Cys-64-Cys-65 decreased the activity of hydrogenase to levels no higher than 30% of those of the parental strain. H2 oxidation with the alternate electron acceptors methylene blue and benzyl viologen was decreased. H2 evolution and exchange activities were also affected. Cys-64 possibly substitutes for either Cys-62 or Cys-65, allowing for partial activity. Mutation of the codons for Cys-294 and Cys-297 to Ser codons resulted in no hydrogenase activity. The results are consistent with alterations of the ligands of FeS clusters in the HoxK subunit of hydrogenase [corrected]. Images PMID:8501046

  4. Structure-Guided Design and Optimization of Small Molecules Targeting the Protein–Protein Interaction between the von Hippel–Lindau (VHL) E3 Ubiquitin Ligase and the Hypoxia Inducible Factor (HIF) Alpha Subunit with in Vitro Nanomolar Affinities

    PubMed Central

    2014-01-01

    E3 ubiquitin ligases are attractive targets in the ubiquitin–proteasome system, however, the development of small-molecule ligands has been rewarded with limited success. The von Hippel–Lindau protein (pVHL) is the substrate recognition subunit of the VHL E3 ligase that targets HIF-1α for degradation. We recently reported inhibitors of the pVHL:HIF-1α interaction, however they exhibited moderate potency. Herein, we report the design and optimization, guided by X-ray crystal structures, of a ligand series with nanomolar binding affinities. PMID:25166285

  5. Gradual processing of the ITS1 from the nucleolus to the cytoplasm during synthesis of the human 18S rRNA.

    PubMed

    Preti, Milena; O'Donohue, Marie-Françoise; Montel-Lehry, Nathalie; Bortolin-Cavaillé, Marie-Line; Choesmel, Valérie; Gleizes, Pierre-Emmanuel

    2013-04-01

    Defects in ribosome biogenesis trigger stress response pathways, which perturb cell proliferation and differentiation in several genetic diseases. In Diamond-Blackfan anemia (DBA), a congenital erythroblastopenia, mutations in ribosomal protein genes often interfere with the processing of the internal transcribed spacer 1 (ITS1), the mechanism of which remains elusive in human cells. Using loss-of-function experiments and extensive RNA analysis, we have defined the precise position of the endonucleolytic cleavage E in the ITS1, which generates the 18S-E intermediate, the last precursor to the 18S rRNA. Unexpectedly, this cleavage is followed by 3'-5' exonucleolytic trimming of the 18S-E precursor during nuclear export of the pre-40S particle, which sets a new mechanism for 18S rRNA formation clearly different from that established in yeast. In addition, cleavage at site E is also followed by 5'-3' exonucleolytic trimming of the ITS1 by exonuclease XRN2. Perturbation of this step on knockdown of the large subunit ribosomal protein RPL26, which was recently associated to DBA, reveals the putative role of a highly conserved cis-acting sequence in ITS1 processing. These data cast new light on the original mechanism of ITS1 elimination in human cells and provide a mechanistic framework to further study the interplay of DBA-linked ribosomal proteins in this process.

  6. Morphology, morphogenesis and small subunit rRNA gene sequence of a soil hypotrichous ciliate, Perisincirra paucicirrata (Ciliophora, Kahliellidae), from the shoreline of the Yellow River, North China.

    PubMed

    Li, Fengchao; Xing, Yi; Li, Jiamei; Al-Rasheid, Khaled A S; He, Songke; Shao, Chen

    2013-01-01

    The morphology, morphogenesis, and 18S rRNA gene sequence of a soil hypotrichous ciliate Perisincirra paucicirrata, isolated from north China, were investigated. Perisincirra paucicirrata differs from its congeners in: (1) having a body length to width ratio in vivo of 4:1, (2) its adoral zone occupying between 15% and 25% of the total body length, and (3) the presence of two parabuccal cirri, three left (with 10-16 cirri each) and two right marginal rows (with 14-24 cirri each), and three dorsal kineties. Our study offers a first attempt to begin to map the morphogenetic processes of the genus, which are mainly characterised by the following: the formation of four frontal ventral transverse anlagens for each daughter cell, with the proter's anlage I originating from the reorganised anterior part of the parental paroral; the paroral and endoral anlage developed from the reorganised old endoral and do not contribute the first frontal cirrus; the frontoventral transverse anlage I contributing the left frontal cirrus; anlage II generating the middle frontal and the buccal cirri; anlage III developing the right frontal cirrus and the anterior parabuccal cirrus; and anlage IV contributing the posterior parabuccal cirrus. As an additional contribution, we judge that the inner one or the two right rows of P. kahli and P. longicirrata are marginal rows. Phylogenetic analysis based on SSU rDNA sequences suggests that Perisincirra is related to sporadotrichids, but provides no credible evidence for its taxonomic position.

  7. Crystal structures of TM0549 and NE1324—two orthologs of E. coli AHAS isozyme III small regulatory subunit

    PubMed Central

    Petkowski, Janusz J.; Chruszcz, Maksymilian; Zimmerman, Matthew D.; Zheng, Heping; Skarina, Tatiana; Onopriyenko, Olena; Cymborowski, Marcin T.; Koclega, Katarzyna D.; Savchenko, Alexei; Edwards, Aled; Minor, Wladek

    2007-01-01

    Crystal structures of two orthologs of the regulatory subunit of acetohydroxyacid synthase III (AHAS, EC 2.2.1.6) from Thermotoga maritima (TM0549) and Nitrosomonas europea (NE1324) were determined by single-wavelength anomalous diffraction methods with the use of selenomethionine derivatives at 2.3 Å and 2.5 Å, respectively. TM0549 and NE1324 share the same fold, and in both proteins the polypeptide chain contains two separate domains of a similar size. Each protein contains a C-terminal domain with ferredoxin-type fold and an N-terminal ACT domain, of which the latter is characteristic for several proteins involved in amino acid metabolism. The ferredoxin domain is stabilized by a calcium ion in the crystal structure of NE1324 and by a Mg(H2O)6 2+ ion in TM0549. Both TM0549 and NE1324 form dimeric assemblies in the crystal lattice. PMID:17586771

  8. [Mg2+ ions affect the structure of the central domain of the 18S rRNA in the vicinity of the ribosomal protein S13 binding site].

    PubMed

    Ivanov, A V; Malygin, A A; Karpova, G G

    2013-01-01

    It is known that Mg2+ ions at high concentrations stabilize the structure of the 16S rRNA in a conformation favorable for binding to the ribosomal proteins in the course of the eubacterial 30S ribosomal subunits assembly in vitro. Effect of Mg2+ on the formation of the 18S rRNA structure at the 40S subunit assembly remains poorly explored. In this paper, we show that the sequentional increase of the Mg2+ concentration from 0.5 mM to 20 mM leads to a significant decrease of the affinity of recombinant human ribosomal protein S13 (rpS13e) to a RNA transcript corresponding to the central domain fragment of the 18S rRNA (18SCD). The regions near the rpS13e binding site in 18SCD (including the nucleotides of helices H20 and H22), whose availabilities to hydroxyl radicals were dependent on the Mg2+ concentration, were determined. It was found that increase of the concentrations of Mg2+ results in the enhanced accessibilities of nucleotides G933-C937 and C1006-A1009 in helix H22 and reduces those of nucleotides A1023, A1024, and A1028-S1026 in the helix H20. Comparison of the results obtained with the crystallographic data on the structure of the central domain of 18S rRNA in the 40S ribosomal subunit led to conclusion that increase of Mg2+ concentrations results in the reorientation of helices H20 and H24 relatively helices H22 and H23 to form a structure, in which these helices are positioned the same way as in 40S subunits. Hence, saturation of the central domain of 18S rRNA with coordinated Mg2+ ions causes the same changes in its structure as rpS13e binding does, and leads to decreasing of this domain affinity to the protein.

  9. The Subunit Structure of Benzylsuccinate Synthase†

    PubMed Central

    Li, Lei; Patterson, Dustin P.; Fox, Christel C.; Lin, Brian; Coschigano, Peter W.; Marsh, E. Neil G.

    2010-01-01

    Benzylsuccinate synthase is a member of the glycyl radical family of enzymes. It catalyzes the addition of toluene to fumarate to form benzylsuccinate as the first step in the anaerobic pathway of toluene fermentation. The enzyme comprises three subunits α, β and γ that in Thauera Aromatica T1 strain are encoded by the tutD, tutG and tutF genes respectively. The large α-subunit contains the essential glycine and cysteine residues that are conserved in all glycyl radical enzymes. However, the function of the small β- and γ-subunits has remained unclear. We have over-expressed all three subunits of benzylsuccinate synthase in E. coli, both individually and in combination. Co-expression of the γ-subunit (but not the β-subunit) is essential for efficient expression of the α-subunit. The benzylsuccinate synthase complex lacking the glycyl radical could be purified as an α2β2γ2 hexamer by nickel-affinity chromatography through a ‘His6’ affinity tag engineered onto the C-terminus of the α-subunit. Unexpectedly, BSS was found to contain two iron-sulfur clusters, one associated with the β-subunit and the other with the γ-subunit that appear to be necessary for the structural integrity of the complex. The spectroscopic properties of these clusters suggest that they are most likely [4Fe-4S] clusters. Removal of iron with chelating agents results in dissociation of the complex; similarly a mutant γ-subunit lacking the [4Fe-4S] cluster is unable to stabilize the α-subunit when the proteins are co-expressed. PMID:19159265

  10. Rotating proton pumping ATPases: subunit/subunit interactions and thermodynamics.

    PubMed

    Nakanishi-Matsui, Mayumi; Sekiya, Mizuki; Futai, Masamitsu

    2013-03-01

    In this article, we discuss single molecule observation of rotational catalysis by E. coli ATP synthase (F-ATPase) using small gold beads. Studies involving a low viscous drag probe showed the stochastic properties of the enzyme in alternating catalytically active and inhibited states. The importance of subunit interaction between the rotor and the stator, and thermodynamics of the catalysis are also discussed. "Single Molecule Enzymology" is a new trend for understanding enzyme mechanisms in biochemistry and physiology.

  11. Investigation of molluscan phylogeny on the basis of 18S rRNA sequences.

    PubMed

    Winnepenninckx, B; Backeljau, T; De Wachter, R

    1996-12-01

    The 18S rRNA sequences of 12 molluscs, representing the extant classes Gastropoda, Bivalvia, Polyplacophora, Scaphopoda, and Caudofoveata, were determined and compared with selected known 18S rRNA sequences of Metazoa, including other Mollusca. These data do not provide support for a close relationship between Platyhelminthes (Turbellaria) and Mollusca, but rather suggest that the latter group belongs to a clade of eutrochozoan coelomates. The 18S rRNA data fail to recover molluscan, bivalve, or gastropod monophyly. However, the branching pattern of the eutrochozoan phyla and classes is unstable, probably due to the explosive Cambrian radiation during which these groups arose. Similarly, the 18S rRNA data do not provide a reliable signal for the molluscan interclass relationships. Nevertheless, we obtained strong preliminary support for phylogenetic inferences at more restricted taxonomic levels, such as the monophyly of Polyplacophora, Caenogastropoda, Euthyneura, Heterodonta, and Arcoida.

  12. Detection of Babesia microti parasites by highly sensitive 18S rRNA reverse transcription PCR.

    PubMed

    Hanron, Amelia E; Billman, Zachary P; Seilie, Annette M; Chang, Ming; Murphy, Sean C

    2017-03-01

    Babesia are increasingly appreciated as a cause of transfusion-transmitted infection. Sensitive methods are needed to screen blood products. We report herein that B. microti 18S rRNA is over 1,000-fold more abundant than its coding genes, making reverse transcription PCR (RT-PCR) much more sensitive than PCR. Babesia 18S rRNA may be useful for screening the blood supply.

  13. PCR-based diversity estimates of artificial and environmental 18S rRNA gene libraries.

    PubMed

    Potvin, Marianne; Lovejoy, Connie

    2009-01-01

    Environmental clone libraries constructed using small subunit ribosomal RNA (rRNA) or other gene-specific primers have become the standard molecular approach for identifying microorganisms directly from their environment. This technique includes an initial polymerase chain reaction (PCR) amplification step of a phylogenetically useful marker gene using universal primers. Although it is acknowledged that such primers introduce biases, there have been few studies if any to date systematically examining such bias in eukaryotic microbes. We investigated some implications of such bias by constructing clone libraries using several universal primer pairs targeting rRNA genes. Firstly, we constructed artificial libraries using a known mix of small cultured pelagic arctic algae with representatives from five major lineages and secondly we investigated environmental samples using several primer pairs. No primer pair retrieved all of the original algae in the artificial clone libraries and all showed a favorable bias toward the dinoflagellate Polarella glacialis and a bias against the prasinophyte Micromonas and a pennate diatom. Several other species were retrieved by only one primer pair tested. Despite this, sequences from nine environmental libraries were diverse and contained representatives from all major eukaryotic clades expected in marine samples. Further, libraries from the same sample grouped together using Bray-Curtis clustering, irrespective of primer pairs. We conclude that environmental PCR-based techniques are sufficient to compare samples, but the total diversity will probably always be underestimated and relative abundance estimates should be treated with caution.

  14. 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

  15. Sodium channel auxiliary subunits.

    PubMed

    Tseng, Tsai-Tien; McMahon, Allison M; Johnson, Victoria T; Mangubat, Erwin Z; Zahm, Robert J; Pacold, Mary E; Jakobsson, Eric

    2007-01-01

    Voltage-gated ion channels are well known for their functional roles in excitable tissues. Excitable tissues rely on voltage-gated ion channels and their auxiliary subunits to achieve concerted electrical activity in living cells. Auxiliary subunits are also known to provide functional diversity towards the transport and biogenesis properties of the principal subunits. Recent interests in pharmacological properties of these auxiliary subunits have prompted significant amounts of efforts in understanding their physiological roles. Some auxiliary subunits can potentially serve as drug targets for novel analgesics. Three families of sodium channel auxiliary subunits are described here: beta1 and beta3, beta2 and beta4, and temperature-induced paralytic E (TipE). While sodium channel beta-subunits are encoded in many animal genomes, TipE has only been found exclusively in insects. In this review, we present phylogenetic analyses, discuss potential evolutionary origins and functional data available for each of these subunits. For each family, we also correlate the functional specificity with the history of evolution for the individual auxiliary subunits.

  16. 18S Ribosomal RNA Evaluation as Preanalytical Quality Control for Animal DNA

    PubMed Central

    Meli, Marina L.; Novacco, Marilisa; Borel, Nicole

    2016-01-01

    The 18S ribosomal RNA (rRNA) gene is present in all eukaryotic cells. In this study, we evaluated the use of this gene to verify the presence of PCR-amplifiable host (animal) DNA as an indicator of sufficient sample quality for quantitative real-time PCR (qPCR) analysis. We compared (i) samples from various animal species, tissues, and sample types, including swabs; (ii) multiple DNA extraction methods; and (iii) both fresh and formalin-fixed paraffin-embedded (FFPE) samples. Results showed that 18S ribosomal RNA gene amplification was possible from all tissue samples evaluated, including avian, reptile, and FFPE samples and most swab samples. A single swine rectal swab, which showed sufficient DNA quantity and the demonstrated lack of PCR inhibitors, nonetheless was negative by 18S qPCR. Such a sample specifically illustrates the improvement of determination of sample integrity afforded by inclusion of 18S rRNA gene qPCR analysis in addition to spectrophotometric analysis and the use of internal controls for PCR inhibition. Other possible applications for the described 18S rRNA qPCR are preselection of optimal tissue specimens for studies or preliminary screening of archived samples prior to acceptance for biobanking projects. PMID:27672657

  17. First report of an intraerythrocytic small piroplasm in wild Iberian lynx (Lynx pardinus).

    PubMed

    Luaces, Inés; Aguirre, Enara; García-Montijano, Marino; Velarde, Jorge; Tesouro, Miguel A; Sánchez, Celia; Galka, Margarita; Fernández, Pilar; Sainz, Angel

    2005-10-01

    A wild injured Iberian lynx (Lynx pardinus) was taken from the Sierra Morena population. During the health check small intraerythrocytic piroplasms, morphologically indistinguishable from other feline piroplasms, were observed in Wright-Giemsa-stained blood films. Amplification by polymerase chain reaction of a portion of the 18S nuclear small subunit (NSS) rRNA gene and sequencing revealed similarity of the unknown organism with sequences obtained from Pallas's cat from Mongolia and from a domestic cat in Spain. In a retrospective (1993-2003) study of 50 Iberian lynx tissue samples, no amplifications of the 18S NSS rRNA gene of the organism were obtained. This is the first report of a naturally occurring erythroparasitemia in the Iberian lynx and the first documented case of naturally occurring piroplasm infection in a free-ranging felid from Europe.

  18. Compositional properties and thermal adaptation of 18S rRNA in vertebrates

    PubMed Central

    Varriale, Annalisa; Torelli, Giuseppe; Bernardi, Giorgio

    2008-01-01

    In order to investigate the influence of temperature on the GC level of the paired sequences of ribosomal 18S RNAs in vertebrates, we have studied their base composition in cold- and warm-blooded vertebrates using a stem-by-stem comparison. We observed that a number of stems of 18S ribosomal RNAs (rRNAs) are variable among species and that the majority of such stems are GC richer in warm-blooded than in cold-blooded vertebrates. We also constructed the secondary structures of the 18S rRNAs of a polar fish, a marsupial, and a monotreme to compare them with those of temperate/tropical fishes and of eutherians, respectively. In these cases, differences similar to those already mentioned were found. We conclude that there is a correlation between stem stability and body temperature even within the relatively limited temperature range of vertebrates. PMID:18567811

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

    PubMed

    Schnare, Murray N; Gray, Michael W

    2011-10-14

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

  20. Phylogeny of chloromonas (chlorophyceae): A study of 18S ribosomal RNA gene sequences

    SciTech Connect

    Buchheim, M.A.; Buchheim, J.A.; Chapman, R.L.

    1997-04-01

    The unicellular, biflagellate genus Chloromonas differs from its ally, Chlamydomonas, primarily by the absence of pyrenoids in the vegetative stage of the former. As with most green flagellate genera, little is known about phylogenetic affinities within and among Chloromonas species. Phylogenetic analyses of nuclear-encoded small-subunit ribosomal RNA gene sequences demonstrate that a sampling of five Chloromonas taxa, obtained from major culture collections, do not form a monophyletic group. However, only three of these isolates, Chloromonas clathrata, Chloromonas serbinowi, and Chloromonas rosae, are diagnosable morphologically as Chloromonas species by the absence of a pyrenoid in the vegetative stage. The three diagnosable Chloromonas taxa form an alliance with two pyrenoid-bearing chlamydomonads, Chlamydomonas augustae and Chlamydomonas macrostellata. With the exception of Chloromonas serbinowi, which represents the basal lineage within the clade, each of the diagnosable Chloromonas taxa and their pyrenoid-bearing Chlamydomonas allies were isolated originally from mountain soils, snow, or cold peat. These observations suggest that hibitat, independent of pyrenoid status, may be most closely linked to the natural history of this clade of chlamydomonad flagellates. 51 refs., 3 figs., 3 tabs.

  1. Identification of the 18S-ribosomal-DNA genotypes of Acanthamoeba isolates from the Philippines.

    PubMed

    Rivera, W L; Adao, D E V

    2008-12-01

    Cyst morphology has been commonly used to identify the free-living amoeba Acanthamoeba to subgenus level. A more accurate and consistent method, based on the sequence analysis of the gene coding for the amoeba's small-subunit ribosomal RNA (Rns), has, however, been developed. There have been no attempts to identify the Acanthamoeba genotypes circulating in the Philippines. In this study, therefore, the ASA.S1 region of the Rns gene from 17 Acanthamoeba isolates, collected from soil, water and contact-lens storage cases in different regions of the Philippines, was sequenced. After the isolates were genotyped, using the BLAST program, their phylogenetic positions relative to known Acanthamoeba isolates were determined. For this, the model-based (GTR + Gamma) neighbour-joining, maximum-likelihood and Bayesian-inference analyses and the non-model-based maximum-parsimony analysis were used. All but two of the isolates were identified as the T5 or T4 genotypes, which are probably common in soil, water and contact-lens cases across the Philippines. The only other genotypes identified were T15 (as a single isolate from a contact-lens case) and T3 (as a single soil isolate).

  2. Taxonomic resolutions based on 18S rRNA genes: a case study of subclass copepoda.

    PubMed

    Wu, Shu; Xiong, Jie; Yu, Yuhe

    2015-01-01

    Biodiversity studies are commonly conducted using 18S rRNA genes. In this study, we compared the inter-species divergence of variable regions (V1-9) within the copepod 18S rRNA gene, and tested their taxonomic resolutions at different taxonomic levels. Our results indicate that the 18S rRNA gene is a good molecular marker for the study of copepod biodiversity, and our conclusions are as follows: 1) 18S rRNA genes are highly conserved intra-species (intra-species similarities are close to 100%); and could aid in species-level analyses, but with some limitations; 2) nearly-whole-length sequences and some partial regions (around V2, V4, and V9) of the 18S rRNA gene can be used to discriminate between samples at both the family and order levels (with a success rate of about 80%); 3) compared with other regions, V9 has a higher resolution at the genus level (with an identification success rate of about 80%); and 4) V7 is most divergent in length, and would be a good candidate marker for the phylogenetic study of Acartia species. This study also evaluated the correlation between similarity thresholds and the accuracy of using nuclear 18S rRNA genes for the classification of organisms in the subclass Copepoda. We suggest that sample identification accuracy should be considered when a molecular sequence divergence threshold is used for taxonomic identification, and that the lowest similarity threshold should be determined based on a pre-designated level of acceptable accuracy.

  3. Purification of the small mechanosensitive channel of Escherichia coli (MscS): the subunit structure, conduction, and gating characteristics in liposomes

    NASA Technical Reports Server (NTRS)

    Sukharev, Sergei

    2002-01-01

    The small mechanosensitive channel, MscS, is a part of the turgor-driven solute efflux system that protects bacteria from lysis in the event of osmotic downshift. It has been identified in Escherichia coli as a product of the orphan yggB gene, now called mscS (Levina et al., 1999, EMBO J. 18:1730). Here I show that that the isolated 31-kDa MscS protein is sufficient to form a functional mechanosensitive channel gated directly by tension in the lipid bilayer. MscS-6His complexes purified in the presence of octylglucoside and lipids migrate in a high-resolution gel-filtration column as particles of approximately 200 kDa. Consistent with that, the protein cross-linking patterns predict a hexamer. The channel reconstituted in soybean asolectin liposomes was activated by pressures of 20-60 mm Hg and displayed the same asymmetric I-V curve and slight anionic preference as in situ. At the same time, the single-channel conductance is proportional to the buffer conductivity in a wide range of salt concentrations. The rate of channel activation in response to increasing pressure gradient across the patch was slower than the rate of closure in response to decreasing steps of pressure gradient. Therefore, the open probability curves were recorded with descending series of pressures. Determination of the curvature of patches by video imaging permitted measurements of the channel activity as a function of membrane tension (gamma). Po(gamma) curves had the midpoint at 5.5 +/- 0.1 dyne/cm and gave estimates for the energy of opening DeltaG = 11.4 +/- 0.5 kT, and the transition-related area change DeltaA = 8.4 +/- 0.4 nm(2) when fitted with a two-state Boltzmann model. The correspondence between channel properties in the native and reconstituted systems is discussed.

  4. The SRP9/14 subunit of the signal recognition particle (SRP) is present in more than 20-fold excess over SRP in primate cells and exists primarily free but also in complex with small cytoplasmic Alu RNAs.

    PubMed Central

    Bovia, F; Fornallaz, M; Leffers, H; Strub, K

    1995-01-01

    The heterodimeric protein SRP9/14 bound to the Alu sequences of SRP RNA is essential for the translational control function of the signal recognition particle (SRP). The Alu RNAs of primate cells are believed to be derived from SRP RNA and have been shown to bind to an SRP14-related protein in vitro. We have used antibodies to characterize SRP9/14 and examine its association with small RNAs in vivo. Although SRP9 proteins are the same size in both rodent and primate cells, SRP14 subunits are generally larger in primate cells. An additional alanine-rich domain at the C-terminus accounts for the larger size of one human isoform. Although the other four SRP proteins are largely assembled into SRP in both rodent and primate cells, we found that the heterodimer SRP9/14 is present in 20-fold excess over SRP in primate cells. An increased synthesis rate of both proteins may contribute to their accumulation. The majority of the excess SRP9/14 is cytoplasmic and does not appear to be bound to any small RNAs; however, a significant fraction of a small cytoplasmic Alu RNA is complexed with SRP9/14 in a 8.5 S particle. Our findings that there is a large excess of SRP9/14 in primate cells and that Alu RNAs are bound to SRP9/14 in vivo suggest that this heterodimeric protein may play additional roles in the translational control of gene expression and/or Alu transcript metabolism. Images PMID:7542942

  5. Granulomatous prostatitis due to Cryptococcus neoformans: diagnostic usefulness of special stains and molecular analysis of 18S rDNA.

    PubMed

    Wada, R; Nakano, N; Yajima, N; Yoneyama, T; Wakasaya, Y; Murakami, C; Yamato, K; Yagihashi, S

    2008-01-01

    A 57-year-old Japanese man complained of pain on micturition. The prostate was of normal size but hard. Transrectal needle biopsy demonstrated granulomatous prostatitis with small focal abscesses. Staining with periodic acid-Schiff, Grocott's methenamine silver and Fontana-Masson revealed yeast-form fungus in the granulomas. The mucoid capsule of the fungus stained with mucicarmine. PCR specific for cryptococcal 18S rDNA using DNA extracted from the pathological specimen was positive, and the sequence was homologous to Cryptococcus neoformans. A diagnosis of cryptococcal granulomatous prostatitis was made. The patient was then found to suffer from meningitis and lung abscess, and was treated with amphotericin B and flucytosine. Careful histological and molecular studies are beneficial to reach the correct diagnosis and to prevent an unfavorable outcome of disseminated cryptococcosis.

  6. Vorticella Linnaeus, 1767 (Ciliophora, Oligohymenophora, Peritrichia) is a grade not a clade: redefinition of Vorticella and the families Vorticellidae and Astylozoidae using molecular characters derived from the gene coding for small subunit ribosomal RNA.

    PubMed

    Sun, Ping; Clamp, John; Xu, Dapeng; Kusuoka, Yasushi; Miao, Wei

    2012-01-01

    Recent phylogenetic analyses of the peritrich genus Vorticella have suggested that it might be paraphyletic, with one Vorticella species - Vorticella microstoma grouping with the swimming peritrichs Astylozoon and Opisthonecta in a distant clade. These results were based on very limited taxon sampling and thus could not be accepted as conclusive evidence for revising the generic classification. We tested paraphyly of the genus Vorticella by making a new analysis with a broad range of samples from three continents that yielded 52 new sequences of the gene coding for small subunit rRNA. Our results, together with the available sequences in Genbank, form a comprehensive set of data for the genus Vorticella. Analyses of these data showed that Vorticella microstoma morphotypes, Astylozoon, and Opisthonecta form a well-supported, monophyletic clade, that is distinct from and basal to the family Vorticellidae containing other species of Vorticella. Paraphyly of the genus Vorticella and family Vorticellidae was strongly confirmed by these results. Furthermore, the two clades of Vorticella identified by the SSU rRNA gene are so genetically diverse whereas the genetic distances within the one containing Vorticella microstoma morphotypes, Astylozoon, and Opisthonecta were so slight, which marked it as a separate family that must be defined by molecular characters in the absence of unifying morphological and morphogenetic characters. An emended characterization and status of the genus Vorticella, the families Vorticellidae and Astylozoidae are presented and discussed.

  7. Prevalence of microsporidiosis due to Enterocytozoon bieneusi and Encephalitozoon (Septata) intestinalis among patients with AIDS-related diarrhea: determination by polymerase chain reaction to the microsporidian small-subunit rRNA gene.

    PubMed

    Coyle, C M; Wittner, M; Kotler, D P; Noyer, C; Orenstein, J M; Tanowitz, H B; Weiss, L M

    1996-11-01

    Microsporidia are emerging as opportunistic pathogens in patients with AIDS. Enterocytozoon bieneusi and Encephalitozoon (Septata) intestinalis have been implicated in enteric infections in AIDS patients with chronic diarrhea, a wasting syndrome, and malabsorption. We used the polymerase chain reaction (PCR) and primers that amplify the conserved regions of the small-subunit rRNA (SSU-rRNA) gene of E. bieneusi and E. intestinalis in tissue specimens from HIV-infected patients with and without diarrhea to examine the association between microsporidia and diarrhea in patients with AIDS. Tissue specimens were obtained from 68 patients with AIDS and diarrhea (mean CD4 lymphocyte count, 21/mm3) and 43 AIDS patients without diarrhea (mean CD4 lymphocyte count, 60/mm3). By means of PCR with use of the SSU-rRNA primers specific for E. bieneusi and E. intestinalis, we found that 44% of patients with diarrhea were infected with microsporidia, whereas only 2.3% of the patients without diarrhea were infected with microsporidia (P < .001). There was a clear association between the presence of microsporidia and diarrhea. In addition, the SSU-rRNA primers proved to be sensitive and specific when used in this clinical setting.

  8. Changes in Growth CO2 Result in Rapid Adjustments of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Small Subunit Gene Expression in Expanding and Mature Leaves of Rice1

    PubMed Central

    Gesch, Russ W.; Boote, Kenneth J.; Vu, Joseph C.V.; Hartwell Allen, L.; Bowes, George

    1998-01-01

    The accumulation of soluble carbohydrates resulting from growth under elevated CO2 may potentially signal the repression of gene activity for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcS). To test this hypothesis we grew rice (Oryza sativa L.) under ambient (350 μL L−1) and high (700 μL L−1) CO2 in outdoor, sunlit, environment-controlled chambers and performed a cross-switching of growth CO2 concentration at the late-vegetative phase. Within 24 h, plants switched to high CO2 showed a 15% and 23% decrease in rbcS mRNA, whereas plants switched to ambient CO2 increased 27% and 11% in expanding and mature leaves, respectively. Ribulose-1,5-bisphosphate carboxylase/oxygenase total activity and protein content 8 d after the switch increased up to 27% and 20%, respectively, in plants switched to ambient CO2, but changed very little in plants switched to high CO2. Plants maintained at high CO2 showed greater carbohydrate pool sizes and lower rbcS transcript levels than plants kept at ambient CO2. However, after switching growth CO2 concentration, there was not a simple correlation between carbohydrate and rbcS transcript levels. We conclude that although carbohydrates may be important in the regulation of rbcS expression, changes in total pool size alone could not predict the rapid changes in expression that we observed. PMID:9765537

  9. Morphology and small subunit (SSU) rRNA gene sequence of the new brackish water ciliate Neobakuella flava n. g., n. sp. (Ciliophora, Spirotricha, Bakuellidae) and SSU rRNA gene sequences of six additional hypotrichs from Korea.

    PubMed

    Li, Liqiong; Khan, Sadia Nawroz; Ji, Daode; Shin, Mann Kyoon; Berger, Helmut

    2011-01-01

    The morphology and the small subunit (SSU) rRNA gene sequence of the hypotrich Neobakuella flava n. g., n. sp. from the estuary of the Taehwagang River (Ulsan, South Korea) were investigated. The three frontal cirri, the composition of the midventral complex of cirral pairs and rows, and the simple dorsal kinety pattern of three bipolar kineties assign it to the urostyloid taxon Bakuellidae. The increased number of buccal and parabuccal cirri, the presence of transverse cirri, and more than one left marginal row, as well as the lack of caudal cirri separate Neobakuella n. g. from the other bakuellids. Neobakuella flava n. sp. has many 0.3 μm sized green and/or yellow usually dark-green cortical granules and some sparsely distributed, 2 × 1 μm sized grass green with yellowish shimmer granules. The gene sequence data indicate a close relationship with Diaxonella and a distinct separation from the bakuellid Metaurostylopsis and parabirojimid Parabirojimia. The SSU rRNA gene sequences of four further urostyloids (i.e. Diaxonella pseudorubra, Anteholosticha monilata, Metaurostylopsis struederkypkeae, Pseudourostyla cristata) and two stylonychines (i.e. Sterkiella cavicola, Sterkiella histriomuscorum) from Korea were analyzed. Anteholosticha monilata, type of the genus, is clearly separated from the Holosticha clade, supporting the morphological separation from Holosticha. Sterkiella cavicola, type of Sterkiella, clusters within the stylonychines and is obviously closely related with S. histriomuscorum.

  10. The gene for human U2 snRNP auxiliary factor small 35-kDa subunit (U2AF1) maps to the progressive myoclonus epilepsy (EPM1) critical region on chromosome 21q22.3

    SciTech Connect

    Lalioti, M.D.; Rossier, C.; Antonarakis, S.E.

    1996-04-15

    We used targeted exon trapping to clone portions of genes from human chromosome 21q22.3. One trapped sequence showed complete homology with the cDNA of human U2AF{sup 35} (M96982; HGM-approved nomenclature U2AF1), which encodes for the small 35-kDa subunit of the U2 snRNP auxiliary factor. Using the U2AF1 cDNA as a probe, we mapped this gene to cosmid Q15D2, a P1, and YAC 350F7 of the Chumakov et al. contig, close to the cystathionine-{beta}-synthase gene (CBS) on 21q22.3. This localization was confirmed by PCR using oligonucleotides from the 3{prime} UTR and by FISH. As U2AF1 associated with a number of different factors during mRNA splicing, overexpression in trisomy 21 individuals could contribute to some Down syndrome phenotypes by interfering with the splicing process. Furthermore, because this gene maps in the critical region for the progressive myoclonus epilepsy I locus (EPM1), mutation analysis will be carried out in patients to evaluate the potential role of U2AF1 as a candidate for EPM1. 24 refs., 1 fig.

  11. Silencing expression of the catalytic subunit of DNA-dependent protein kinase by small interfering RNA sensitizes human cells for radiation-induced chromosome damage, cell killing, and mutation

    NASA Technical Reports Server (NTRS)

    Peng, Yuanlin; Zhang, Qinming; Nagasawa, Hatsumi; Okayasu, Ryuichi; Liber, Howard L.; Bedford, Joel S.

    2002-01-01

    Targeted gene silencing in mammalian cells by RNA interference (RNAi) using small interfering RNAs (siRNAs) was recently described by Elbashir et al. (S. M. Elbashir et al., Nature (Lond.), 411: 494-498, 2001). We have used this methodology in several human cell strains to reduce expression of the Prkdc (DNA-PKcs) gene coding for the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) that is involved in the nonhomologous end joining of DNA double-strand breaks. We have also demonstrated a radiosensitization for several phenotypic endpoints of radiation damage. In low-passage normal human fibroblasts, siRNA knock-down of DNA-PKcs resulted in a reduced capacity for restitution of radiation-induced interphase chromosome breaks as measured by premature chromosome condensation, an increased yield of acentric chromosome fragments at the first postirradiation mitosis, and an increased radiosensitivity for cell killing. For three strains of related human lymphoblasts, DNA-PKcs-targeted siRNA transfection resulted in little or no increase in radiosensitivity with respect to cell killing, a 1.5-fold decrease in induced mutant yield in TK6- and p53-null NH32 cells, but about a 2-fold increase in induced mutant yield in p53-mutant WTK1 cells at both the hypoxanthine quanine phosphoribosyl transferase (hprt) and the thymidine kinase loci.

  12. Structural dynamics and ssDNA binding activity of the three N-terminal domains of the large subunit of Replication Protein A from small angle X-ray scattering

    SciTech Connect

    Pretto, Dalyir I.; Tsutakawa, Susan; Brosey, Chris A.; Castillo, Amalchi; Chagot, Marie-Eve; Smith, Jarrod A.; Tainer, John A.; Chazin, Walter J.

    2010-03-11

    Replication Protein A (RPA) is the primary eukaryotic ssDNA binding protein utilized in diverse DNA transactions in the cell. RPA is a heterotrimeric protein with seven globular domains connected by flexible linkers, which enable substantial inter-domain motion that is essential to its function. Small angle X-ray scattering (SAXS) experiments on two multi-domain constructs from the N-terminus of the large subunit (RPA70) were used to examine the structural dynamics of these domains and their response to the binding of ssDNA. The SAXS data combined with molecular dynamics simulations reveal substantial interdomain flexibility for both RPA70AB (the tandem high affinity ssDNA binding domains A and B connected by a 10-residue linker) and RPA70NAB (RPA70AB extended by a 70-residue linker to the RPA70N protein interaction domain). Binding of ssDNA to RPA70NAB reduces the interdomain flexibility between the A and B domains, but has no effect on RPA70N. These studies provide the first direct measurements of changes in orientation of these three RPA domains upon binding ssDNA. The results support a model in which RPA70N remains structurally independent of RPA70AB in the DNA bound state and therefore freely available to serve as a protein recruitment module.

  13. Taenia spp.: 18S rDNA microsatellites for molecular systematic diagnosis.

    PubMed

    Foronda, P; Casanova, J C; Martinez, E; Valladares, B; Feliu, C

    2005-06-01

    The 18S rDNA gene of adult worms of Taenia parva found in Genetta genetta in the Iberian Peninsula and larval stages of T. pisiformis from the wild rabbit (Oryctolagus cuniculus) in Tenerife (Canary Islands) were amplified and sequenced. The sequences of the 18S rDNA gene of T. parva (1768 bp) and T. pisiformis (1760 bp) are reported for the first time (GenBank accession nos. AJ555167-AJ555168 and AJ555169-AJ555170, respectively). In 168 alignment positions microsatellites in the 18S rDNA of both taxa were detected for the first time (TGC in T. parva and TGCT in T. pisiformis) and differences in their sequences with different repetition numbers were observed. The use of nucleotide sequences of this gene in the resolution of systematic problems in cestodes is discussed with reference to the systematic status of Taenia spp. and mainly in human taeniids such as T. solium, T. saginata, and Asian human isolates of Taenia.

  14. Molecular systematics of Volvocales (Chlorophyceae, Chlorophyta) based on exhaustive 18S rRNA phylogenetic analyses.

    PubMed

    Nakada, Takashi; Misawa, Kazuharu; Nozaki, Hisayoshi

    2008-07-01

    The taxonomy of Volvocales (Chlorophyceae, Chlorophyta) was traditionally based solely on morphological characteristics. However, because recent molecular phylogeny largely contradicts the traditional subordinal and familial classifications, no classification system has yet been established that describes the subdivision of Volvocales in a manner consistent with the phylogenetic relationships. Towards development of a natural classification system at and above the generic level, identification and sorting of hundreds of sequences based on subjective phylogenetic definitions is a significant step. We constructed an 18S rRNA gene phylogeny based on 449 volvocalean sequences collected using exhaustive BLAST searches of the GenBank database. Many chimeric sequences, which can cause fallacious phylogenetic trees, were detected and excluded during data collection. The results revealed 21 strongly supported primary clades within phylogenetically redefined Volvocales. Phylogenetic classification following PhyloCode was proposed based on the presented 18S rRNA gene phylogeny along with the results of previous combined 18S and 26S rRNA and chloroplast multigene analyses.

  15. 18S rRNA gene sequencing identifies a novel species of Henneguya parasitizing the gills of the channel catfish (Ictaluridae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the southeastern United States, the channel catfish Ictalurus punctatus is a host to at least eight different species of myxozoan parasites belonging to the genus Henneguya, four of which have been characterized molecularly using sequencing of the small subunit ribosomal RNA gene (SSU rRNA). Howe...

  16. Ribosomal and mitochondrial DNA analysis of Trichuridae nematodes of carnivores and small mammals.

    PubMed

    Guardone, Lisa; Deplazes, Peter; Macchioni, Fabio; Magi, Marta; Mathis, Alexander

    2013-10-18

    Several species of Trichuridae nematodes can infect dogs, cats and wild mammals. The diagnosis of these infections relies on the microscopic identification of eggs which are characterized by a similar "lemon" shape and polar plugs in all Trichuridae. Thus, morphological diagnosis to species level is challenging. The use of biomolecular diagnostic methods is desirable but very little genetic data are known from Trichuridae of carnivores and small mammals. The aim of this work was to genetically characterize several species of Trichuridae that can affect dogs, cats and wild mammals, as a basis to develop molecular diagnostic tests. Specimens (adult worms or eggs) of Eucoleus aerophilus (syn. Capillaria aerophila), Eucoleus boehmi (syn. Capillaria boehmi), Pearsonema plica (syn. Capillaria plica), Aonchotheca putorii (syn. Capillaria putorii), Calodium hepaticum (syn. Capillaria hepatica), Calodium splenaecum (syn. Capillaria splenaeca) and Trichuris vulpis were obtained from carcasses of red foxes, feces of dogs, the liver of a vole and from the spleen of Crocidura sp. Parts of the small subunit rRNA (18S rRNA) gene and of the mitochondrial cytochrome c oxidase subunit I (cox 1 mtDNA) gene were amplified from the above mentioned nematodes, yielding the first 18S rRNA gene sequences of all the capillariid nematodes and the first cox 1 mtDNA sequences of E. boehmi, P. plica, C. hepaticum, A. putorii and T. vulpis. The 18S rRNA gene is highly conserved among the different species and not suitable as a target for specific diagnostic oligonucleotides. However, these sequences contribute to a better understanding of the complex taxonomic relations among Trichuridae. Indeed, a dendrogram based on the 18S rRNA gene locus supports the latest taxonomic revision. Interspecies divergence was much higher at the cox 1 mtDNA gene locus, rendering it suitable for DNA barcoding and particularly valuable in resolving closely related species. Furthermore, the mitochondrial genetic

  17. The Proteasome Subunit Rpn8 Interacts with the Small Nucleolar RNA Protein (snoRNP) Assembly Protein Pih1 and Mediates Its Ubiquitin-independent Degradation in Saccharomyces cerevisiae.

    PubMed

    Paci, Alexandr; Liu, Peter X H; Zhang, Lingjie; Zhao, Rongmin

    2016-05-27

    Pih1 is a scaffold protein of the Rvb1-Rvb2-Tah1-Pih1 (R2TP) protein complex, which is conserved in fungi and animals. The chaperone-like activity of the R2TP complex has been implicated in the assembly of multiple protein complexes, such as the small nucleolar RNA protein complex. However, the mechanism of the R2TP complex activity in vivo and the assembly of the complex itself are still largely unknown. Pih1 is an unstable protein and tends to aggregate when expressed alone. The C-terminal fragment of Pih1 contains multiple destabilization factors and acts as a degron when fused to other proteins. In this study, we investigated Pih1 interactors and identified a specific interaction between Pih1 and the proteasome subunit Rpn8 in yeast Saccharomyces cerevisiae when HSP90 co-chaperone Tah1 is depleted. By analyzing truncation mutants, we identified that the C-terminal 30 amino acids of Rpn8 are sufficient for the binding to Pih1 C terminus. With in vitro and in vivo degradation assays, we showed that the Pih1 C-terminal fragment Pih1(282-344) is able to induce a ubiquitin-independent degradation of GFP. Additionally, we demonstrated that truncation of the Rpn8 C-terminal disordered region does not affect proteasome assembly but specifically inhibits the degradation of the GFP-Pih1(282-344) fusion protein in vivo and Pih1 in vitro We propose that Pih1 is a ubiquitin-independent proteasome substrate, and the direct interaction with Rpn8 C terminus mediates its proteasomal degradation.

  18. Characterization of the Dominant and Rare Members of a Young Hawaiian Soil Bacterial Community with Small-Subunit Ribosomal DNA Amplified from DNA Fractionated on the Basis of Its Guanine and Cytosine Composition

    PubMed Central

    Nüsslein, Klaus; Tiedje, James M.

    1998-01-01

    The small-subunit ribosomal DNA (rDNA) diversity was found to be very high in a Hawaiian soil community that might be expected to have lower diversity than the communities in continental soils because the Hawaiian soil is geographically isolated and only 200 years old, is subjected to a constant climate, and harbors low plant diversity. Since an underlying community structure could not be revealed by analyzing the total eubacterial rDNA, we first fractionated the DNA on the basis of guanine-plus-cytosine (G+C) content by using bis-benzimidazole and equilibrium centrifugation and then analyzed the bacterial rDNA amplified from a fraction with a high biomass (63% G+C fraction) and a fraction with a low biomass (35% G+C fraction). The rDNA clone libraries were screened by amplified rDNA restriction analysis to determine phylotype distribution. The dominant biomass reflected by the 63% G+C fraction contained several dominant phylotypes, while the community members that were less successful (35% G+C fraction) did not show dominance but there was a very high diversity of phylotypes. Nucleotide sequence analysis revealed taxa belonging to the groups expected for the G+C contents used. The dominant phylotypes in the 63% G+C fraction were members of the Pseudomonas, Rhizobium-Agrobacterium, and Rhodospirillum assemblages, while all of the clones sequenced from the 35% G+C fraction were affiliated with several Clostridium assemblages. The two-step rDNA analysis used here uncovered more diversity than can be detected by direct rDNA analysis of total community DNA. The G+C separation step is also a way to detect some of the less dominant organisms in a community. PMID:9546163

  19. Evaluation of PCR amplification bias by terminal restriction fragment length polymorphism analysis of small-subunit rRNA and mcrA genes by using defined template mixtures of methanogenic pure cultures and soil DNA extracts.

    PubMed

    Lueders, Tillmann; Friedrich, Michael W

    2003-01-01

    Terminal restriction fragment length polymorphism (T-RFLP) analysis is a widely used method for profiling microbial community structure in different habitats by targeting small-subunit (SSU) rRNA and also functional marker genes. It is not known, however, whether relative gene frequencies of individual community members are adequately represented in post-PCR amplicon frequencies as shown by T-RFLP. In this study, precisely defined artificial template mixtures containing genomic DNA of four different methanogens in various ratios were prepared for subsequent T-RFLP analysis. PCR amplicons were generated from defined mixtures targeting not only the SSU rRNA but also the methyl-coenzyme M reductase (mcrA/mrtA) genes of methanogens. Relative amplicon frequencies of microorganisms were quantified by comparing fluorescence intensities of characteristic terminal restriction fragments. SSU ribosomal DNA (rDNA) template ratios in defined template mixtures of the four-membered community were recovered absolutely by PCR-T-RFLP analysis, which demonstrates that the T-RFLP analysis evaluated can give a quantitative view of the template pool. SSU rDNA-targeted T-RFLP analysis of a natural community was found to be highly reproducible, independent of PCR annealing temperature, and unaffected by increasing PCR cycle numbers. Ratios of mcrA-targeted T-RFLP analysis were biased, most likely by PCR selection due to the degeneracy of the primers used. Consequently, for microbial community analyses, each primer system used should be evaluated carefully for possible PCR bias. In fact, such bias can be detected by using T-RFLP analysis as a tool for the precise quantification of the PCR product pool.

  20. Protein synthesis by ribosomes with tethered subunits.

    PubMed

    Orelle, Cédric; Carlson, Erik D; Szal, Teresa; Florin, Tanja; Jewett, Michael C; Mankin, Alexander S

    2015-08-06

    The ribosome is a ribonucleoprotein machine responsible for protein synthesis. In all kingdoms of life it is composed of two subunits, each built on its own ribosomal RNA (rRNA) scaffold. The independent but coordinated functions of the subunits, including their ability to associate at initiation, rotate during elongation, and dissociate after protein release, are an established model of protein synthesis. Furthermore, the bipartite nature of the ribosome is presumed to be essential for biogenesis, since dedicated assembly factors keep immature ribosomal subunits apart and prevent them from translation initiation. Free exchange of the subunits limits the development of specialized orthogonal genetic systems that could be evolved for novel functions without interfering with native translation. Here we show that ribosomes with tethered and thus inseparable subunits (termed Ribo-T) are capable of successfully carrying out protein synthesis. By engineering a hybrid rRNA composed of both small and large subunit rRNA sequences, we produced a functional ribosome in which the subunits are covalently linked into a single entity by short RNA linkers. Notably, Ribo-T was not only functional in vitro, but was also able to support the growth of Escherichia coli cells even in the absence of wild-type ribosomes. We used Ribo-T to create the first fully orthogonal ribosome-messenger RNA system, and demonstrate its evolvability by selecting otherwise dominantly lethal rRNA mutations in the peptidyl transferase centre that facilitate the translation of a problematic protein sequence. Ribo-T can be used for exploring poorly understood functions of the ribosome, enabling orthogonal genetic systems, and engineering ribosomes with new functions.

  1. Optical and electrical stability of viral-templated copper sulfide (Cu1.8S) films

    NASA Astrophysics Data System (ADS)

    Shahriar Zaman, Mohammed; Bernard Grajeda, Gabriel; Haberer, Elaine D.

    2014-04-01

    The optical and electrical stabilities of viral-templated non-stoichiometric copper sulfide, digenite (Cu1.8S) films were investigated. The films were composed of large agglomerates of randomly aligned Cu1.8S-coated M13 filamentous phage. Free carrier optical absorption associated with localized surface plasmon resonance (LSPR) was observed in the near infrared spectral region, and the films were electrically active, displaying a linear current-voltage relationship. Under ambient conditions, the magnitude of the LSPR absorption increased, following a power law relationship with time, and the electrical resistance of viral-templated films decreased significantly. In contrast, the resistance of films stored under low oxygen, low humidity conditions experienced a smaller reduction in electrical resistance. Changes in optical and electrical film properties under ambient conditions were associated with an increase in free carrier concentration within the copper chalcogenide material due to oxygen exposure. X-ray photoelectron spectroscopy was used to relate this increase in free carrier concentration to compositional changes on the viral-templated material surface.

  2. Loop-mediated isothermal amplification targeting 18S ribosomal DNA for rapid detection of Acanthamoeba.

    PubMed

    Yang, Hye-Won; Lee, Yu-Ran; Inoue, Noboru; Jha, Bijay Kumar; Danne, Dinzouna-Boutamba Sylvatrie; Kim, Hong-Kyun; Lee, Junhun; Goo, Youn-Kyoung; Kong, Hyun-Hee; Chung, Dong-Il; Hong, Yeonchul

    2013-06-01

    Amoebic keratitis (AK) caused by Acanthamoeba is one of the most serious corneal infections. AK is frequently misdiagnosed initially as viral, bacterial, or fungal keratitis, thus ensuring treatment delays. Accordingly, the early detection of Acanthamoeba would contribute significantly to disease management and selection of an appropriate anti-amoebic therapy. Recently, the loop-mediated isothermal amplification (LAMP) method has been applied to the clinical diagnosis of a range of infectious diseases. Here, we describe a rapid and efficient LAMP-based method targeting Acanthamoeba 18S rDNA gene for the detection of Acanthamoeba using clinical ocular specimens in the diagnosis of AK. Acanthamoeba LAMP assays detected 11 different strains including all AK-associated species. The copy number detection limit for a positive signal was 10 DNA copies of 18S rDNA per reaction. No cross-reactivity with the DNA of fungi or other protozoa was observed. The sensitivity of LAMP assay was higher than those of Nelson primer PCR and JDP primer PCR. In the present study, LAMP assay based on directly heat-treated samples was found to be as efficient at detecting Acanthamoeba as DNA extracted using a commercial kit, whereas PCR was only effective when commercial kit-extracted DNA was used. This study showed that the devised Acanthamoeba LAMP assay could be used to diagnose AK in a simple, sensitive, and specific manner.

  3. Detecting morphological convergence in true fungi, using 18S rRNA gene sequence data.

    PubMed

    Berbee, M L; Taylor, J W

    1992-01-01

    For the true fungi, phylogenetic relationships inferred from 18S ribosomal DNA sequence data agree with morphology when (1) the fungi exhibit diagnostic morphological characters, (2) the sequence-based phylogenetic groups are statistically supported, and (3) the ribosomal DNA evolves at roughly the same rate in the lineages being compared. 18S ribosomal RNA gene sequence data and biochemical data provide a congruent definition of true fungi. Sequence data support the traditional fungal subdivisions Ascomycotina and Basidiomycotina. In conflict with morphology, some zygomycetes group with chytrid water molds rather than with other terrestrial fungi, possibly owing to unequal rates of nucleotide substitutions among zygomycete lineages. Within the ascomycetes, the taxonomic consequence of simple or reduced morphology has been a proliferation of mutually incongruent classification systems. Sequence data provide plausible resolution of relationships for some cases where reduced morphology has created confusion. For example, phylogenetic trees from rDNA indicate that those morphologically simple ascomycetes classified as yeasts are polyphyletic and that forcible spore discharge was lost convergently from three lineages of ascomycetes producing flask-like fruiting bodies.

  4. Loop-Mediated Isothermal Amplification Targeting 18S Ribosomal DNA for Rapid Detection of Acanthamoeba

    PubMed Central

    Yang, Hye-Won; Lee, Yu-Ran; Inoue, Noboru; Jha, Bijay Kumar; Danne, Dinzouna-Boutamba Sylvatrie; Kim, Hong-Kyun; Lee, Junhun; Goo, Youn-Kyoung; Kong, Hyun-Hee; Chung, Dong-Il

    2013-01-01

    Amoebic keratitis (AK) caused by Acanthamoeba is one of the most serious corneal infections. AK is frequently misdiagnosed initially as viral, bacterial, or fungal keratitis, thus ensuring treatment delays. Accordingly, the early detection of Acanthamoeba would contribute significantly to disease management and selection of an appropriate anti-amoebic therapy. Recently, the loop-mediated isothermal amplification (LAMP) method has been applied to the clinical diagnosis of a range of infectious diseases. Here, we describe a rapid and efficient LAMP-based method targeting Acanthamoeba 18S rDNA gene for the detection of Acanthamoeba using clinical ocular specimens in the diagnosis of AK. Acanthamoeba LAMP assays detected 11 different strains including all AK-associated species. The copy number detection limit for a positive signal was 10 DNA copies of 18S rDNA per reaction. No cross-reactivity with the DNA of fungi or other protozoa was observed. The sensitivity of LAMP assay was higher than those of Nelson primer PCR and JDP primer PCR. In the present study, LAMP assay based on directly heat-treated samples was found to be as efficient at detecting Acanthamoeba as DNA extracted using a commercial kit, whereas PCR was only effective when commercial kit-extracted DNA was used. This study showed that the devised Acanthamoeba LAMP assay could be used to diagnose AK in a simple, sensitive, and specific manner. PMID:23864737

  5. Substitution of Azotobacter vinelandii hydrogenase small-subunit cysteines by serines can create insensitivity to inhibition by O2 and preferentially damages H2 oxidation over H2 evolution.

    PubMed Central

    McTavish, H; Sayavedra-Soto, L A; Arp, D J

    1995-01-01

    Mutants in which conserved cysteines 294, 297 or 64 and 65 of the Azotobacter vinelandii hydrogenase small subunit were replaced by serines were studied. Cysteines 294 and 297 are homologous to cysteines 246 and 249 of the Desulfovibrio gigas hydrogenase, and these cysteines are ligands to the [3Fe-4S] clusters (A. Volbeda, M.-H. Charon, C. Piras, E. C. Hatchikian, M. Frey, and J. C. Fontecilla-Camps, Nature (London) 373:580-587, 1995). Cysteine 65 is homologous to cysteine 20 of the D. gigas hydrogenase, and this cysteine is a ligand to the proximal [4Fe-4S] cluster. All three mutants retained some hydrogenase activity. All three mutants studied had H2 oxidation-to-H2 evolution activity ratios with whole cells of approximately 1.5, compared with 46 for the wild type. The changes preferentially deplete H2 oxidation activity, while having less effect on evolution. The K64,65C-->S hydrogenase was partially purified and had a specific activity for the evolution reaction that was 22% that of the wild type, while the oxidation-specific activity was 2% that of the wild type. Because cysteine 65 provides a ligand to the proximal [4Fe-4S] cluster, this cluster can be altered without entirely eliminating enzyme activity. Likewise, the detection of H2 evolution and H2 oxidation activities with whole cells and membranes of the K294C-->S and K297C-->S mutants indicates that the [3Fe-4S] cluster can also be altered or possibly eliminated without entirely eliminating enzyme activity. Membranes with K294C-->S or K297C-->S hydrogenase were uninhibited by O2 in H2 oxidation and uninhibited by H2 in H2 evolution. Wild-type membranes and membranes with K64,65C-->S hydrogenase were both sensitive to these inhibitors. These data indicate that the [3Fe-4S] cluster controls the reversible inhibition of hydrogenase activity by O2 or H2. PMID:7608067

  6. 18S rRNA suggests that Entoprocta are protostomes, unrelated to Ectoprocta.

    PubMed

    Mackey, L Y; Winnepenninckx, B; De Wachter, R; Backeljau, T; Emschermann, P; Garey, J R

    1996-05-01

    The Ento- and Ectoprocta are sometimes placed together in the Bryozoa, which have variously been regarded as proto- or deuterostomes. However, Entoprocta have also been allied to the pseudocoelomates, while Ectoprocta are often united with the Brachiopoda and Phoronida in the (super)phylum Lophophorata. Hence, the phylogenetic relationships of these taxa are still much debated. We determined complete 18S rRNA sequences of two entoprocts, an ectoproct, an inarticulate brachiopod, a phoronid, two annelids, and a platyhelminth. Phylogenetic analyses of these data show that (1) entoprocts and lophophorates have spiralian, protostomous affinities, (2) Ento- and Ectoprocta are not sister taxa, (3) phoronids and brachiopods form a monophyletic clade, and (4) neither Ectoprocta or Annelida appear to be monophyletic. Both deuterostomous and pseudocoelomate features may have arisen at least two times in evolutionary history. These results advocate a Spiralia-Radialia-based classification rather than one based on the Protostomia-Deuterostomia concept.

  7. Phylogenetic relationships of Spiruromorpha from birds of prey based on 18S rDNA.

    PubMed

    Honisch, M; Krone, O

    2008-06-01

    A total of 153 free-ranging birds from Germany belonging to 15 species were examined for nematodes in their digestive and respiratory tracts. In 51.7% of the birds 14 different nematode species were found: the intestinal ascarids Porrocaecum depressum and P. angusticolle, the strongylid Hovorkonema variegatum, which inhabits the trachea and bronchi, the hairworms Eucoleus dispar and Capillaria tenuissima isolated from the digestive system, the spirurid nematodes Cyrnea leptoptera, C. mansioni, C. seurati, Microtetrameres cloacitectus, Physaloptera alata, P. apivori, Synhimantus hamatus and S. laticeps, which inhabit the proventriculus and gizzard of the raptors, and the spirurid nematode Serratospiculum tendo, which lives in the air sacs. To revise their systematic positions the ribosomal 18S gene regions of the nematode species were analysed and a phylogenetic tree was constructed. The molecular data confirmed the morphological systematics, except the spirurid family Physalopteridae, which grouped together with the Acuariidae.

  8. 18S ribosomal DNA genotypes of Acanthamoeba species isolated from contact lens cases in the Philippines.

    PubMed

    Rivera, Windell L; Adao, Davin Edric V

    2009-10-01

    This study was carried out to document the genotypes of Acanthamoeba present in contact lens cases from 50 randomly selected contact lens wearers living in Quezon City, Metro Manila, Philippines. Acanthamoeba species were isolated from eight (16%) in 50 contact lens cases examined. We analyzed partial 18S ribosomal DNA (Rns) sequences of the eight isolates and found that the sequence differences were sufficient to distinguish the genotypes. After the isolates were genotyped, using the Basic Local Alignment Search Tool program, their phylogenetic positions relative to known Acanthamoeba isolates were determined. The model-based (GTR+Gamma+Iota) neighbor-joining, maximum likelihood, and Bayesian inference analyses, as well as the non-model-based maximum parsimony analysis were used. Results showed that of the eight isolates, six were Rns genotype T5 while two were Rns genotype T4. This present study indicates that genotype T5 is also a common contaminant in contact lens storage cases.

  9. Novelty in phylogeny of gastrotricha: evidence from 18S rRNA gene.

    PubMed

    Wirz, A; Pucciarelli, S; Miceli, C; Tongiorgi, P; Balsamo, M

    1999-11-01

    Gastrotricha form a phylum which is crucial for defining the origin of pseudocoelomates, in that they share a number of characters with Rotifera and Nematoda but also with acoelomates, and even the evolutionary relationships within the phylum are anything but defined. For this reason the first extensive molecular data on Gastrotricha from the 18S rRNA sequences of both orders have been obtained and analyzed. Sequence analyses show that the phylum Gastrotricha is strictly monophyletic along an evolutionary line quite distinct from that of both Rotifera and Nematoda. A new view of the evolutionary history of the phylum Gastrotricha is put forward, in which Chaetonotida, and not Macrodasyida, are the most primitive forms of the group, contrary to the commonly held view. A polyphyletic origin of aschelminthes is supported, and the misleading term pseudocoelomates should be discarded.

  10. The phylogenetic status of arthropods, as inferred from 18S rRNA sequences.

    PubMed

    Turbeville, J M; Pfeifer, D M; Field, K G; Raff, R A

    1991-09-01

    Partial 18S rRNA sequences of five chelicerate arthropods plus a crustacean, myriapod, insect, chordate, echinoderm, annelid, and platyhelminth were compared. The sequence data were used to infer phylogeny by using a maximum-parsimony method, an evolutionary-distance method, and the evolutionary-parsimony method. The phylogenetic inferences generated by maximum-parsimony and distance methods support both monophyly of the Arthropoda and monophyly of the Chelicerata within the Arthropoda. These results are congruent with phylogenies based on rigorous cladistic analyses of morphological characters. Results support the inclusion of the Arthropoda within a spiralian or protostome coelomate clade that is the sister group of a deuterostome clade, refuting the hypothesis that the arthropods represent the "primitive" sister group of a protostome coelomate clade. Bootstrap analyses and consideration of all trees within 1% of the length of the most parsimonious tree suggest that relationships between the nonchelicerate arthropods and relationships within the chelicerate clade cannot be reliably inferred with the partial 18S rRNA sequence data. With the evolutionary-parsimony method, support for monophyly of the Arthropoda is found in the majority of the combinations analyzed if the coelomates are used as "outgroups." Monophyly of the Chelicerata is supported in most combinations assessed. Our analyses also indicate that the evolutionary-parsimony method, like distance and parsimony, may be biased by taxa with long branches. We suggest that a previous study's inference of the Arthropoda as paraphyletic may be the result of (a) having two few arthropod taxa available for analysis and (b) including long-branched taxa.

  11. Chromosome Mapping of 18S Ribosomal RNA Genes in Eleven Hypostomus Species (Siluriformes, Loricariidae): Diversity Analysis of the Sites.

    PubMed

    Rubert, Marceléia; da Rosa, Renata; Zawadzki, Claudio H; Mariotto, Sandra; Moreira-Filho, Orlando; Giuliano-Caetano, Lucia

    2016-08-01

    We investigated the chromosomal distribution of 18S ribosomal DNA (rDNA) in different populations of 11 species of Hypostomus collected in important Brazilian basins, namely South Atlantic, Upper Paraná, and Paraguay applying the fluorescence in situ hybridization (FISH). Hypostomus cochliodon, Hypostomus commersoni, Hypostomus hermanni, Hypostomus regani, Hypostomus albopunctatus, Hypostomus paulinus, Hypostomus aff. paulinus, Hypostomus iheringii, and Hypostomus mutucae presented multiple 18S rDNA sites while Hypostomus strigaticeps and Hypostomus nigromaculatus exhibited a single pair of chromosomes with 18S rDNA sites. The studied species presented variations in the number and position of these sites. The results accomplished were similar to those obtained by the analysis of AgNORs, revealing the same interspecific variability. Each species exhibited distinctive patterns of AgNOR and 18S rDNA distribution, which can be considered cytogenetic markers in each species of the genus and help improve the discussions on the phylogeny of the group.

  12. Analysis of Fungal Diversity in the Wheat Rhizosphere by Sequencing of Cloned PCR-Amplified Genes Encoding 18S rRNA and Temperature Gradient Gel Electrophoresis

    PubMed Central

    Smit, Eric; Leeflang, Paula; Glandorf, Boet; Dirk van Elsas, Jan; Wernars, Karel

    1999-01-01

    Like bacteria, fungi play an important role in the soil ecosystem. As only a small fraction of the fungi present in soil can be cultured, conventional microbiological techniques yield only limited information on the composition and dynamics of fungal communities in soil. DNA-based methods do not depend on the culturability of microorganisms, and therefore they offer an attractive alternative for the study of complex fungal community structures. For this purpose, we designed various PCR primers that allow the specific amplification of fungal 18S-ribosomal-DNA (rDNA) sequences, even in the presence of nonfungal 18S rDNA. DNA was extracted from the wheat rhizosphere, and 18S rDNA gene banks were constructed in Escherichia coli by cloning PCR products generated with primer pairs EF4-EF3 (1.4 kb) and EF4-fung5 (0.5 kb). Fragments of 0.5 kb from the cloned inserts were sequenced and compared to known rDNA sequences. Sequences from all major fungal taxa were amplified by using both primer pairs. As predicted by computer analysis, primer pair EF4-EF3 appeared slightly biased to amplify Basidiomycota and Zygomycota, whereas EF4-fung5 amplified mainly Ascomycota. The 61 clones that were sequenced matched the sequences of 24 different species in the Ribosomal Database Project (RDP) database. Similarity values ranged from 0.676 to 1. Temperature gradient gel electrophoresis (TGGE) analysis of the fungal community in the wheat rhizosphere of a microcosm experiment was carried out after amplification of total DNA with both primer pairs. This resulted in reproducible, distinctive fingerprints, confirming the difference in amplification specificity. Clear banding patterns were obtained with soil and rhizosphere samples by using both primer sets in combination. By comparing the electrophoretic mobility of community fingerprint bands to that of the bands obtained with separate clones, some could be tentatively identified. While 18S-rDNA sequences do not always provide the taxonomic

  13. 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.

  14. Dynamic regulation of β1 subunit trafficking controls vascular contractility.

    PubMed

    Leo, M Dennis; Bannister, John P; Narayanan, Damodaran; Nair, Anitha; Grubbs, Jordan E; Gabrick, Kyle S; Boop, Frederick A; Jaggar, Jonathan H

    2014-02-11

    Ion channels composed of pore-forming and auxiliary subunits control physiological functions in virtually all cell types. A conventional view is that channels assemble with their auxiliary subunits before anterograde plasma membrane trafficking of the protein complex. Whether the multisubunit composition of surface channels is fixed following protein synthesis or flexible and open to acute and, potentially, rapid modulation to control activity and cellular excitability is unclear. Arterial smooth muscle cells (myocytes) express large-conductance Ca(2+)-activated potassium (BK) channel α and auxiliary β1 subunits that are functionally significant modulators of arterial contractility. Here, we show that native BKα subunits are primarily (∼95%) plasma membrane-localized in human and rat arterial myocytes. In contrast, only a small fraction (∼10%) of total β1 subunits are located at the cell surface. Immunofluorescence resonance energy transfer microscopy demonstrated that intracellular β1 subunits are stored within Rab11A-postive recycling endosomes. Nitric oxide (NO), acting via cGMP-dependent protein kinase, and cAMP-dependent pathways stimulated rapid (≤1 min) anterograde trafficking of β1 subunit-containing recycling endosomes, which increased surface β1 almost threefold. These β1 subunits associated with surface-resident BKα proteins, elevating channel Ca(2+) sensitivity and activity. Our data also show that rapid β1 subunit anterograde trafficking is the primary mechanism by which NO activates myocyte BK channels and induces vasodilation. In summary, we show that rapid β1 subunit surface trafficking controls functional BK channel activity in arterial myocytes and vascular contractility. Conceivably, regulated auxiliary subunit trafficking may control ion channel activity in a wide variety of cell types.

  15. Highly divergent 18S rRNA gene paralogs in a Cryptosporidium genotype from eastern chipmunks (Tamias striatus)1

    PubMed Central

    Stenger, Brianna L.S.; Clark, Mark E.; Kváč, Martin; Khan, Eakalak; Giddings, Catherine W.; Dyer, Neil W.; Schultz, Jessie L.; McEvoy, John M.

    2015-01-01

    Cryptosporidium is an apicomplexan parasite that causes the disease cryptosporidiosis in humans, livestock, and other vertebrates. Much of the knowledge on Cryptosporidium diversity is derived from 18S rRNA gene (18S rDNA) phylogenies. Eukaryote genomes generally have multiple 18S rDNA copies that evolve in concert, which is necessary for the accurate inference of phylogenetic relationships. However, 18S rDNA copies in some genomes evolve by a birth-and-death process that can result in sequence divergence among copies. Most notably, divergent 18S rDNA paralogs in the apicomplexan Plasmodium share only 89–95% sequence similarity, encode structurally distinct rRNA molecules, and are expressed at different life cycle stages. In the present study, Cryptosporidium 18S rDNA was amplified from 28/72 (38.9%) eastern chipmunks (Tamias striatus). Phylogenetic analyses showed the co-occurrence of two 18S rDNA types, Type A and Type B, in 26 chipmunks, and Type B clustered with a sequence previously identified as Cryptosporidium chipmunk genotype II. Types A and B had a sister group relationship but shared less than 93% sequence similarity. In contrast, actin and heat shock protein 70 gene sequences were homogeneous in samples with both Types A and B present. It was therefore concluded that Types A and B are divergent 18S rDNA paralogs in Cryptosporidium chipmunk genotype II. Substitution patterns in Types A and B were consistent with functionally constrained evolution; however, Type B evolved more rapidly than Type A and had a higher G+C content (46.3% versus 41.0%). Oocysts of Cryptosporidium chipmunk genotype II measured 4.17 μm (3.73–5.04 μm) × 3.94 μm (3.50–4.98 μm) with a length-to-width ratio of 1.06 ± 0.06 μm, and infection occurred naturally in the jejunum, cecum, and colon of eastern chipmunks. The findings of this study have implications for the use of 18S rDNA sequences to infer phylogenetic relationships. PMID:25772204

  16. Radiolaria Divided into Polycystina and Spasmaria in Combined 18S and 28S rDNA Phylogeny

    PubMed Central

    Dolven, Jane K.; Ose, Randi F.; Klaveness, Dag; Kristensen, Tom; Bjørklund, Kjell R.; Shalchian-Tabrizi, Kamran

    2011-01-01

    Radiolarians are marine planktonic protists that belong to the eukaryote supergroup Rhizaria together with Foraminifera and Cercozoa. Radiolaria has traditionally been divided into four main groups based on morphological characters; i.e. Polycystina, Acantharia, Nassellaria and Phaeodaria. But recent 18S rDNA phylogenies have shown that Phaeodaria belongs within Cerocozoa, and that the previously heliozoan group Taxopodida should be included in Radiolaria. 18S rDNA phylogenies have not yet resolved the sister relationship between the main Radiolaria groups, but nevertheless suggests that Spumellaria, and thereby also Polycystina, are polyphyletic. Very few sequences other than 18S rDNA have so far been generated from radiolarian cells, mostly due to the fact that Radiolaria has been impossible to cultivate and single cell PCR has been hampered by low success rate. Here we have therefore investigated the mutual evolutionary relationship of the main radiolarian groups by using the novel approach of combining single cell whole genome amplification with targeted PCR amplification of the 18S and 28S rDNA genes. Combined 18S and 28S phylogeny of sequences obtained from single cells shows that Radiolaria is divided into two main lineages: Polycystina (Spumellaria+Nassellaria) and Spasmaria (Acantharia+Taxopodida). Further we show with high support that Foraminifera groups within Radiolaria supporting the Retaria hypothesis. PMID:21853146

  17. ITS1 sequence variabilities correlate with 18S rDNA sequence types in the genus Acanthamoeba (Protozoa: Amoebozoa).

    PubMed

    Köhsler, Martina; Leitner, Brigitte; Blaschitz, Marion; Michel, Rolf; Aspöck, Horst; Walochnik, Julia

    2006-01-01

    The subgenus classification of the ubiquitously spread and potentially pathogenic acanthamoebae still poses a great challenge. Fifteen 18S rDNA sequence types (T1-T15) have been established, but the vast majority of isolates fall into sequence type T4, and so far, there is no means to reliably differentiate within T4. In this study, the first internal transcribed spacer (ITS1), a more variable region than the 18S rRNA gene, was sequenced, and the sequences of 15 different Acanthamoeba isolates were compared to reveal if ITS1 sequence variability correlates with 18S rDNA sequence typing and if the ITS1 sequencing allows a differentiation within T4. It was shown that the variability in ITS1 is tenfold higher than in the 18S rDNA, and that ITS1 clusters correlate with the 18S rDNA clusters and thus corroborate the Acanthamoeba sequence type system. Moreover, high sequence dissimilarities and distinctive microsatellite patterns could enable a more detailed differentiation within T4.

  18. The subunit composition and function of mammalian cytochrome c oxidase.

    PubMed

    Kadenbach, Bernhard; Hüttemann, Maik

    2015-09-01

    Cytochrome c oxidase (COX) from mammals and birds is composed of 13 subunits. The three catalytic subunits I-III are encoded by mitochondrial DNA, the ten nuclear-coded subunits (IV, Va, Vb, VIa, VIb, VIc, VIIa, VIIb, VIIc, VIII) by nuclear DNA. The nuclear-coded subunits are essentially involved in the regulation of oxygen consumption and proton translocation by COX, since their removal or modification changes the activity and their mutation causes mitochondrial diseases. Respiration, the basis for ATP synthesis in mitochondria, is differently regulated in organs and species by expression of tissue-, developmental-, and species-specific isoforms for COX subunits IV, VIa, VIb, VIIa, VIIb, and VIII, but the holoenzyme in mammals is always composed of 13 subunits. Various proteins and enzymes were shown, e.g., by co-immunoprecipitation, to bind to specific COX subunits and modify its activity, but these interactions are reversible, in contrast to the tightly bound 13 subunits. In addition, the formation of supercomplexes with other oxidative phosphorylation complexes has been shown to be largely variable. The regulatory complexity of COX is increased by protein phosphorylation. Up to now 18 phosphorylation sites have been identified under in vivo conditions in mammals. However, only for a few phosphorylation sites and four nuclear-coded subunits could a specific function be identified. Research on the signaling pathways leading to specific COX phosphorylations remains a great challenge for understanding the regulation of respiration and ATP synthesis in mammalian organisms. This article reviews the function of the individual COX subunits and their isoforms, as well as proteins and small molecules interacting and regulating the enzyme.

  19. Pharmacological inhibition of PAR2 with the pepducin P2pal-18S protects mice against acute experimental biliary pancreatitis

    PubMed Central

    Michael, E. S.; Kuliopulos, A.; Covic, L.; Steer, M. L.

    2013-01-01

    Pancreatic acinar cells express proteinase-activated receptor-2 (PAR2) that is activated by trypsin-like serine proteases and has been shown to exert model-specific effects on the severity of experimental pancreatitis, i.e., PAR2−/− mice are protected from experimental acute biliary pancreatitis but develop more severe secretagogue-induced pancreatitis. P2pal-18S is a novel pepducin lipopeptide that targets and inhibits PAR2. In studies monitoring PAR2-stimulated intracellular Ca2+ concentration changes, we show that P2pal-18S is a full PAR2 inhibitor in acinar cells. Our in vivo studies show that P2pal-18S significantly reduces the severity of experimental biliary pancreatitis induced by retrograde intraductal bile acid infusion, which mimics injury induced by endoscopic retrograde cholangiopancreatography (ERCP). This reduction in pancreatitis severity is observed when the pepducin is given before or 2 h after bile acid infusion but not when it is given 5 h after bile acid infusion. Conversely, P2pal-18S increases the severity of secretagogue-induced pancreatitis. In vitro studies indicate that P2pal-18S protects acinar cells against bile acid-induced injury/death, but it does not alter bile acid-induced intracellular zymogen activation. These studies are the first to report the effects of an effective PAR2 pharmacological inhibitor on pancreatic acinar cells and on the severity of experimental pancreatitis. They raise the possibility that a pepducin such as P2pal-18S might prove useful in the clinical management of patients at risk for developing severe biliary pancreatitis such as occurs following ERCP. PMID:23275617

  20. Pharmacological inhibition of PAR2 with the pepducin P2pal-18S protects mice against acute experimental biliary pancreatitis.

    PubMed

    Michael, E S; Kuliopulos, A; Covic, L; Steer, M L; Perides, G

    2013-03-01

    Pancreatic acinar cells express proteinase-activated receptor-2 (PAR2) that is activated by trypsin-like serine proteases and has been shown to exert model-specific effects on the severity of experimental pancreatitis, i.e., PAR2(-/-) mice are protected from experimental acute biliary pancreatitis but develop more severe secretagogue-induced pancreatitis. P2pal-18S is a novel pepducin lipopeptide that targets and inhibits PAR2. In studies monitoring PAR2-stimulated intracellular Ca(2+) concentration changes, we show that P2pal-18S is a full PAR2 inhibitor in acinar cells. Our in vivo studies show that P2pal-18S significantly reduces the severity of experimental biliary pancreatitis induced by retrograde intraductal bile acid infusion, which mimics injury induced by endoscopic retrograde cholangiopancreatography (ERCP). This reduction in pancreatitis severity is observed when the pepducin is given before or 2 h after bile acid infusion but not when it is given 5 h after bile acid infusion. Conversely, P2pal-18S increases the severity of secretagogue-induced pancreatitis. In vitro studies indicate that P2pal-18S protects acinar cells against bile acid-induced injury/death, but it does not alter bile acid-induced intracellular zymogen activation. These studies are the first to report the effects of an effective PAR2 pharmacological inhibitor on pancreatic acinar cells and on the severity of experimental pancreatitis. They raise the possibility that a pepducin such as P2pal-18S might prove useful in the clinical management of patients at risk for developing severe biliary pancreatitis such as occurs following ERCP.

  1. Comparison of Sanger and next generation sequencing performance for genotyping Cryptosporidium isolates at the 18S rRNA and actin loci.

    PubMed

    Paparini, Andrea; Gofton, Alexander; Yang, Rongchang; White, Nicole; Bunce, Michael; Ryan, Una M

    2015-01-01

    Cryptosporidium is an important enteric pathogen that infects a wide range of humans and animals. Rapid and reliable detection and characterisation methods are essential for understanding the transmission dynamics of the parasite. Sanger sequencing, and high-throughput sequencing (HTS) on an Ion Torrent platform, were compared with each other for their sensitivity and accuracy in detecting and characterising 25 Cryptosporidium-positive human and animal faecal samples. Ion Torrent reads (n = 123,857) were obtained at both 18S rRNA and actin loci for 21 of the 25 samples. Of these, one isolate at the actin locus (Cattle 05) and three at the 18S rRNA locus (HTS 10, HTS 11 and HTS 12), suffered PCR drop-out (i.e. PCR failures) when using fusion-tagged PCR. Sanger sequences were obtained for both loci for 23 of the 25 samples and showed good agreement with Ion Torrent-based genotyping. Two samples both from pythons (SK 02 and SK 05) produced mixed 18S and actin chromatograms by Sanger sequencing but were clearly identified by Ion Torrent sequencing as C. muris. One isolate (SK 03) was typed as C. muris by Sanger sequencing but was identified as a mixed C. muris and C. tyzzeri infection by HTS. 18S rRNA Type B sequences were identified in 4/6 C. parvum isolates when deep sequenced but were undetected in Sanger sequencing. Sanger was cheaper than Ion Torrent when sequencing a small numbers of samples, but when larger numbers of samples are considered (n = 60), the costs were comparative. Fusion-tagged amplicon based approaches are a powerful way of approaching mixtures, the only draw-back being the loss of PCR efficiency on low-template samples when using primers coupled to MID tags and adaptors. Taken together these data show that HTS has excellent potential for revealing the "true" composition of species/types in a Cryptosporidium infection, but that HTS workflows need to be carefully developed to ensure sensitivity, accuracy and contamination are

  2. Sequencing and characterization of full-length sequence of 18S rRNA gene from the reniform nematode

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The 18S rRNA gene is fundamental to cellular and organismal protein synthesis and because of its stable persistence through generations it is also used in phylogenetic analysis among taxa. Variation within this gene is rare but it has been observed in few metazoan species. For the first time, we h...

  3. Eukaryotic diversity in premise drinking water using 18S rDNA sequencing: implications for health risks

    EPA Science Inventory

    The goal of this study was to characterize microbial eukaryotes over a 12 month period, so as to provide insight into the occurrence of potentially important predators and bacterial hosts in hot and cold premise plumbing. Nearly 6,300 partial (600 bp) 18S rRNA gene sequences from...

  4. Ribosome Subunit Stapling for Orthogonal Translation in E.  coli.

    PubMed

    Fried, Stephen D; Schmied, Wolfgang H; Uttamapinant, Chayasith; Chin, Jason W

    2015-10-19

    The creation of orthogonal large and small ribosomal subunits, which interact with each other but not with endogenous ribosomal subunits, would extend our capacity to create new functions in the ribosome by making the large subunit evolvable. To this end, we rationally designed a ribosomal RNA that covalently links the ribosome subunits via an RNA staple. The stapled ribosome is directed to an orthogonal mRNA, allowing the introduction of mutations into the large subunit that reduce orthogonal translation, but have minimal effects on cell growth. Our approach provides a promising route towards orthogonal subunit association, which may enable the evolution of key functional centers in the large subunit, including the peptidyl-transferase center, for unnatural polymer synthesis in cells.

  5. Redox-sensitive extracellular gates formed by auxiliary beta subunits of calcium-activated potassium channels.

    PubMed

    Zeng, Xu-Hui; Xia, Xiao-Ming; Lingle, Christopher J

    2003-06-01

    An important step to understanding ion channels is identifying the structural components that act as the gates to ion movement. Here we describe a new channel gating mechanism, produced by the beta3 auxiliary subunits of Ca2+-activated, large-conductance BK-type K+ channels when expressed with their pore-forming alpha subunits. BK beta subunits have a cysteine-rich extracellular segment connecting two transmembrane segments, with small cytosolic N and C termini. The extracellular segments of the beta3 subunits form gates to block ion permeation, providing a mechanism by which current can be rapidly diminished upon cellular repolarization. Furthermore, this gating mechanism is abolished by reduction of extracellular disulfide linkages, suggesting that endogenous mechanisms may regulate this gating behavior. The results indicate that auxiliary beta subunits of BK channels reside sufficiently close to the ion permeation pathway defined by the alpha subunits to influence or block access of small molecules to the permeation pathway.

  6. Assembly of in vitro synthesized large subunits into ribulose-bisphosphate carboxylase/oxygenase. Formation and discharge of an L8-like species.

    PubMed

    Hubbs, A E; Roy, H

    1993-06-25

    Ribulose-bisphosphate carboxylase/oxygenase (Rubisco) from higher plants consists of eight approximately 53-kDa large subunits and eight approximately 14-kDa small subunits. Cytosolic ribosomes synthesize the small subunits as precursors, which enter the chloroplast, undergo proteolytic processing, and assemble with large subunits. Large subunits, synthesized in the chloroplast, first form a complex with the chloroplast chaperonin 60 (Cpn60(14)). In the presence of ATP, large subunits dissociate from Cpn60(14) and assemble into Rubisco. We now describe partial characterization of a new species, Z, containing radiotracer-labeled, newly synthesized pea Rubisco large subunits. Rubisco assembly occurs in low salt in the presence of small subunits and ATP. As with Rubisco assembly, the formation of Z is ATP-dependent and is inhibited by high chloride. Once formed, Z is stable except in high chloride. Z does not appear to interact directly with small subunits. However, after Z formation, Rubisco assembly occurs in an ATP-independent reaction that requires KCl and small subunits. These results are consistent with the hypothesis that Z is a large subunit containing structure that can contribute large subunits to Rubisco under appropriate conditions. Z shares some physical characteristics with reported cyanobacterial L8 core particles. However, formation of Rubisco from Z in the absence of ATP and the presence of small subunits appears to require conditions that otherwise destabilize Z.

  7. The nuclear 18S ribosomal RNA gene as a source of phylogenetic information in the genus Taenia.

    PubMed

    Yan, Hongbin; Lou, Zhongzi; Li, Li; Ni, Xingwei; Guo, Aijiang; Li, Hongmin; Zheng, Yadong; Dyachenko, Viktor; Jia, Wanzhong

    2013-03-01

    Most species of the genus Taenia are of considerable medical and veterinary significance. In this study, complete nuclear 18S rRNA gene sequences were obtained from seven members of genus Taenia [Taenia multiceps, Taenia saginata, Taenia asiatica, Taenia solium, Taenia pisiformis, Taenia hydatigena, and Taenia taeniaeformis] and a phylogeny inferred using these sequences. Most of the variable sites fall within the variable regions, V1-V5. We show that sequences from the nuclear 18S ribosomal RNA gene have considerable promise as sources of phylogenetic information within the genus Taenia. Furthermore, given that almost all the variable sites lie within defined variable portions of that gene, it will be appropriate and economical to sequence only those regions for additional species of Taenia.

  8. Gene cloning of the 18S rRNA of an ancient viable moss from the permafrost of northeastern Siberia

    NASA Astrophysics Data System (ADS)

    Marsic, Damien; Hoover, Richard B.; Gilichinsky, David A.; Ng, Joseph D.

    1999-12-01

    A moss plant dating as much as 40,000 years old was collected from the permafrost of the Kolyma Lowlands of Northeastern Siberia. The plant tissue was revived and cultured for the extraction of its genomic DNA. Using the polymerase chain reaction technique, the 18S ribosomal RNA gene was cloned and its sequence studied. Comparative sequence analysis of the cloned ribosomal DNA to other known 18S RNA showed very high sequence identity and was revealed to be closest to the moss specie, Aulacomnium turgidum. The results of this study also show the ability of biological organisms to rest dormant in deep frozen environments where they can be revived and cultured under favorable conditions. This is significant in the notion that celestial icy bodies can be media to preserve biological function and genetic material during long term storage or transport.

  9. Typification of virulent and low virulence Babesia bigemina clones by 18S rRNA and rap-1c.

    PubMed

    Thompson, C; Baravalle, M E; Valentini, B; Mangold, A; Torioni de Echaide, S; Ruybal, P; Farber, M; Echaide, I

    2014-06-01

    The population structure of original Babesia bigemina isolates and reference strains with a defined phenotypic profile was assessed using 18S rRNA and rap-1c genes. Two reference strains, BbiS2P-c (virulent) and BbiS1A-c (low virulence), were biologically cloned in vitro. The virulence profile of the strains and clones was assessed in vivo. One fully virulent and one low-virulence clone were mixed in identical proportions to evaluate their growth efficiency in vitro. Each clone was differentiated by two microsatellites and the gene gp45. The 18S rRNA and rap-1c genes sequences from B. bigemina biological clones and their parental strains, multiplied exclusively in vivo or in vitro, were compared with strain JG-29. The virulence of clones derived from the BbiS2P-c strain was variable. Virulent clone Bbi9P1 grew more efficiently in vitro than did the low-virulence clone Bbi2A1. The haplotypes generated by the nucleotide polymorphism, localized in the V4 region of the 18S rRNA, allowed the identification of three genotypes. The rap-1c haplotypes allowed defining four genotypes. Parental and original strains were defined by multiple haplotypes identified in both genes. The rap-1c gene, analyzed by high-resolution melting (HRM), allowed discrimination between two genotypes according to their phenotype, and both were different from JG-29. B. bigemina biological clones made it possible to define the population structure of isolates and strains. The polymorphic regions of the 18S rRNA and rap-1c genes allowed the identification of different subpopulations within original B. bigemina isolates by the definition of several haplotypes and the differentiation of fully virulent from low virulence clones.

  10. 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.

  11. Molecular hybridization of iodinated 4S, 5S, and 18S + 28S RNA to salamander chromosomes

    PubMed Central

    1976-01-01

    4S, 5S, AND 18S + 28S RNA from the newt Taricha granulosa granulosa were iodinated in vitro with carrier-free 125I and hybridized to the denatured chromosomes of Taricha granulosa and Batrachoseps weighti. Iodinated 18S + 28S RNA hybridizes to the telomeric region on the shorter arm of chromosome 2 and close to the centromere on the shorter arm of chromosome 9 from T. granulosa. On this same salamander the label produced by the 5S RNA is located close to or on the centromere of chromosome 7 and the iodinated 4S RNA labels the distal end of the longer arm of chromosome 5. On the chromosomes of B. wrighti, 18S + 28S RNA hybridizes close to the centromeric region on the longer arm of the largest chromosome. Two centromeric sites are hybridized by the iodinated 5S RNA. After hybridization with iodinated 4S RNA, label is found near the end of the shorter arm of chromosome 3. It is concluded that both ribosomal and transfer RNA genes are clustered in the genome of these two salamanders. PMID:944187

  12. Immunological inter-strain crossreactivity correlated to 18S rDNA sequence types in Acanthamoeba spp.

    PubMed

    Walochnik, J; Obwaller, A; Aspöck, H

    2001-02-01

    Various species of the genus Acanthamoeba have been described as potential pathogens; however, differentiation of acanthamoebae remains problematic. The genus has been divided into 12 18S rDNA sequence types, most keratitis causing strains exhibiting sequence type T4. We recently isolated a keratitis causing Acanthamoeba strain showing sequence type T6, but being morphologically identical to a T4 strain. The aim of our study was to find out, whether the 18S rDNA sequence based identification correlates to immunological differentiation. The protein and antigen profiles of the T6 isolate and three reference Acanthamoeba strains were investigated using two sera from Acanthamoeba keratitis patients and one serum from an asymptomatic individual. It was shown, that the T6 strain produces a distinctly different immunological pattern, while patterns within T4 were identical. Affinity purified antibodies were used to further explore immunological cross-reactivity between sequence types. Altogether, the results of our study support the Acanthamoeba 18S rDNA sequence type classification in the investigated strains.

  13. An updated 18S rRNA phylogeny of tunicates based on mixture and secondary structure models

    PubMed Central

    Tsagkogeorga, Georgia; Turon, Xavier; Hopcroft, Russell R; Tilak, Marie-Ka; Feldstein, Tamar; Shenkar, Noa; Loya, Yossi; Huchon, Dorothée; Douzery, Emmanuel JP; Delsuc, Frédéric

    2009-01-01

    Background Tunicates have been recently revealed to be the closest living relatives of vertebrates. Yet, with more than 2500 described species, details of their evolutionary history are still obscure. From a molecular point of view, tunicate phylogenetic relationships have been mostly studied based on analyses of 18S rRNA sequences, which indicate several major clades at odds with the traditional class-level arrangements. Nonetheless, substantial uncertainty remains about the phylogenetic relationships and taxonomic status of key groups such as the Aplousobranchia, Appendicularia, and Thaliacea. Results Thirty new complete 18S rRNA sequences were acquired from previously unsampled tunicate species, with special focus on groups presenting high evolutionary rate. The updated 18S rRNA dataset has been aligned with respect to the constraint on homology imposed by the rRNA secondary structure. A probabilistic framework of phylogenetic reconstruction was adopted to accommodate the particular evolutionary dynamics of this ribosomal marker. Detailed Bayesian analyses were conducted under the non-parametric CAT mixture model accounting for site-specific heterogeneity of the evolutionary process, and under RNA-specific doublet models accommodating the occurrence of compensatory substitutions in stem regions. Our results support the division of tunicates into three major clades: 1) Phlebobranchia + Thaliacea + Aplousobranchia, 2) Appendicularia, and 3) Stolidobranchia, but the position of Appendicularia could not be firmly resolved. Our study additionally reveals that most Aplousobranchia evolve at extremely high rates involving changes in secondary structure of their 18S rRNA, with the exception of the family Clavelinidae, which appears to be slowly evolving. This extreme rate heterogeneity precluded resolving with certainty the exact phylogenetic placement of Aplousobranchia. Finally, the best fitting secondary-structure and CAT-mixture models suggest a sister

  14. Trends in Nasal Subunit Reconstruction by Facial Plastic and Reconstructive Surgeons.

    PubMed

    Larrabee, Yuna C; Phillips, David J; Sclafani, Anthony P

    2017-02-01

    To determine if facial plastic and reconstructive surgeons still adhere to the classic nasal subunit principle as described by Burget and Menick. Observational survey. A Weill Cornell Medicine institutional review board approved electronic survey that was sent via e-mail to active members of the American Academy of Facial Plastic and Reconstructive Surgery (AAFPRS). The survey consisted of 32 multiple-choice questions pertaining to the operative management of small (22-30%), medium (50-58%), and large (75-81%) defects of each subunit of the nose, as well as demographic, provider, and practice characteristics. There were 111 responses to the survey (10.1% response rate). Ninety-eight percent of respondents reported familiarity with the subunit principle, and 59.6% considered the subunit principle in greater than 90% of cases. Almost three-quarters (70.4%) of respondents felt the subunit principle should be applied but could be modified based on the particular nasal defect, whereas 28.7% felt it was only sometimes helpful and was not mandatory for successful nasal reconstruction. Large defects of the tip and ala are generally treated by excision of the remaining subunit (79.4 and 80.6%, respectively). Fewer surgeons would excise the remaining subunit for large defects of the dorsum (39.8%), sidewall (38.8%), and soft tissue facet (18.4%). Simple repair without additional excision was the treatment of choice for small defects of the tip (58.2%), ala (59.2%), sidewall (65%), dorsum (68%), and soft tissue facet (71.8%). However, in many small- (up to 32%) and medium- (up to 51%) sized defects of the tip, ala, sidewall, and dorsum, respondents reported partial subunit excision. The majority of AAFPRS members abide to the classical subunit principle by completely excising the remaining subunit for large defects of the tip and ala. Many surgeons modify the subunit principle in small and medium defects.

  15. The ribosomal subunit assembly line

    PubMed Central

    Dlakić, Mensur

    2005-01-01

    Recent proteomic studies in Saccharomyces cerevisiae have identified nearly 200 proteins, other than the structural ribosomal proteins, that participate in the assembly of ribosomal subunits and their transport from the nucleus. In a separate line of research, proteomic studies of mature plant ribosomes have revealed considerable variability in the protein composition of individual ribosomes. PMID:16207363

  16. Applied genomics: data mining reveals species-specific malaria diagnostic targets more sensitive than 18S rRNA.

    PubMed

    Demas, Allison; Oberstaller, Jenna; DeBarry, Jeremy; Lucchi, Naomi W; Srinivasamoorthy, Ganesh; Sumari, Deborah; Kabanywanyi, Abdunoor M; Villegas, Leopoldo; Escalante, Ananias A; Kachur, S Patrick; Barnwell, John W; Peterson, David S; Udhayakumar, Venkatachalam; Kissinger, Jessica C

    2011-07-01

    Accurate and rapid diagnosis of malaria infections is crucial for implementing species-appropriate treatment and saving lives. Molecular diagnostic tools are the most accurate and sensitive method of detecting Plasmodium, differentiating between Plasmodium species, and detecting subclinical infections. Despite available whole-genome sequence data for Plasmodium falciparum and P. vivax, the majority of PCR-based methods still rely on the 18S rRNA gene targets. Historically, this gene has served as the best target for diagnostic assays. However, it is limited in its ability to detect mixed infections in multiplex assay platforms without the use of nested PCR. New diagnostic targets are needed. Ideal targets will be species specific, highly sensitive, and amenable to both single-step and multiplex PCRs. We have mined the genomes of P. falciparum and P. vivax to identify species-specific, repetitive sequences that serve as new PCR targets for the detection of malaria. We show that these targets (Pvr47 and Pfr364) exist in 14 to 41 copies and are more sensitive than 18S rRNA when utilized in a single-step PCR. Parasites are routinely detected at levels of 1 to 10 parasites/μl. The reaction can be multiplexed to detect both species in a single reaction. We have examined 7 P. falciparum strains and 91 P. falciparum clinical isolates from Tanzania and 10 P. vivax strains and 96 P. vivax clinical isolates from Venezuela, and we have verified a sensitivity and specificity of ∼100% for both targets compared with a nested 18S rRNA approach. We show that bioinformatics approaches can be successfully applied to identify novel diagnostic targets and improve molecular methods for pathogen detection. These novel targets provide a powerful alternative molecular diagnostic method for the detection of P. falciparum and P. vivax in conventional or multiplex PCR platforms.

  17. Molecular phylogenetics of subclass Peritrichia (Ciliophora: Oligohymenophorea) based on expanded analyses of 18S rRNA sequences.

    PubMed

    Utz, Laura R P; Eizirik, Eduardo

    2007-01-01

    Phylogenetic relationships among peritrich ciliates remain unclear in spite of recent progress. To expand the analyses performed in previous studies, and to statistically test hypotheses of monophyly, we analyzed a broad sample of 18s rRNA sequences (including 15 peritrich genera), applying a conservative alignment strategy and several phylogenetic approaches. The main results are that: (i) the monophyly of Peritrichia cannot be rejected; (ii) the two main clades of Sessilida do not correspond to formally recognized taxa; (iii) the monophyly of genera Vorticella and Epistylis is significantly rejected; and (iv) morphological structures commonly used in peritrich taxonomy may be evolutionarily labile.

  18. Protein Expression of Proteasome Subunits in Elderly Patients with Schizophrenia

    PubMed Central

    Scott, Madeline R; Rubio, Maria D; Haroutunian, Vahram; Meador-Woodruff, James H

    2016-01-01

    The ubiquitin proteasome system (UPS) is a major regulator of protein processing, trafficking, and degradation. While protein ubiquitination is utilized for many cellular processes, one major function of this system is to target proteins to the proteasome for degradation. In schizophrenia, studies have found UPS transcript abnormalities in both blood and brain, and we have previously reported decreased protein expression of ubiquitin-associated proteins in brain. To test whether the proteasome is similarly dysregulated, we measured the protein expression of proteasome catalytic subunits as well as essential subunits from proteasome regulatory complexes in 14 pair-matched schizophrenia and comparison subjects in superior temporal cortex. We found decreased expression of Rpt1, Rpt3, and Rpt6, subunits of the 19S regulatory particle essential for ubiquitin-dependent degradation by the proteasome. Additionally, the α subunit of the 11S αβ regulatory particle, which enhances proteasomal degradation of small peptides and unfolded proteins, was also decreased. Haloperidol-treated rats did not have altered expression of these subunits, suggesting the changes we observed in schizophrenia are likely not due to chronic antipsychotic treatment. Interestingly, expression of the catalytic subunits of both the standard and immunoproteasome were unchanged, suggesting the abnormalities we observed may be specific to the complexed state of the proteasome. Aging has significant effects on the proteasome, and several subunits (20S β2, Rpn10, Rpn13, 11Sβ, and 11Sγ) were significantly correlated with subject age. These data provide further evidence of dysfunction of the ubiquitin-proteasome system in schizophrenia, and suggest that altered proteasome activity may be associated with the pathophysiology of this illness. PMID:26202105

  19. Primers to block the amplification of symbiotic apostome ciliate 18S rRNA gene in a PCR-based copepod diet study

    NASA Astrophysics Data System (ADS)

    Yi, Xiaoyan; Zhang, Huan; Liu, Guangxing

    2014-05-01

    Pelagic copepods play an important role in the marine food web. However, a full understanding of the ecological status of this zooplankton group depends on the careful study of their natural diets. In previous PCR-based copepod diet studies, we found many apostome ciliates that live symbiotically under the exoskeleton of the copepods, and their sequences were often over-represented in the 18S rRNA gene (18S rDNA) libraries. As a first step to address this issue, we designed three apostome ciliate 18S rDNA blocking primers, and tested their blocking efficiency against apostome ciliate 18s rDNA under various PCR conditions. Using a semi-quantitative PCR method, we optimized the conditions to efficiently amplify the 18S rDNA of the prey while simultaneously excluding the symbiotic apostome ciliates. This technique will facilitate PCR-based diet studies of copepods and other zooplankton in their natural environments.

  20. Identification of binding sites on the regulatory A subunit of protein phosphatase 2A for the catalytic C subunit and for tumor antigens of simian virus 40 and polyomavirus.

    PubMed Central

    Ruediger, R; Roeckel, D; Fait, J; Bergqvist, A; Magnusson, G; Walter, G

    1992-01-01

    Protein phosphatase 2A is composed of three subunits: the catalytic subunit C and two regulatory subunits, A and B. The A subunit consists of 15 nonidentical repeats and has a rodlike shape. It is associated with the B and C subunits as well as with the simian virus 40 small T, polyomavirus small T, and polyomavirus medium T tumor antigens. We determined the binding sites on subunit A for subunit C and tumor antigens by site-directed mutagenesis of A. Twenty-four N- and C-terminal truncations and internal deletions of A were assayed by coimmunoprecipitation for their ability to bind C and tumor antigens. It was found that C binds to repeats 11 to 15 at the C terminus of A, whereas T antigens bind to overlapping but distinct regions of the N terminus. Simian virus 40 small T binds to repeats 3 to 6, and polyomavirus small T and medium T bind to repeats 2 to 8. The data suggest cooperativity between C and T antigens in binding to A. This is most apparent for medium T antigen, which can only bind to those A subunit molecules that provide the entire binding region for the C subunit. We infer from our results that B also binds to N-terminal repeats. A model of the small T/medium T/B-A-C complexes is presented. Images PMID:1328865

  1. Identification of cephalopod species from the North and Baltic Seas using morphology, COI and 18S rDNA sequences

    NASA Astrophysics Data System (ADS)

    Gebhardt, Katharina; Knebelsberger, Thomas

    2015-09-01

    We morphologically analyzed 79 cephalopod specimens from the North and Baltic Seas belonging to 13 separate species. Another 29 specimens showed morphological features of either Alloteuthis mediaor Alloteuthis subulata or were found to be in between. Reliable identification features to distinguish between A. media and A. subulata are currently not available. The analysis of the DNA barcoding region of the COI gene revealed intraspecific distances (uncorrected p) ranging from 0 to 2.13 % (average 0.1 %) and interspecific distances between 3.31 and 22 % (average 15.52 %). All species formed monophyletic clusters in a neighbor-joining analysis and were supported by bootstrap values of ≥99 %. All COI haplotypes belonging to the 29 Alloteuthis specimens were grouped in one cluster. Neither COI nor 18S rDNA sequences helped to distinguish between the different Alloteuthis morphotypes. For species identification purposes, we recommend the use of COI, as it showed higher bootstrap support of species clusters and less amplification and sequencing failure compared to 18S. Our data strongly support the assumption that the genus Alloteuthis is only represented by a single species, at least in the North Sea. It remained unclear whether this species is A. subulata or A. media. All COI sequences including important metadata were uploaded to the Barcode of Life Data Systems and can be used as reference library for the molecular identification of more than 50 % of the cephalopod fauna known from the North and Baltic Seas.

  2. Phylogenetic Relationships Among Xiphinema and Xiphidorus Nematode Species from Brazil Inferred from 18S rDNA Sequences

    PubMed Central

    Oliveira, Claudio M. G.; Hübschen, Judith; Brown, Derek J. F.; Ferraz, Luiz C. C. B.; Wright, Frank; Neilson, Roy

    2004-01-01

    Maximum likelihood trees produced from 18S rDNA sequences separated 14 Xiphinema and five Xiphidorus nematode species from Brazil into distinct groups that concurred with their current morphological taxonomic status. Species belonging to the X. americanum group (X. brevicolle, X. diffusum, X. oxycaudatum, and X. peruvianum) formed a single group that was clearly separated from the other Xiphinema species. As with previous taxonomic studies that noted only minor morphological differences between putative X. americanum group species, separation of these species based upon 18S rDNA sequences was inconclusive. Thus it is probable that instead of comprising distinct species, the X. americanum group may in fact represent numerous morphotypes with large inter- and intra- population morphological variability that may be environmentally driven. Within the cluster representing non X. americanum group species, there was little statistical support to clearly separate species. However, three subgroups, comprising (i) the X. setariae/vulgare complex, (ii) X. ifacolum and X. paritaliae, and (iii) X. brasiliense and X. ensiculiferum were well resolved. PMID:19262801

  3. Optical and electrical stability of viral-templated copper sulfide (Cu{sub 1.8}S) films

    SciTech Connect

    Shahriar Zaman, Mohammed; Bernard Grajeda, Gabriel; Haberer, Elaine D.

    2014-04-14

    The optical and electrical stabilities of viral-templated non-stoichiometric copper sulfide, digenite (Cu{sub 1.8}S) films were investigated. The films were composed of large agglomerates of randomly aligned Cu{sub 1.8}S-coated M13 filamentous phage. Free carrier optical absorption associated with localized surface plasmon resonance (LSPR) was observed in the near infrared spectral region, and the films were electrically active, displaying a linear current-voltage relationship. Under ambient conditions, the magnitude of the LSPR absorption increased, following a power law relationship with time, and the electrical resistance of viral-templated films decreased significantly. In contrast, the resistance of films stored under low oxygen, low humidity conditions experienced a smaller reduction in electrical resistance. Changes in optical and electrical film properties under ambient conditions were associated with an increase in free carrier concentration within the copper chalcogenide material due to oxygen exposure. X-ray photoelectron spectroscopy was used to relate this increase in free carrier concentration to compositional changes on the viral-templated material surface.

  4. Early diagnosis of Exophiala CAPD peritonitis by 18S ribosomal RNA gene sequencing and its clinical significance.

    PubMed

    Lau, Susanna K P; Woo, Patrick C Y; Chiu, Siu-kau; Leung, Kit-wah; Yung, Raymond W H; Yuen, Kwok-yung

    2003-06-01

    Phenotypic identification of fungi in clinical microbiology laboratories is often difficult and late, especially for slow growing and rarely encountered fungi. We describe the application of 18S ribosomal RNA (rRNA) gene sequencing in the early diagnosis of a case of Exophiala peritonitis. A yeast-like fungus was isolated from the dialysate fluid of a 66-year-old man undergoing continuous ambulatory peritoneal dialysis. It grew slowly after 12 days of incubation to yield mature cultures to permit recognition of microscopic features resembling those of Exophiala, a dematiacerous mold. 18S rRNA gene sequencing provided results 12 days earlier than phenotypic identification and revealed 15 base difference (0.9%) between the isolate and Exophiala sp. strain GHP 1205 (GenBank Accession no. AJ232954), indicating that the isolate most closely resembles a strain of Exophiala species. The patient responded to 4 weeks of intravenous amphotericin B therapy. Early identification of the fungus was important for the choice of anti-fungal regimen. As opportunistic fungal infections in immunocompromised patients are globally emerging problems, the development of molecular techniques for fungal identification is crucial for early diagnosis and appropriate treatment.

  5. Distribution of 18S rDNA sites and absence of the canonical TTAGG insect telomeric repeat in parasitoid Hymenoptera.

    PubMed

    Gokhman, Vladimir E; Anokhin, Boris A; Kuznetsova, Valentina G

    2014-08-01

    Karyotypes of six species belonging to three main clades of parasitoid Hymenoptera, the superfamilies Ichneumonoidea (Ichneumonidae: Ichneumon amphibolus), Cynipoidea (Cynipidae: Diplolepis rosae) and Chalcidoidea (Eurytomidae: Eurytoma robusta, Eu. serratulae and Eu. compressa, and Torymidae: Torymus bedeguaris) were studied using FISH with 18S rDNA and telomeric (TTAGG)n probes. Haploid karyotypes of D. rosae, Eu. robusta and Eu. serratulae carried the only 18S rDNA hybridization signal, whereas those of I. amphibolus and Eu. compressa carried three and two rDNA clusters respectively. In addition, three rDNA sites were visualized in the aneuploid female of T. bedeguaris. The number of rDNA clusters in parasitoid Hymenoptera generally correlates to the chromosome number. Apart from the overwhelming majority of the studied species of aculeate Hymenoptera, no hybridization signals were obtained from FISH with the telomeric (TTAGG)n probe in the examined parasitoid species. These data suggest absence of the canonical (TTAGG)n insect telomeric motif in the Ichneumonoidea, Cynipoidea and Chalcidoidea, and perhaps in parasitoid Hymenoptera in general.

  6. Genetic diversity of Cryptosporidium in fish at the 18S and actin loci and high levels of mixed infections.

    PubMed

    Yang, Rongchang; Palermo, Cindy; Chen, Linda; Edwards, Amanda; Paparini, Andrea; Tong, Kaising; Gibson-Kueh, Susan; Lymbery, Alan; Ryan, Una

    2015-12-15

    Cryptosporidium is an enteric parasite that infects humans and a wide range of animals. Relatively little is known about the epidemiology and taxonomy of Cryptosporidium in fish. In the present study, a total of 775 fish, belonging to 46 species and comprising ornamental fish, marine fish and freshwater fish were screened for the prevalence of Cryptosporidium by PCR. The overall prevalence of Cryptosporidium in fish was 5.3% (41/775), with prevalences ranging from 1.5 to 100% within individual host species. Phylogenetic analysis of these Cryptosporidium isolates as well as 14 isolates from previous studies indicated extensive genetic diversity as well as evidence for mixed infections. At the 18S locus the following species were identified; Cryptosporidium molnari-like genotype (n=14), Cryptosporidium huwi (n=8), piscine genotype 2 (n=4), piscine genotype 3-like (n=1), piscine genotype 4 (n=2), piscine genotype 5 (n=13), piscine genotype 5-like (n=1) and five novel genotypes (n=5). At the actin locus, species identification agreed with the 18S locus for only 52.3% of isolates sequenced, indicating high levels of mixed infections. Future studies will need to employ both morphological characterization and deep sequencing amplicon-based technologies to better understand the epidemiological and phylogenetic relationships of piscine-derived Cryptosporidium species and genotypes, particularly when mixed infections are detected.

  7. Phylogenetic Analysis of the Spider Mite Sub-Family Tetranychinae (Acari: Tetranychidae) Based on the Mitochondrial COI Gene and the 18S and the 5′ End of the 28S rRNA Genes Indicates That Several Genera Are Polyphyletic

    PubMed Central

    Matsuda, Tomoko; Morishita, Maiko; Hinomoto, Norihide; Gotoh, Tetsuo

    2014-01-01

    The spider mite sub-family Tetranychinae includes many agricultural pests. The internal transcribed spacer (ITS) region of nuclear ribosomal RNA genes and the cytochrome c oxidase subunit I (COI) gene of mitochondrial DNA have been used for species identification and phylogenetic reconstruction within the sub-family Tetranychinae, although they have not always been successful. The 18S and 28S rRNA genes should be more suitable for resolving higher levels of phylogeny, such as tribes or genera of Tetranychinae because these genes evolve more slowly and are made up of conserved regions and divergent domains. Therefore, we used both the 18S (1,825–1,901 bp) and 28S (the 5′ end of 646–743 bp) rRNA genes to infer phylogenetic relationships within the sub-family Tetranychinae with a focus on the tribe Tetranychini. Then, we compared the phylogenetic tree of the 18S and 28S genes with that of the mitochondrial COI gene (618 bp). As observed in previous studies, our phylogeny based on the COI gene was not resolved because of the low bootstrap values for most nodes of the tree. On the other hand, our phylogenetic tree of the 18S and 28S genes revealed several well-supported clades within the sub-family Tetranychinae. The 18S and 28S phylogenetic trees suggest that the tribes Bryobiini, Petrobiini and Eurytetranychini are monophyletic and that the tribe Tetranychini is polyphyletic. At the genus level, six genera for which more than two species were sampled appear to be monophyletic, while four genera (Oligonychus, Tetranychus, Schizotetranychus and Eotetranychus) appear to be polyphyletic. The topology presented here does not fully agree with the current morphology-based taxonomy, so that the diagnostic morphological characters of Tetranychinae need to be reconsidered. PMID:25289639

  8. High protists diversity in the plankton of sulfurous lakes and lagoons examined by 18s rRNA gene sequence analyses.

    PubMed

    Triadó-Margarit, Xavier; Casamayor, Emilio O

    2015-12-01

    Diversity of small protists was studied in sulfidic and anoxic (euxinic) stratified karstic lakes and coastal lagoons by 18S rRNA gene analyses. We hypothesized a major sulfide effect, reducing protist diversity and richness with only a few specialized populations adapted to deal with low-redox conditions and high-sulfide concentrations. However, genetic fingerprinting suggested similar ecological diversity in anoxic and sulfurous than in upper oxygen rich water compartments with specific populations inhabiting euxinic waters. Many of them agreed with genera previously identified by microscopic observations, but also new and unexpected groups were detected. Most of the sequences matched a rich assemblage of Ciliophora (i.e., Coleps, Prorodon, Plagiopyla, Strombidium, Metopus, Vorticella and Caenomorpha, among others) and algae (mainly Cryptomonadales). Unidentified Cercozoa, Fungi, Stramenopiles and Discoba were recurrently found. The lack of GenBank counterparts was higher in deep hypolimnetic waters and appeared differentially allocated in the different taxa, being higher within Discoba and lower in Cryptophyceae. A larger number of populations than expected were specifically detected in the deep sulfurous waters, with unknown ecological interactions and metabolic capabilities.

  9. Further evidence for the variability of the 18S rDNA loci in the family Tingidae (Hemiptera, Heteroptera)

    PubMed Central

    Golub, Natalia V.; Golub, Viktor B.; Kuznetsova, Valentina G.

    2016-01-01

    Abstract As of now, within the lace bug family Tingidae (Cimicomorpha), only 1.5% of the species described have been cytogenetically studied. In this paper, male karyotypes of Stephanitis caucasica, Stephanitis pyri, Physatocheila confinis, Lasiacantha capucina, Dictyla rotundata and Dictyla echii were studied using FISH mapping with an 18S rDNA marker. The results show variability: the major rDNA sites are predominantly located on a pair of autosomes but occasionally on the X and Y chromosomes. All currently available data on the distribution of the major rDNA in the Tingidae karyotypes are summarized and shortly discussed. Our main concern is to clarify whether the chromosomal position of rDNA loci can contribute to resolving the phylogenetic relationships among the Tingidae taxa. PMID:28123675

  10. Phylogenetic relationships of the Culicomorpha inferred from 18S and 5.8S ribosomal DNA sequences. (Diptera:Nematocera).

    PubMed

    Miller, B R; Crabtree, M B; Savage, H M

    1997-05-01

    We investigated the evolutionary origins of the mosquito family Culicidae by examination of 18S and 5.8S ribosomal gene sequence divergence. Phylogenetic analyses demonstrated that within the infraorder Culicomorpha, taxa in the families Corethrellidae, Chaoboridae and Culicidae formed a monophyletic group; there was support for a sister relationship between this lineage and a representative of the Chironomidae. A chaoborid midge was the closest relative of the mosquitoes. Taxa from four genera of mosquitoes formed a monophyletic group; lack of a spacer in the 5.8S gene was unique to members of the Culicidae. A member of the genus Anopheles formed the most basal lineage among the mosquitoes analysed. Phylogenetic relationships were unresolved for representatives in the families Dixidae, Simuliidae and Ceratopogonidae.

  11. Molecular Diversity of Eukaryotes in Municipal Wastewater Treatment Processes as Revealed by 18S rRNA Gene Analysis

    PubMed Central

    Matsunaga, Kengo; Kubota, Kengo; Harada, Hideki

    2014-01-01

    Eukaryotic communities involved in sewage treatment processes have been investigated by morphological identification, but have not yet been well-characterized using molecular approaches. In the present study, eukaryotic communities were characterized by constructing 18S rRNA gene clone libraries. The phylogenetic affiliations of a total of 843 clones were Alveolata, Fungi, Rhizaria, Euglenozoa, Stramenopiles, Amoebozoa, and Viridiplantae as protozoans and Rotifera, Gastrotricha, and Nematoda as metazoans. Sixty percent of the clones had <97% sequence identity to described eukaryotes, indicating the greater diversity of eukaryotes than previously recognized. A core OTU closely related to Epistylis chrysemydis was identified, and several OTUs were shared by 4–8 libraries. Members of the uncultured lineage LKM11 in Cryptomycota were predominant fungi in sewage treatment processes. This comparative study represents an initial step in furthering understanding of the diversity and role of eukaryotes in sewage treatment processes. PMID:25491751

  12. Analysis of 18S rRNA gene sequences suggests significant molecular differences between Macrodasyida and Chaetonotida (Gastrotricha).

    PubMed

    Manylov, Oleg G; Vladychenskaya, Natalia S; Milyutina, Irina A; Kedrova, Olga S; Korokhov, Nikolai P; Dvoryanchikov, Gennady A; Aleshin, Vladimir V; Petrov, Nikolai B

    2004-03-01

    Partial 18S rRNA gene sequences of four macrodasyid and one chaetonotid gastrotrichs were obtained and compared with the available sequences of other gastrotrich species and representatives of various metazoan phyla. Contrary to the earlier molecular data, the gastrotrich sequences did not comprise a monophyletic group but formed two distinct clades, corresponding to the Macrodasyida and Chaetonotida, with the basal position occupied by the sequences of Tetranchyroderma sp. and Xenotrichula sp., respectively. Depending on the taxon sampling and methods of analysis, the two clades were separated by various combinations of clades Rotifera, Gnathostomulida, and Platyhelminthes, and never formed a clade with Nematoda. Thus, monophyly of the Gastrotricha is not confirmed by analysis of the presently available molecular data.

  13. Molecular diversity of eukaryotes in municipal wastewater treatment processes as revealed by 18S rRNA gene analysis.

    PubMed

    Matsunaga, Kengo; Kubota, Kengo; Harada, Hideki

    2014-01-01

    Eukaryotic communities involved in sewage treatment processes have been investigated by morphological identification, but have not yet been well-characterized using molecular approaches. In the present study, eukaryotic communities were characterized by constructing 18S rRNA gene clone libraries. The phylogenetic affiliations of a total of 843 clones were Alveolata, Fungi, Rhizaria, Euglenozoa, Stramenopiles, Amoebozoa, and Viridiplantae as protozoans and Rotifera, Gastrotricha, and Nematoda as metazoans. Sixty percent of the clones had <97% sequence identity to described eukaryotes, indicating the greater diversity of eukaryotes than previously recognized. A core OTU closely related to Epistylis chrysemydis was identified, and several OTUs were shared by 4-8 libraries. Members of the uncultured lineage LKM11 in Cryptomycota were predominant fungi in sewage treatment processes. This comparative study represents an initial step in furthering understanding of the diversity and role of eukaryotes in sewage treatment processes.

  14. Molecular phylogeny of extant gymnosperms and seed plant evolution: analysis of nuclear 18S rRNA sequences.

    PubMed

    Chaw, S M; Zharkikh, A; Sung, H M; Lau, T C; Li, W H

    1997-01-01

    To study the evolutionary relationships among the four living gymnosperm orders and the interfamilial relationships in each order, a set of 65 nuclear 18S rRNA sequences from ferns, gymnosperms, and angiosperms was analyzed using the neighbor-joining and maximum-parsimony methods. With Selaginella as the outgroup, the analysis strongly indicates that the seed plants form a monophyletic group with the ferns as a sister group. Within the seed plants the angiosperms are clearly a monophyletic group. Although the bootstrap support for the monophyly of the gymnosperm clade is moderate, the monophyly is further supported by its lack of angiosperm-specific indels. Within the gymnosperms there appear to be three monophyletic clades: Cycadales-Ginkgoales, Gnetales, and Coniferales. The cycad-ginkgo clade is the earliest gymnosperm lineage. Given the strong support for the sister group relationship between Gnetales and Coniferales, it is unlikely that Gnetales is a sister group of the angiosperms, contrary to the view of many plant taxonomists. Within Coniferales, Pinaceae is monophyletic and basal to the remaining conifer families, among which there are three monophyletic clades: Phyllocladaceae-Podocarpaceae, Araucariaceae, and Sciadopityaceae-Taxaceae-Cephalotaxaceae-Taxodiacea e-Cupressaceae. Within the latter clade, Sciadopityaceae may be an outgroup to the other four families. Among the angiosperms, no significant cluster at the level of subclass was found, but there was evidence that Nymphaeaceae branched off first. Within the remaining angiosperms, the monocots included in this study are nested and form a monophyletic group. This study attests to the utility of nuclear 18S rRNA sequences in addressing relationships among living gymnosperms. Considerable variation in substitution rates was observed among the ferns and seed plants.

  15. Sequence heterogeneity in the 18S rRNA gene in Theileria equi from horses presented in Switzerland.

    PubMed

    Liu, Qin; Meli, Marina L; Zhang, Yi; Meili, Theres; Stirn, Martina; Riond, Barbara; Weibel, Beatrice; Hofmann-Lehmann, Regina

    2016-05-15

    A reverse line blot (RLB) hybridization assay was adapted and applied for equine blood samples collected at the animal hospital of the University of Zurich to determine the presence of piroplasms in horses in Switzerland. A total of 100 equine blood samples were included in the study. The V4 hypervariable region of the 18S rRNA gene was amplified by polymerase chain reaction and analyzed using the RLB assay. Samples from seven horses hybridized to a Theileria/Babesia genus-specific and a Theileria genus-specific probe. Of these, two hybridized also to the Theileria equi-specific probe. The other five positive samples did not hybridize to any of the species-specific probes, suggesting the presence of unrecognized Theileria variants or genotypes. The 18S rRNA gene of the latter five samples were sequenced and found to be closely related to T. equi isolated from horses in Spain (AY534822) and China (KF559357) (≥98.4% identity). Four of the seven horses that tested positive had a documented travel history (France, Italy, and Spain) or lived abroad (Hungary). The present study adds new insight into the presence and sequence heterogeneity of T. equi in Switzerland. The results prompt that species-specific probes must be designed in regions of the gene unique to T. equi. Of note, none of the seven positive horses were suspected of having Theileria infection at the time of presentation to the clinic. Clinicians should be aware of the possibility of equine piroplasma infections outside of endemic areas and in horses without signs of piroplasmosis.

  16. 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

  17. DEVELOPING AN APICOMPLEXAN DNA BARCODOING SYSTEM TO DETECT BLOOD PARASITES OF SMALL CORAL REEF FISHES.

    PubMed

    Sikkel, Paul; Renoux, Lance P; Dolan, Maureen; Smit, Nico; Cook, Courtney

    2017-04-10

    Apicomplexan parasites are obligate parasites of many species of vertebrates. To date, there is very limited understanding of these parasites in the most diverse group of vertebrates, actinopterygian fishes. While DNA barcoding targeting the eukaryotic 18S small subunit (SSU) rRNA gene sequence has been useful in identifying apicomplexans in tetrapods, identification of apicomplexans infecting fishes has relied solely on morphological identification by microscopy. In this study, a DNA barcoding method was developed that targets the 18S rRNA gene primers for identification apicomplexans parasitizing certain actinopterygian fishes. A lead primer set was selected showing no cross reactivity to the overwhelming abundant host DNA and successfully confirmed 34 of the 41 (82.9%) microscopically verified parasitized fish blood samples analyzed in this study. Furthermore, this DNA barcoding method identified four additional samples that screened negative for parasitemia suggesting this molecular method may provide improved sensitivity over morphological characterization by microscopy. In addition, this PCR screening method for fish apicomplexans using Whatman FTA preserved DNA was tested in efforts leading to a more simplified field collection, transport and sample storage method as well as a streamlining sample processing important for DNA barcoding large sample sets.

  18. "Silent" Amino Acid Residues at Key Subunit Interfaces Regulate the Geometry of Protein Nanocages.

    PubMed

    Zhang, Shengli; Zang, Jiachen; Zhang, Xiaorong; Chen, Hai; Mikami, Bunzo; Zhao, Guanghua

    2016-11-22

    Rendering the geometry of protein-based assemblies controllable remains challenging. Protein shell-like nanocages represent particularly interesting targets for designed assembly. Here, we introduce an engineering strategy-key subunit interface redesign (KSIR)-that alters a natural subunit-subunit interface by selective deletion of a small number of "silent" amino acid residues (no participation in interfacial interactions) into one that triggers the generation of a non-native protein cage. We have applied KSIR to construct a non-native 48-mer nanocage from its native 24-mer recombinant human H-chain ferritin (rHuHF). This protein is a heteropolymer composed of equal numbers of two different subunits which are derived from one polypeptide. This strategy has allowed the study of conversion between protein nanocages with different geometries by re-engineering key subunit interfaces and the demonstration of the important role of the above-mentioned specific residues in providing geometric specificity for protein assembly.

  19. ATP-induced helicase slippage reveals highly coordinated subunits

    NASA Astrophysics Data System (ADS)

    Wang, Michelle D.

    2012-02-01

    Helicases are vital enzymes that carry out strand separation of duplex nucleic acids during replication, repair and recombination. T7 helicase, a model hexameric motor, has been observed to use dTTP, but not ATP, to unwind dsDNA as it translocates along ssDNA. Whether and how different subunits of the helicase coordinate their chemo-mechanical activities and DNA binding during translocation is still under debate. Here we address this question using a single-molecule approach to monitor helicase unwinding. We found that T7 helicase does in fact unwind dsDNA in the presence of ATP and that the unwinding rate is even faster than that with dTTP. However, unwinding was repeatedly interrupted by sudden slippage events, ultimately preventing unwinding over a substantial distance. This behaviour was greatly reduced with the supplement of a small amount of dTTP. These findings presented an opportunity to use nucleotide mixtures to investigate helicase subunit coordination. Our results support a model where nearly all subunits coordinate their chemo-mechanical activities and DNA binding. Such subunit coordination may be general to many ring-shaped helicases and reveals a potential mechanism for regulation of DNA unwinding during replication.

  20. Monitoring the mycobiota during Greco di Tufo and Aglianico wine fermentation by 18S rRNA gene sequencing.

    PubMed

    De Filippis, Francesca; La Storia, Antonietta; Blaiotta, Giuseppe

    2017-05-01

    Spontaneous alcoholic fermentation of grape must is a complex process, carried out by indigenous yeast populations arising from the vineyard or the winery environment and therefore representing an autochthonous microbial terroir of the production area. Microbial diversity at species and biotype level is extremely important in order to develop the composite and typical flavour profile of DOCG (Appellation of Controlled and Guaranteed Origin) wines. In this study, we monitored fungal populations involved in spontaneous fermentations of Aglianico and Greco di Tufo grape must by high-throughput sequencing (HTS) of 18S rRNA gene amplicons. We firstly proposed an alternative/addition to ITS as target gene in HTS studies and highlighted consistency between the culture-dependent and -independent approaches. A complex mycobiota was found at the beginning of the fermentation, mainly characterized by non-Saccharomyces yeasts and several moulds, with differences between the two types of grapes. Moreover, Interdelta patterns revealed a succession of several Saccharomyces cerevisiae biotypes and a high genetic diversity within this species.

  1. [Molecular phylogeny of gastrotricha based on 18S rRNA genes comparison: rejection of hypothesis of relatedness with nematodes].

    PubMed

    Petrov, N B; Pegova, A N; Manylov, O G; Vladychenskaia, N S; Miuge, N S; Aleshin, V V

    2007-01-01

    Gastrotrichs are meiobenthic free-living aquatic worms whose phylogenetic and intra-group relationships remain unclear despite some attempts to resolve them on the base of morphology or molecules. In this study we analysed complete sequences of the 18S rRNA gene of 15 taxa (8 new and 7 published) to test numerous hypotheses on gastrotrich phylogeny and to verify whether controversial interrelationships from previous molecular data could be due to the short region available for analysis and the poor taxa sampling. Data were analysed using both maximum likelihood and Bayesian inference. Results obtained suggest that gastrotrichs, together with Gnathostomulida, Plathelminthes, Syndermata (Rotifera + Acanthocephala), Nemertea and Lophotrochozoa, comprise a clade Spiralia. Statistical tests reject phylogenetic hypotheses regarding Gastrotricha as close relatives of Nematoda and other Ecdysozoa or placing them at the base of bilaterian tree close to acoels and nemertodermatides. Within Gastrotricha, Chaetonotida and Macrodasyida comprise two well supported clades. Our analysis confirmed the monophyly of the Chaetonotidae and Xenotrichulidae within Chaetonida as well as Turbanellidae and Thaumastodermatidae within Macrodasyida. Mesodasys is a sister group of the Turbanellidae, and Lepidodasyidae appears to be a polyphyletic group as Cephalodasys forms a separate lineage at the base of macrodasyids, whereas Lepidodasys groups with Neodasys between Thaumastodermatidae and Turbanellidae. To infer a more reliable Gastrotricha phylogeny many species and additional genes should be involved in future analyses.

  2. Phylogeny of the sundews, Drosera (Droseraceae), based on chloroplast rbcL and nuclear 18S ribosomal DNA Sequences.

    PubMed

    Rivadavia, Fernando; Kondo, Katsuhiko; Kato, Masahiro; Hasebe, Mitsuyasu

    2003-01-01

    The sundew genus Drosera consists of carnivorous plants with active flypaper traps and includes nearly 150 species distributed mainly in Australia, Africa, and South America, with some Northern Hemisphere species. In addition to confused intrageneric classification of Drosera, the intergeneric relationships among the Drosera and two other genera in the Droseraceae with snap traps, Dionaea and Aldrovanda, are problematic. We conducted phylogenetic analyses of DNA sequences of the chloroplast rbcL gene for 59 species of Drosera, covering all sections except one. These analyses revealed that five of 11 sections, including three monotypic sections, are polyphyletic. Combined rbcL and 18S rDNA sequence data were used to infer phylogenetic relationships among Drosera, Dionaea, and Aldrovanda. This analysis revealed that all Drosera species form a clade sister to a clade including Dionaea and Aldrovanda, suggesting that the snap traps of Aldrovanda and Dionaea are homologous despite their morphological differences. MacClade reconstructions indicated that multiple episodes of aneuploidy occurred in a clade that includes mainly Australian species, while the chromosome numbers in the other clades are not as variable. Drosera regia, which is native to South Africa, and most species native to Australia, were clustered basally, suggesting that Drosera originated in Africa or Australia. The rbcL tree indicates that Australian species expanded their distribution to South America and then to Africa. Expansion of distribution to the Northern Hemisphere from the Southern Hemispere occurred in a few different lineages.

  3. Phylogeny of Intestinal Ciliates, Including Charonina ventriculi, and Comparison of Microscopy and 18S rRNA Gene Pyrosequencing for Rumen Ciliate Community Structure Analysis

    PubMed Central

    Devente, Savannah R.; Kirk, Michelle R.; Seedorf, Henning; Dehority, Burk A.

    2015-01-01

    The development of high-throughput methods, such as the construction of 18S rRNA gene clone or pyrosequencing libraries, has allowed evaluation of ciliate community composition in hundreds of samples from the rumen and other intestinal habitats. However, several genera of mammalian intestinal ciliates have been described based only on morphological features and, to date, have not been identified using molecular methods. Here, we isolated single cells of one of the smallest but widely distributed intestinal ciliates, Charonina ventriculi, and sequenced its 18S rRNA gene. We verified the sequence in a full-cycle rRNA approach using fluorescence in situ hybridization and thereby assigned an 18S rRNA gene sequence to this species previously known only by its morphology. Based on its full-length 18S rRNA gene sequence, Charonina ventriculi was positioned within the phylogeny of intestinal ciliates in the subclass Trichostomatia. The taxonomic framework derived from this phylogeny was used for taxonomic assignment of trichostome ciliate 18S rRNA gene sequence data stemming from high-throughput amplicon pyrosequencing of rumen-derived DNA samples. The 18S rRNA gene-based ciliate community structure was compared to that obtained from microscopic counts using the same samples. Both methods allowed identification of dominant members of the ciliate communities and classification of the rumen ciliate community into one of the types first described by Eadie in 1962. Notably, each method is associated with advantages and disadvantages. Microscopy is a highly accurate method for evaluation of total numbers or relative abundances of different ciliate genera in a sample, while 18S rRNA gene pyrosequencing represents a valuable alternative for comparison of ciliate community structure in a large number of samples from different animals or treatment groups. PMID:25616800

  4. Sequence alignment of 18S ribosomal RNA and the basal relationships of Adephagan beetles: evidence for monophyly of aquatic families and the placement of Trachypachidae.

    PubMed

    Shull, V L; Vogler, A P; Baker, M D; Maddison, D R; Hammond, P M

    2001-01-01

    Current hypotheses regarding family relationships in the suborder Adephaga (Coleoptera) are conflicting. Here we report full-length 18S ribosomal RNA sequences of 39 adephagans and 13 outgroup taxa. Data analysis focused on the impact of sequence alignment on tree topology, using two principally different approaches. Tree alignments, which seek to minimize indels and substitutions on the tree in a single step, as implemented in an approximate procedure by the computer program POY, were contrasted with a more traditional procedure based on alignments followed by phylogenetic inference based on parsimony, likelihood, and distance analyses. Despite substantial differences between the procedures, phylogenetic conclusions regarding basal relationships within Adephaga and relationships between the four suborders of Coleoptera were broadly similar. The analysis weakly supports monophyly of Adephaga, with Polyphaga usually as its sister, and the two small suborders Myxophaga and Archostemata basal to them. In some analyses, however, Polyphaga was reconstructed as having arisen from within Hydradephaga. Adephaga generally split into two monophyletic groups, corresponding to the terrestrial Geadephaga and the aquatic Hydradephaga, as initially proposed by Crowson in 1955, consistent with a single colonization of the aquatic environment by adephagan ancestors and contradicting the recent proposition of three independent invasions. A monophyletic Hydradephaga is consistently, though not strongly, supported under most analyses, and a parametric bootstrapping test significantly rejects an hypothesis of nonmonophyly. The enigmatic Trachypachidae, which exhibit many similarities to aquatic forms but whose species are entirely terrestrial, were usually recovered as a basal lineage within Geadephaga. Strong evidence opposes the view that terrestrial trachypachids are related to the dytiscoid water beetles.

  5. Cloning and expression of the gene encoding catalytic subunit of thermostable glucose dehydrogenase from Burkholderia cepacia in Escherichia coli.

    PubMed

    Inose, Ken; Fujikawa, Masako; Yamazaki, Tomohiko; Kojima, Katsuhiro; Sode, Koji

    2003-02-21

    We have cloned a 1620-nucleotide gene encoding the catalytic subunit (alpha subunit) of a thermostable glucose dehydrogenase (GDH) from Burkholderia cepacia. The FAD binding motif was found in the N-terminal region of the alpha subunit. The deduced primary structure of the alpha subunit showed about 48% identity to the catalytic subunits of sorbitol dehydrogenase (SDH) from Gluconobacter oxydans and 2-keto-D-gluconate dehydrogenases (2KGDH) from Erwinia herbicola and Pantoea citrea. The alpha subunit of B. cepacia was expressed in Escherichia coli in its active water-soluble form, showing maximum dye-mediated GDH activity at 70 degrees C, retaining high thermal stability. A putative open reading frame (ORF) of 507 nucleotides was also found upstream of the alpha subunit encoding an 18-kDa peptide, designated as gamma subunit. The deduced primary structure of gamma subunit showed about 30% identity to the small subunits of the SDH from G. oxydans and 2KGDHs from E. herbicola and P. citrea.

  6. 28 CFR 51.6 - Political subunits.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Political subunits. 51.6 Section 51.6 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) PROCEDURES FOR THE ADMINISTRATION OF SECTION 5 OF THE VOTING RIGHTS ACT OF 1965, AS AMENDED General Provisions § 51.6 Political subunits. All...

  7. Preparation of a one-subunit cytochrome oxidase from Paracoccus denitrificans: spectral analysis and enzymatic activity.

    PubMed

    Müller, M; Schlapfer, B; Azzi, A

    1988-09-20

    Cytochrome c oxidase was isolated from Paracoccus denitrificans as a two-subunit enzyme. Chymotrypsin-catalyzed proteolysis reduced the molecular weight of each subunit by about 8000. The spectral properties of this preparation, as well as its Km for cytochrome c(1.7 muM), remained unchanged with respect to the native enzyme. Vmax was reduced by about 55% when assayed in Triton X-100 or in Triton X-100 supplemented with asolectin. Following further proteolysis by Staphylococcus aureus V8 protease, subunit I remained unchanged as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas subunit II was split into small peptides. These were removed by ion-exchange high-performance liquid chromatography. The one-subunit enzyme had an apparent molecular weight of 43,000. The reduction of molecular weight was also confirmed by the diminution of the ultraviolet/Soret absorption ratio. This value was 1.8-2.1 for the native enzyme and 1.3-1.5 for the one-subunit enzyme. The spectral properties (including the spectrum CO reduced minus reduced) were not modified by the proteolytic treatment, indicating that cytochromes a and a3 were present in equal amounts. The lack of spectral alteration and the known close association of the copper B atom with cytochrome a3 suggest that copper B is also contained within the one-subunit enzyme. The Km of the one-subunit oxidase was similar to that of the two-subunit enzyme; Vmax was decreased by about 50%. The activity of the one-subunit oxidase had a salt-dependent maximum at 30 mM KCl, almost identical with that of the undigested enzyme, and was inhibited by micromolar concentrations of KCN.

  8. Characterization of the vaginal fungal flora in pregnant diabetic women by 18S rRNA sequencing.

    PubMed

    Zheng, N-N; Guo, X-C; Lv, W; Chen, X-X; Feng, G-F

    2013-08-01

    Pregnancy and diabetes are regarded as individual risk factors for vaginal candidiasis. The high prevalence of vaginal candidiasis in pregnant diabetic women can be explained by disruption of the balance of the vaginal normal flora. However, little is known about the overall structure and composition of the vaginal fungal flora in pregnant diabetic women. In the present study, the diversity and richness of the vaginal fungal flora in healthy non-pregnant women (group HN), healthy pregnant women (group HP), women with gestational diabetes mellitus (group GDM), and pregnant women with diabetes mellitus type I (group T1DM) were investigated using an 18S rRNA gene clone library method. Our data demonstrated that the composition of the vaginal fungal flora in the four groups could be divided into two phyla (Ascomycetes, 20/26, and Basidiomycetes, 6/26). The most predominant vaginal fungal species belonged to the Candida and Saccharomyces genera, uncultured fungi, and a large number of low-abundance taxa that were unrecorded or underrepresented in previous studies using cultivation-dependent methods. Variation in operational taxonomic units (OTUs) between the study cohorts was generally high in the clone libraries, as 9, 13, 17, and 20 phylotypes were identified in groups HN, HP, GDM, and T1DM, respectively. The Shannon indices of groups GDM and T1DM (with poorer glycemic control) were significantly higher compared to groups HN and HP (p < 0.05). The data presented here revealed an increased diversity and varied composition of the vaginal fungal flora in pregnant diabetic women and demonstrated that poor glycemic control might be associated with disturbances in the vaginal fungal flora.

  9. 17(R),18(S)-Epoxyeicosatetraenoic Acid, A Potent Eicosapentaenoic Acid (EPA)-Derived Regulator of Cardiomyocyte Contraction: Structure-Activity Relationships and Stable Analogs

    PubMed Central

    Falck, John R.; Wallukat, Gerd; Puli, Narender; Goli, Mohan; Arnold, Cosima; Konkel, Anne; Rothe, Michael; Fischer, Robert; Müller, Dominik N.; Schunck, Wolf-Hagen

    2011-01-01

    17(R),18(S)-Epoxyeicosatetraenoic acid [17(R),18(S)-EETeTr], a cytochrome P450 epoxygenase metabolite of eicosapentaenoic acid (EPA), exerts negative chronotropic effects and protects neonatal rat cardiomyocytes against Ca2+-overload with an EC50 ~1–2 nM. Structure-activity studies revealed a cis-Δ11,12- or Δ14,15-olefin and a 17(R),18(S)-epoxide are minimal structural elements for anti-arrhythmic activity whereas antagonist activity was often associated with the combination of a Δ14,15-olefin and a 17(S),18(R)-epoxide. Compared with natural material, the agonist and antagonist analogs are chemically and metabolically more robust and several show promise as templates for future development of clinical candidates. PMID:21591683

  10. Outside-in recrystallization of ZnS-Cu1.8 S hollow spheres with interdispersed lattices for enhanced visible light solar hydrogen generation.

    PubMed

    Zhu, Ting; Nuo Peh, Connor Kang; Hong, Minghui; Ho, Ghim Wei

    2014-09-01

    For the first time an earth-abundant and nontoxic ZnS-Cu(1.8) S hybrid photocatalyst has been engineered with well-defined nanosheet hollow structures by a template-engaged method. In contrast to conventional surface coupling and loading, the unique outside-in recrystallization promotes co-precipitation of ZnS and Cu(1.8) S into homogeneous interdispersed lattices, hence forming a hybrid semiconductor with visible responsive photocatalytic activity. The as-derived ZnS-Cu(1.8) S semiconductor alloy is tailored into a hierarchical hollow structure to provide readily accessible porous shells and interior spaces for effective ion transfer/exchange. Notably, this synergistic morphology, interface and crystal lattice engineering, aim towards the design of novel nanocatalysts for various sustainable environmental and energy applications.

  11. Cytoplasmically made subunits of yeast mitochondrial F1-ATPase and cytochrome c oxidase are synthesized as individual precursors, not as polyproteins.

    PubMed

    Lewin, A S; Gregor, I; Mason, T L; Nelson, N; Schatz, G

    1980-07-01

    At least three subunits of yeast mitochondrial F1-ATPase (ATP phosphohydrolase, EC 3.6.1.3) and at least two subunits of cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) are synthesized outside the mitochondria and imported into the organelles as individual precursors that are between 2000 and 6000 daltons larger than the mature subunits. These precursors were shown to be primary translation products. Therefore, neither the five F1 subunits nor the four small cytochrome c oxidase subunits are synthesized as a single polyprotein.

  12. New record of Apoholosticha sinica (Ciliophora, Urostylida) from the UK: morphology, 18S rRNA gene phylogeny and notes on morphogenesis.

    PubMed

    Hu, Xiaozhong; Fan, Yangbo; Warren, Alan

    2015-08-01

    The benthic urostylid ciliate Apoholosticha sinicaFan et al., 2014 was isolated from a salt marsh at Blakeney, UK, and reinvestigated using light microscopy and small-subunit rRNA gene sequencing. Morphologically, it corresponds well with the original description. Several stages of divisional morphogenesis and physiological reorganization were also observed from which the following could be deduced: (i) the oral apparatus is completely newly built in the proter; (ii) frontal-ventral-transverse cirral anlage II does not produce a buccal cirrus; (iii) each of the posteriormost three or four anlagen contributes one transverse cirrus at its posterior end; (iv) a row of frontoterminal cirri originates from the rearmost frontal-ventral-transverse cirral anlage; (v) the last midventral row is formed from the penultimate frontal-ventral-transverse cirral anlage. Based on new data, two diagnostic features were added to the genus definition: (i) the midventral complex is composed of midventral pairs and midventral row and (ii) pretransverse ventral cirri are absent. Based on a combination of morphological and morphogenetic data, the genus Apoholosticha is assigned to the recently erected subfamily Nothoholostichinae Paiva et al., 2014, which is consistent with sequence comparison and phylogenetic analyses based on SSU rRNA gene data. It is also concluded that this benthic species, previously reported only from China, is not an endemic form.

  13. Polymorphisms in the 18S rDNA gene of Cystoisospora belli and clinical features of cystoisosporosis in HIV-infected patients.

    PubMed

    Resende, Deisy V; Pedrosa, André L; Correia, Dalmo; Cabrine-Santos, Marlene; Lages-Silva, Eliane; Meira, Wendell S F; Oliveira-Silva, Márcia B

    2011-03-01

    Intraspecific variability among Cystoisospora belli isolates and its clinical implications in human cystoisosporosis have not been established. In this study, the restriction fragment length polymorphisms in a 1.8-kb amplicon of the small subunit ribosomal DNA (SSU rDNA) of the parasite was investigated in 20 C. belli-positive stool samples obtained from 15 HIV-infected patients. Diarrheic syndrome was observed in all patients with cystoisosporosis and the number of diarrheic episodes per patient during hospitalization ranged from 1 to 26 (mean of 9.64 ± 9.30), with a mean duration of 2 to 12 days (mean of 5.90 ± 3 days). Three restriction profiles (RF) were generated with MboII digestion, which were named RFI, RFII, and RFIII. Two isolates obtained from a patient with extraintestinal cystoisosporosis showed distinct restriction profiles with MboII. This study demonstrates that patients can be infected with different C. belli genotypes, and this information may be useful for identifying new C. belli genotypes infecting humans.

  14. Chloroplast development at low temperatures requires a homolog of DIM1, a yeast gene encoding the 18S rRNA dimethylase.

    PubMed Central

    Tokuhisa, J G; Vijayan, P; Feldmann, K A; Browse, J A

    1998-01-01

    Poikilothermic organisms require mechanisms that allow survival at chilling temperatures (2 to 15 degreesC). We have isolated chilling-sensitive mutants of Arabidopsis, a plant that is very chilling resistant, and are characterizing them to understand the genes involved in chilling resistance. The T-DNA-tagged mutant paleface1 (pfc1) grows normally at 22 degrees C but at 5 degrees C exhibits a pattern of chilling-induced chlorosis consistent with a disruption of chloroplast development. Genomic DNA flanking the T-DNA was cloned and used to isolate wild-type genomic and cDNA clones. The PFC1 transcript is present at a low level in wild-type plants and was not detected in pfc1 plants. Wild-type Arabidopsis expressing antisense constructs of PFC1 grew normally at 22 degrees C but showed chilling-induced chlorosis, confirming that the gene is essential for low-temperature development of chloroplasts. The deduced amino acid sequence of PFC1 has identity with rRNA methylases found in bacteria and yeast that modify specific adenosines of pre-rRNA transcripts. The pfc1 mutant does not have these modifications in the small subunit rRNA of the plastid. PMID:9596631

  15. Limitations of metazoan 18S rRNA sequence data: implications for reconstructing a phylogeny of the animal kingdom and inferring the reality of the Cambrian explosion.

    PubMed

    Abouheif, E; Zardoya, R; Meyer, A

    1998-10-01

    We document the phylogenetic behavior of the 18S rRNA molecule in 67 taxa from 28 metazoan phyla and assess the effects of among-site rate variation on reconstructing phylogenies of the animal kingdom. This empirical assessment was undertaken to clarify further the limits of resolution of the 18S rRNA gene as a phylogenetic marker and to address the question of whether 18S rRNA phylogenies can be used as a source of evidence to infer the reality of a Cambrian explosion. A notable degree of among-site rate variation exists between different regions of the 18S rRNA molecule, as well as within all classes of secondary structure. There is a significant negative correlation between inferred number of nucleotide substitutions and phylogenetic information, as well as with the degree of substitutional saturation within the molecule. Base compositional differences both within and between taxa exist and, in certain lineages, may be associated with long branches and phylogenetic position. Importantly, excluding sites with different degrees of nucleotide substitution significantly influences the topology and degree of resolution of maximum-parsimony phylogenies as well as neighbor-joining phylogenies (corrected and uncorrected for among-site rate variation) reconstructed at the metazoan scale. Together, these data indicate that the 18S rRNA molecule is an unsuitable candidate for reconstructing the evolutionary history of all metazoan phyla, and that the polytomies, i.e., unresolved nodes within 18S rRNA phylogenies, cannot be used as a single or reliable source of evidence to support the hypothesis of a Cambrian explosion.

  16. Detection and characterization of fungal infections of Ammophila arenaria (marram grass) roots by denaturing gradient gel electrophoresis of specifically amplified 18s rDNA.

    PubMed Central

    Kowalchuk, G A; Gerards, S; Woldendorp, J W

    1997-01-01

    Marram grass (Ammophila arenaria L.), a sand-stabilizing plant species in coastal dune areas, is affected by a specific pathosystem thought to include both plant-pathogenic fungi and nematodes. To study the fungal component of this pathosystem, we developed a method for the cultivation-independent detection and characterization of fungi infecting plant roots based on denaturing gradient gel electrophoresis (DGGE) of specifically amplified DNA fragments coding for 18S rRNA (rDNA). A nested PCR strategy was employed to amplify a 569-bp region of the 18S rRNA gene, with the addition of a 36-bp GC clamp, from fungal isolates, from roots of test plants infected in the laboratory, and from field samples of marram grass roots from both healthy and degenerating stands from coastal dunes in The Netherlands. PCR products from fungal isolates were subjected to DGGE to examine the variation seen both between different fungal taxa and within a single species. DGGE of the 18S rDNA fragments could resolve species differences from fungi used in this study yet was unable to discriminate between strains of a single species. The 18S rRNA genes from 20 isolates of fungal species previously recovered from A. arenaria roots were cloned and partially sequenced to aid in the interpretation of DGGE data. DGGE patterns recovered from laboratory plants showed that this technique could reliably identify known plant-infecting fungi. Amplification products from field A. arenaria roots also were analyzed by DGGE, and the major bands were excised, reamplified, sequenced, and subjected to phylogenetic analysis. Some recovered 18S rDNA sequences allowed for phylogenetic placement to the genus level, whereas other sequences were not closely related to known fungal 18S rDNA sequences. The molecular data presented here reveal fungal diversity not detected in previous culture-based surveys. PMID:9327549

  17. 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.

  18. Subunit structure of the follitropin receptor

    SciTech Connect

    Shin, J.

    1985-01-01

    Both of the ..cap alpha.. and ..beta.. subunits of intact human follitropin (FSH) were radioiodinated with /sup 125/I-FSH-sodium iodide and chloramine-T, and could be resolved on polyacrylamide gels (SDS-PAGE). The electrophoretic mobility of radioiodinated FSH ..cap alpha.. and ..beta.. subunits as well as the ..cap alpha beta.. dimer changed markedly depending on the concentration of reducing agents. /sup 125/I-FSH (Ka = 1.4 x 10/sup 10/ M/sup -1/), complexes to the receptor on procine granulosa cells or in Triton X-100 extracts, was affinity-crosslinked with a cleavable (nondisulfide) homobifunctional reagent, bis(2-(succinimidooxycarbonyloxy)ethyl)sulfone, solubilized in sodium dodecyl sulfate with or without reducing agents, and electrophoresed. Crosslinked samples revealed three additional bands of slower electrophoretic mobility, corresponding to 65 (unreduced 62), 83 (unreduced 76) and 117 (unreduced 110)kDa, in addition to hormone bands. Formation of the three bands requires the /sup 125/I-FSH hormone to bind specifically to the receptor with subsequent cross-linking. The rate of formation and cleavage of the cross-linked complexes indicated a sequential and incremental addition of 22, 18, and 34 kDa components to the FSH ..cap alpha beta.. dimer. The results of reduction of cross-linked complexes demonstrated the existence of disulfide linkage between the three components. FSH was photoactively derivatized with N-hydroxysuccinimide ester of 4-azidobenzolyl-glycine and radioiodinated for photoaffinity labeling. When derivatized /sup 125/I-FSH (Ka = 1.12 10/sup 10/ M/sup -1/) bound to the cell was photolyzed for cross-linking and resolved on the SDS-PAGE, two new bands (106 and 61 kDa) under reducing condition appeared in addition to the hormone bands. Upon reduction with dithiotheitol and second-dimensional electrophoresis, the unreduced 104 kDa (reduced 106 kDa) band released two small components 31 and 14 kDa.

  19. 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.

  20. Tuning the Biological Activity Profile of Antibacterial Polymers via Subunit Substitution Pattern

    PubMed Central

    2015-01-01

    Binary nylon-3 copolymers containing cationic and hydrophobic subunits can mimic the biological properties of host-defense peptides, but relationships between composition and activity are not yet well understood for these materials. Hydrophobic subunits in previously studied examples have been limited mostly to cycloalkane-derived structures, with cyclohexyl proving to be particularly promising. The present study evaluates alternative hydrophobic subunits that are isomeric or nearly isomeric with the cyclohexyl example; each has four sp3 carbons in the side chains. The results show that varying the substitution pattern of the hydrophobic subunit leads to relatively small changes in antibacterial activity but causes significant changes in hemolytic activity. We hypothesize that these differences in biological activity profile arise, at least in part, from variations among the conformational propensities of the hydrophobic subunits. The α,α,β,β-tetramethyl unit is optimal among the subunits we have examined, providing copolymers with potent antibacterial activity and excellent prokaryote vs eukaryote selectivity. Bacteria do not readily develop resistance to the new antibacterial nylon-3 copolymers. These findings suggest that variation in subunit conformational properties could be generally valuable in the development of synthetic polymers for biological applications. PMID:24601599

  1. Tuning the biological activity profile of antibacterial polymers via subunit substitution pattern.

    PubMed

    Liu, Runhui; Chen, Xinyu; Chakraborty, Saswata; Lemke, Justin J; Hayouka, Zvi; Chow, Clara; Welch, Rodney A; Weisblum, Bernard; Masters, Kristyn S; Gellman, Samuel H

    2014-03-19

    Binary nylon-3 copolymers containing cationic and hydrophobic subunits can mimic the biological properties of host-defense peptides, but relationships between composition and activity are not yet well understood for these materials. Hydrophobic subunits in previously studied examples have been limited mostly to cycloalkane-derived structures, with cyclohexyl proving to be particularly promising. The present study evaluates alternative hydrophobic subunits that are isomeric or nearly isomeric with the cyclohexyl example; each has four sp(3) carbons in the side chains. The results show that varying the substitution pattern of the hydrophobic subunit leads to relatively small changes in antibacterial activity but causes significant changes in hemolytic activity. We hypothesize that these differences in biological activity profile arise, at least in part, from variations among the conformational propensities of the hydrophobic subunits. The α,α,β,β-tetramethyl unit is optimal among the subunits we have examined, providing copolymers with potent antibacterial activity and excellent prokaryote vs eukaryote selectivity. Bacteria do not readily develop resistance to the new antibacterial nylon-3 copolymers. These findings suggest that variation in subunit conformational properties could be generally valuable in the development of synthetic polymers for biological applications.

  2. Subunit mass analysis for monitoring antibody oxidation

    PubMed Central

    Sokolowska, Izabela; Mo, Jingjie; Dong, Jia; Lewis, Michael J.; Hu, Ping

    2017-01-01

    ABSTRACT Methionine oxidation is a common posttranslational modification (PTM) of monoclonal antibodies (mAbs). Oxidation can reduce the in-vivo half-life, efficacy and stability of the product. Peptide mapping is commonly used to monitor the levels of oxidation, but this is a relatively time-consuming method. A high-throughput, automated subunit mass analysis method was developed to monitor antibody methionine oxidation. In this method, samples were treated with IdeS, EndoS and dithiothreitol to generate three individual IgG subunits (light chain, Fd’ and single chain Fc). These subunits were analyzed by reversed phase-ultra performance liquid chromatography coupled with an online quadrupole time-of-flight mass spectrometer and the levels of oxidation on each subunit were quantitated based on the deconvoluted mass spectra using the UNIFI software. The oxidation results obtained by subunit mass analysis correlated well with the results obtained by peptide mapping. Method qualification demonstrated that this subunit method had excellent repeatability and intermediate precision. In addition, UNIFI software used in this application allows automated data acquisition and processing, which makes this method suitable for high-throughput process monitoring and product characterization. Finally, subunit mass analysis revealed the different patterns of Fc methionine oxidation induced by chemical and photo stress, which makes it attractive for investigating the root cause of oxidation. PMID:28106519

  3. Subunit mass analysis for monitoring antibody oxidation.

    PubMed

    Sokolowska, Izabela; Mo, Jingjie; Dong, Jia; Lewis, Michael J; Hu, Ping

    2017-04-01

    Methionine oxidation is a common posttranslational modification (PTM) of monoclonal antibodies (mAbs). Oxidation can reduce the in-vivo half-life, efficacy and stability of the product. Peptide mapping is commonly used to monitor the levels of oxidation, but this is a relatively time-consuming method. A high-throughput, automated subunit mass analysis method was developed to monitor antibody methionine oxidation. In this method, samples were treated with IdeS, EndoS and dithiothreitol to generate three individual IgG subunits (light chain, Fd' and single chain Fc). These subunits were analyzed by reversed phase-ultra performance liquid chromatography coupled with an online quadrupole time-of-flight mass spectrometer and the levels of oxidation on each subunit were quantitated based on the deconvoluted mass spectra using the UNIFI software. The oxidation results obtained by subunit mass analysis correlated well with the results obtained by peptide mapping. Method qualification demonstrated that this subunit method had excellent repeatability and intermediate precision. In addition, UNIFI software used in this application allows automated data acquisition and processing, which makes this method suitable for high-throughput process monitoring and product characterization. Finally, subunit mass analysis revealed the different patterns of Fc methionine oxidation induced by chemical and photo stress, which makes it attractive for investigating the root cause of oxidation.

  4. Are there proteins between the ribosomal subunits? Hot tritium bombardment experiments.

    PubMed

    Yusupov, M M; Spirin, A S

    1986-03-03

    The hot tritium bombardment technique [(1976) Dokl. Akad. Nauk SSSR 228, 1237-1238] was used for studying the surface localization of ribosomal proteins on Escherichia coli ribosomes. The degree of tritium labeling of proteins was considered as a measure of their exposure (surface localization). Proteins S1, S4, S7, S9 and/or S11, S12 and/or L20, S13, S18, S20, S21, L5, L6, L7/L12, L10, L11, L16, L17, L24, L26 and L27 were shown to be the most exposed on the ribosome surface. The sets of exposed ribosomal proteins on the surface of 70 S ribosomes, on the one hand, and the surfaces of 50 S and 30 S ribosomal subunits in the dissociated state, on the other, were compared. It was found that the dissociation of ribosomes into subunits did not result in exposure of additional ribosomal proteins. The conclusion was drawn that proteins are absent from the contacting surfaces of the ribosomal subunits.

  5. Quantitative analysis of dinoflagellates and diatoms community via Miseq sequencing of actin gene and v9 region of 18S rDNA

    PubMed Central

    Guo, Liliang; Sui, Zhenghong; Liu, Yuan

    2016-01-01

    Miseq sequencing and data analysis for the actin gene and v9 region of 18S rDNA of 7 simulated samples consisting of different mixture of dinoflagellates and diatoms were carried out. Not all the species were detectable in all the 18S v9 samples, and sequence percent in all the v9 samples were not consistent with the corresponding cell percent which may suggest that 18S rDNA copy number in different cells of these species differed greatly which result in the large deviation of the amplification. And 18S rDNA amplification of the microalgae was prone to be contaminated by fungus. The amplification of actin gene all was from the dinoflagellates because of its targeted degenerate primers. All the actin sequences of dinoflagellates were detected in the act samples except act4, and sequence percentage of the dinoflagellates in the act samples was not completely consistent with the dinoflagellates percentage of cell samples, but with certain amplification deviations. Indexes of alpha diversity of actin gene sequencing may be better reflection of community structure, and beta diversity analysis could cluster the dinoflagellates samples with identical or similar composition together and was distinguishable with blooming simulating samples at the generic level. Hence, actin gene was more proper than rDNA as the molecular marker for the community analysis of the dinoflagellates. PMID:27721499

  6. Phylogenetic analyses of four species of Ulva and Monostroma grevillei using ITS, rbc L and 18S rDNA sequence data

    NASA Astrophysics Data System (ADS)

    Lin, Zhongheng; Shen, Songdong; Chen, Weizhou; Li, Huihui

    2013-01-01

    Chlorophyta species are common in the southern and northern coastal areas of China. In recent years, frequent green tide incidents in Chinese coastal waters have raised concerns and attracted the attention of scientists. In this paper, we sequenced the 18S rDNA genes, the internal transcribed spacer (ITS) regions and the rbc L genes in seven organisms and obtained 536-566 bp long ITS sequences, 1 377-1 407 bp long rbc L sequences and 1 718-1 761 bp long partial 18S rDNA sequences. The GC base pair content was highest in the ITS regions and lowest in the rbc L genes. The sequencing results showed that the three Ulva prolifera (or U. pertusa) gene sequences from Qingdao and Nan'ao Island were identical. The ITS, 18S rDNA and rbc L genes in U. prolifera and U. pertusa from different sea areas in China were unchanged by geographic distance. U. flexuosa had the least evolutionary distance from U. californica in both the ITS regions (0.009) and the 18S rDNA (0.002). These data verified that Ulva and Enteromorpha are not separate genera.

  7. Comparative evaluation of the nested ITS PCR against the 18S PCR-RFLP in a survey of bovine trypanosomiasis in Kwale County, Kenya.

    PubMed

    Odongo, Steven; Delespaux, Vincent; Ngotho, Maina; Bekkele, Serkalem Mindaye; Magez, Stefan

    2016-09-01

    We compared the nested internal transcribed spacer (ITS) PCR and the 18S PCR-RFLP (restriction-fragment length polymorphism) pan-trypanosome assays in a cross-sectional survey of bovine trypanosomiasis in 358 cattle in Kwale County, Kenya. The prevalence of trypanosomiasis as determined by the nested ITS PCR was 19.6% (70/358) and by 18S PCR-RFLP was 16.8% (60/358). Of the pathogenic trypanosomes detected, the prevalence of Trypanosoma congolense and Trypanosoma vivax was greater than that of Trypanosoma simiae The nested ITS PCR detected 83 parasite events, whereas the 18S PCR-RFLP detected 64; however, overall frequencies of infections and the parasite events detected did not differ between the assays (χ(2) = 0.8, df = 1, p > 0.05 and χ(2) = 2.5, df = 1, p > 0.05, respectively). The kappa statistic (0.8) showed good agreement between the tests. The nested ITS PCR and the 18S PCR-RFLP had comparable sensitivity, although the nested ITS PCR was better at detecting mixed infections (χ(2) = 5.4, df = 1, p < 0.05).

  8. Physical mapping of 5S and 18S-5.8S-26S RNA gene families in polyploid series of Cenchrus ciliaris Linnaeus, 1771 (Poaceae)

    PubMed Central

    Kharrat-Souissi, Amina; Siljak-Yakovlev, Sonja; Pustahija, Fatima; Chaieb, Mohamed

    2012-01-01

    Abstract The Buffelgrass (Cenchrus ciliaris L., Poaceae) is one of the most important pasturage grasses due to its high productivity and good forage qualities. This species possess a high adaptability to bioclimatic constraints of arid zones and may be used for the restoration of degraded arid ecosystems. Tunisian populations present three ploidy levels (4x, 5x and 6x) with a basic chromosome number x=9. This study reported for the first time the distribution of the ribosomal genes (rRNA) for pentaploid and hexaploid cytotypes of Cenchrus ciliaris. Molecular cytogenetic study using double fluorescence in situ hybridization has shown that the two rDNA families, 5S and 18S-5.8S-26S (18S), displayed intraspecific variation in number of loci among different ploidy levels. Each ploidy level was characterized by specific number of both 5S and 18S rDNA loci (two loci in tetraploid, five in pentaploid and six in hexaploid level). For three studied cytotypes (4x, 5x and 6x) all 5S rDNA loci were localized on the subcentromeric region of chromosomes, while 18S loci were situated on the telomeric region of short chromosome arms. Data of the FISH experiments show proportional increase of ribosomal loci number during polyploidization processes. PMID:24260668

  9. Molecular and phylogenetic characterizations of an Eimeria krijgsmanni Yakimoff & Gouseff, 1938 (Apicomplexa: Eimeriidae) mouse intestinal protozoan parasite by partial 18S ribosomal RNA gene sequence analysis.

    PubMed

    Takeo, Toshinori; Tanaka, Tetsuya; Matsubayashi, Makoto; Maeda, Hiroki; Kusakisako, Kodai; Matsui, Toshihiro; Mochizuki, Masami; Matsuo, Tomohide

    2014-08-01

    Previously, we characterized an undocumented strain of Eimeria krijgsmanni by morphological and biological features. Here, we present a detailed molecular phylogenetic analysis of this organism. Namely, 18S ribosomal RNA gene (rDNA) sequences of E. krijgsmanni were analyzed to incorporate this species into a comprehensive Eimeria phylogeny. As a result, partial 18S rDNA sequence from E. krijgsmanni was successfully determined, and two different types, Type A and Type B, that differed by 1 base pair were identified. E. krijgsmanni was originally isolated from a single oocyst, and thus the result show that the two types might have allelic sequence heterogeneity in the 18S rDNA. Based on phylogenetic analyses, the two types of E. krijgsmanni 18S rDNA formed one of two clades among murine Eimeria spp.; these Eimeria clades reflected morphological similarity among the Eimeria spp. This is the third molecular phylogenetic characterization of a murine Eimeria spp. in addition to E. falciformis and E. papillata.

  10. Cornichon proteins determine the subunit composition of synaptic AMPA receptors.

    PubMed

    Herring, Bruce E; Shi, Yun; Suh, Young Ho; Zheng, Chan-Ying; Blankenship, Sabine M; Roche, Katherine W; Nicoll, Roger A

    2013-03-20

    Cornichon-2 and cornichon-3 (CNIH-2/-3) are AMPA receptor (AMPAR) binding proteins that promote receptor trafficking and markedly slow AMPAR deactivation in heterologous cells, but their role in neurons is unclear. Using CNIH-2 and CNIH-3 conditional knockout mice, we find a profound reduction of AMPAR synaptic transmission in the hippocampus. This deficit is due to the selective loss of surface GluA1-containing AMPARs (GluA1A2 heteromers), leaving a small residual pool of synaptic GluA2A3 heteromers. The kinetics of AMPARs in neurons lacking CNIH-2/-3 are faster than those in WT neurons due to the fast kinetics of GluA2A3 heteromers. The remarkably selective effect of CNIHs on the GluA1 subunit is probably mediated by TARP γ-8, which prevents a functional association of CNIHs with non-GluA1 subunits. These results point to a sophisticated interplay between CNIHs and γ-8 that dictates subunit-specific AMPAR trafficking and the strength and kinetics of synaptic AMPAR-mediated transmission.

  11. Molecular Modeling of the Misfolded Insulin Subunit and Amyloid Fibril

    PubMed Central

    Choi, Jay H.; May, Barnaby C.H.; Wille, Holger; Cohen, Fred E.

    2009-01-01

    Abstract Insulin, a small hormone protein comprising 51 residues in two disulfide-linked polypeptide chains, adopts a predominantly α-helical conformation in its native state. It readily undergoes protein misfolding and aggregates into amyloid fibrils under a variety of conditions. Insulin is a unique model system in which to study protein fibrillization, since its three disulfide bridges are retained in the fibrillar state and thus limit the conformational space available to the polypeptide chains during misfolding and fibrillization. Taking into account this unique conformational restriction, we modeled possible monomeric subunits of the insulin amyloid fibrils using β-solenoid folds, namely, the β-helix and β-roll. Both models agreed with currently available biophysical data. We performed molecular dynamics simulations, which allowed some limited insights into the relative structural stability, suggesting that the β-roll subunit model may be more stable than the β-helix subunit model. We also constructed β-solenoid-based insulin fibril models and conducted fiber diffraction simulation to identify plausible fibril architectures of insulin amyloid. A comparison of simulated fiber diffraction patterns of the fibril models to the experimental insulin x-ray fiber diffraction data suggests that the model fibers composed of six twisted β-roll protofilaments provide the most reasonable fit to available experimental diffraction patterns and previous biophysical studies. PMID:20006956

  12. Electrolyte level and blood pH in dogs infected by various 18S RNA strains of Babaesia canis canis on the early stage of babesiosis.

    PubMed

    Adaszek, Łukasz; Górna, Marta; Winiarczyk, Stanisław

    2012-01-01

    The purpose of the studies was to determine electrolyte disturbances and blood pH changes in dogs with babesiosis and possibly show a connection between the Babesia (B.) canis strain causing the infection and the intensity of these irregularities. 40 animals (group 1) with early babesiosis and 40 healthy dogs (group 2) were studied and their blood pH and blood levels of potassium, chlorides; calcium and sodium were determined. At the same time, molecular typing of parasites was carried out to detect which B.canis strain (18S RNA-A or 185 RNA-B) had caused the disease in dogs of group 1. In group 1, four dogs were acidaemic, twelve had normal blood pH, and 24 were alkalaemic. Potassium concentration was below normal in 16 out of 40 dogs (40%) and normal in 24 dogs. Hypochloremia was present in 36 out of 40 dogs; chloride was normal in the remaining four animals. Serum sodium concentration was low in 16 of 40 dogs, normal in 20 of 40 dogs and high in four dogs. Calcium concentration was normal in all 40 dogs. In dogs of group 2 no abnormalities of haematological or blood biochemical parameters were observed. 29 out of the 40 dogs of group 1 were infected with the 18S RNA-A strain and eleven with the 18S RNA-B strain of Babesia canis canis. We did not observe any correlation between the type of strain causing the infection and the electrolyte disturbances in the serum of sick dogs. Hypocalaemia was observed in ten specimen infected with 18S RNA-A and six infected with 18S RNA-B. Additionally, in dogs infected with 18S RNA-A, hypochloraemia (28), hyponatraemia (10), hypernatraemia (2) were observed, as well as blood pH drop (4) or increase (14). The 18S RNA-B-infected dogs suffered from hypochloraemia (8), hyponatraemia (6), hypernatraemia (2) and increase in blood pH (10).The studies conducted did not answer the question of whether the type of electrolyte disturbances in dogs with babesiosis can be connected with the strain of the parasite that induced the disease, as

  13. Introduction of a novel 18S rDNA gene arrangement along with distinct ITS region in the saline water microalga Dunaliella.

    PubMed

    Hejazi, Mohammad A; Barzegari, Abolfazl; Gharajeh, Nahid Hosseinzadeh; Hejazi, Mohammad S

    2010-04-08

    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 mumol 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.

  14. Gene targeting of CK2 catalytic subunits

    PubMed Central

    Lou, David Y.; Toselli, Paul; Landesman-Bollag, Esther; Dominguez, Isabel

    2013-01-01

    Protein kinase CK2 is a highly conserved and ubiquitous serine–threonine kinase. It is a tetrameric enzyme that is made up of two regulatory CK2β subunits and two catalytic subunits, either CK2α/CK2α, CK2α/ CK2α′, or CK2α′/CK2α′. Although the two catalytic subunits diverge in their C termini, their enzymatic activities are similar. To identify the specific function of the two catalytic subunits in development, we have deleted them individually from the mouse genome by homologous recombination. We have previously reported that CK2α′is essential for male germ cell development, and we now demonstrate that CK2α has an essential role in embryogenesis, as mice lacking CK2α die in mid-embryogenesis, with cardiac and neural tube defects. PMID:18594950

  15. Characterization of the Two Intra-Individual Sequence Variants in the 18S rRNA Gene in the Plant Parasitic Nematode, Rotylenchulus reniformis

    PubMed Central

    Nyaku, Seloame T.; Sripathi, Venkateswara R.; Kantety, Ramesh V.; Gu, Yong Q.; Lawrence, Kathy; Sharma, Govind C.

    2013-01-01

    The 18S rRNA gene is fundamental to cellular and organismal protein synthesis and because of its stable persistence through generations it is also used in phylogenetic analysis among taxa. Sequence variation in this gene within a single species is rare, but it has been observed in few metazoan organisms. More frequently it has mostly been reported in the non-transcribed spacer region. Here, we have identified two sequence variants within the near full coding region of 18S rRNA gene from a single reniform nematode (RN) Rotylenchulus reniformis labeled as reniform nematode variant 1 (RN_VAR1) and variant 2 (RN_VAR2). All sequences from three of the four isolates had both RN variants in their sequences; however, isolate 13B had only RN variant 2 sequence. Specific variable base sites (96 or 5.5%) were found within the 18S rRNA gene that can clearly distinguish the two 18S rDNA variants of RN, in 11 (25.0%) and 33 (75.0%) of the 44 RN clones, for RN_VAR1 and RN_VAR2, respectively. Neighbor-joining trees show that the RN_VAR1 is very similar to the previously existing R. reniformis sequence in GenBank, while the RN_VAR2 sequence is more divergent. This is the first report of the identification of two major variants of the 18S rRNA gene in the same single RN, and documents the specific base variation between the two variants, and hypothesizes on simultaneous co-existence of these two variants for this gene. PMID:23593343

  16. Sequence variation identified in the 18S rRNA gene of Theileria mutans and Theileria velifera from the African buffalo (Syncerus caffer).

    PubMed

    Chaisi, Mamohale E; Collins, Nicola E; Potgieter, Fred T; Oosthuizen, Marinda C

    2013-01-16

    The African buffalo (Syncerus caffer) is a natural reservoir host for both pathogenic and non-pathogenic Theileria species. These often occur naturally as mixed infections in buffalo. Although the benign and mildly pathogenic forms do not have any significant economic importance, their presence could complicate the interpretation of diagnostic test results aimed at the specific diagnosis of the pathogenic Theileria parva in cattle and buffalo in South Africa. The 18S rRNA gene has been used as the target in a quantitative real-time PCR (qPCR) assay for the detection of T. parva infections. However, the extent of sequence variation within this gene in the non-pathogenic Theileria spp. of the Africa buffalo is not well known. The aim of this study was, therefore, to characterise the full-length 18S rRNA genes of Theileria mutans, Theileria sp. (strain MSD) and T. velifera and to determine the possible influence of any sequence variation on the specific detection of T. parva using the 18S rRNA qPCR. The reverse line blot (RLB) hybridization assay was used to select samples which either tested positive for several different Theileria spp., or which hybridised only with the Babesia/Theileria genus-specific probe and not with any of the Babesia or Theileria species-specific probes. The full-length 18S rRNA genes from 14 samples, originating from 13 buffalo and one bovine from different localities in South Africa, were amplified, cloned and the resulting recombinants sequenced. Variations in the 18S rRNA gene sequences were identified in T. mutans, Theileria sp. (strain MSD) and T. velifera, with the greatest diversity observed amongst the T. mutans variants. This variation possibly explained why the RLB hybridization assay failed to detect T. mutans and T. velifera in some of the analysed samples.

  17. Verification of false-positive blood culture results generated by the BACTEC 9000 series by eubacterial 16S rDNA and panfungal 18S rDNA directed polymerase chain reaction (PCR).

    PubMed

    Daxboeck, Florian; Dornbusch, Hans Jürgen; Krause, Robert; Assadian, Ojan; Wenisch, Christoph

    2004-01-01

    A small but significant proportion of blood cultures processed by the BACTEC 9000 series systems is signaled positive, while subsequent Gram's stain and culture on solid media yield no pathogens. In this study, 15 "false-positive" vials (7 aerobes, 8 anaerobes) from 15 patients were investigated for the presence of bacteria and fungi by eubacterial 16S rDNA and panfungal 18S rDNA amplification, respectively. All samples turned out negative by both methods. Most patients (7) had neutropenia, which does not support the theory that high leukocyte counts enhance the generation of false-positive results. In conclusion, the results of this study indicate that false-negative results generated by the BACTEC 9000 series are inherent to the automated detection and not due to the growth of fastidious organisms.

  18. Anthranilate synthase subunit organization in Chromobacterium violaceum.

    PubMed

    Carminatti, C A; Oliveira, I L; Recouvreux, D O S; Antônio, R V; Porto, L M

    2008-09-16

    Tryptophan is an aromatic amino acid used for protein synthesis and cellular growth. Chromobacterium violaceum ATCC 12472 uses two tryptophan molecules to synthesize violacein, a secondary metabolite of pharmacological interest. The genome analysis of this bacterium revealed that the genes trpA-F and pabA-B encode the enzymes of the tryptophan pathway in which the first reaction is the conversion of chorismate to anthranilate by anthranilate synthase (AS), an enzyme complex. In the present study, the organization and structure of AS protein subunits from C. violaceum were analyzed using bioinformatics tools available on the Web. We showed by calculating molecular masses that AS in C. violaceum is composed of alpha (TrpE) and beta (PabA) subunits. This is in agreement with values determined experimentally. Catalytic and regulatory sites of the AS subunits were identified. The TrpE and PabA subunits contribute to the catalytic site while the TrpE subunit is involved in the allosteric site. Protein models for the TrpE and PabA subunits were built by restraint-based homology modeling using AS enzyme, chains A and B, from Salmonella typhimurium (PDB ID 1I1Q).

  19. Targeting single-nucleotide polymorphisms in the 18S rRNA gene to differentiate Cyclospora species from Eimeria species by multiplex PCR.

    PubMed

    Orlandi, Palmer A; Carter, Laurenda; Brinker, Anna Marie; da Silva, Alexandre J; Chu, Dan-My; Lampel, Keith A; Monday, Steven R

    2003-08-01

    Cyclospora cayetanensis is a coccidian parasite that causes protracted diarrheal illness in humans. C. cayetanensis is the only species of this genus thus far associated with human illness, although Cyclospora species from other primates have been named. The current method to detect the parasite uses a nested PCR assay to amplify a 294-bp region of the small subunit rRNA gene, followed by restriction fragment length polymorphism (RFLP) or DNA sequence analysis. Since the amplicons generated from C. cayetanensis and Eimeria species are the same size, the latter step is required to distinguish between these different species. The current PCR-RFLP protocol, however, cannot distinguish between C. cayetanensis and these new isolates. The differential identification of such pathogenic and nonpathogenic parasites is essential in assessing the risks to human health from microorganisms that may be potential contaminants in food and water sources. Therefore, to expand the utility of PCR to detect and identify these parasites in a multiplex assay, a series of genus- and species-specific forward primers were designed that are able to distinguish sites of limited sequence heterogeneity in the target gene. The most effective of these unique primers were those that identified single-nucleotide polymorphisms (SNPs) at the 3' end of the primer. Under more stringent annealing and elongation conditions, these SNP primers were able to differentiate between C. cayetanensis, nonhuman primate species of Cyclospora, and Eimeria species. As a diagnostic tool, the SNP PCR protocol described here presents a more rapid and sensitive alternative to the currently available PCR-RFLP detection method. In addition, the specificity of these diagnostic primers removes the uncertainty that can be associated with analyses of foods or environmental sources suspected of harboring potential human parasitic pathogens.

  20. A molecular phylogenetic study of the Palmae (Arecaceae) based on atpB, rbcL, and 18S nrDNA sequences.

    PubMed

    Hahn, William J

    2002-02-01

    Notoriously slow rates of molecular evolution and convergent evolution among some morphological characters have limited phylogenetic resolution for the palm family (Arecaceae). This study adds nuclear DNA (18S SSU rRNA) and chloroplast DNA (cpDNA; atpB and rbcL) sequence data for 65 genera of palms and characterizes molecular variation for each molecule. Phylogenetic relationships were estimated with maximum likelihood and maximum parsimony techniques for the new data and for previously published molecular data for 45 palm genera. Maximum parsimony analysis was also used to compare molecular and morphological data for 33 palm genera. Incongruence among datasets was detected between cpDNA and 18S data and between molecular and morphological data. Most conflict between nuclear and cpDNA data was associated with the genus Nypa. Several taxa showed relatively long branches with 18S data, but phylogenetic resolution of these taxa was essentially the same for 18S and cpDNA data. Base composition bias for 18S that contributed to erroneous phylogenetic resolution in other taxa did not seem to be present in Palmae. Morphological data were incongruent with all molecular data due to apparent morphological homoplasy for Caryoteae, Ceroxyloideae, Iriarteae, and Thrinacinae. Both cpDNA and nuclear 18S data firmly resolved Caryoteae with Borasseae of Coryphoideae, suggesting that at least some morphological characters used to place Caryoteae in Arecoideae are homoplastic. In this study, increased character sampling seems to be more important than increased taxon sampling; a comparison of the full (65-taxon) and reduced (45- and 33-taxon) datasets suggests little difference in core topology but considerably more nodal support with the increased character sample sizes. These results indicate a general trend toward a stable estimate of phylogenetic relationships for the Palmae. Although the 33-taxon topologies are even better resolved, they lack several critical taxa and are

  1. Characteristic views of E. coli and B. stearothermophilus 30S ribosomal subunits in the electron microscope.

    PubMed Central

    van Heel, M; Stöffler-Meilicke, M

    1985-01-01

    Large sets of electron microscopic images of the 30S ribosomal subunits of Bacillus stearothermophilus (914 molecules) and Escherichia coli (422 molecules) were analysed with image processing techniques. Using computer alignment and a new multivariate statistical classification scheme, three predominant views of the subunit were found for both species. These views, which together account for approximately 90% of the population of images, were determined to a reproducible resolution of up to 1.7 nm, thus elucidating many new structural details. The angular spread of the molecular orientations around the three main stable positions is remarkably small (less than 8 degrees). Some of the current models for the small ribosomal subunit are incompatible with our new results. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:3908096

  2. Covalent dimerization of ribulose bisphosphate carboxylase subunits by UV radiation.

    PubMed

    Ferreira, R M; Franco, E; Teixeira, A R

    1996-08-15

    The effect of UV radiation (UV-A, UV-B and UV-C) on ribulose bisphosphate carboxylase from a variety of plant species was examined. The exposition of plant leaves or the pure enzyme to UV radiation produced a UV-dependent accumulation of a +5 kDa polypeptide (P65). Different approaches were utilized to elucidate the origin and structure of P65: electrophoretic and fluorographic analyses of 35S-labelled ribulose bisphosphate carboxylase exposed to UV radiation and immunological experiments using antibodies specific for P65, for the large and small subunits of ribulose bisphosphate carboxylase and for high-molecular-mass aggregates of the enzyme. These studies revealed that P65 is a dimer, formed by the covalent, non-disulphide linkage of one small subunit with one large subunit of ribulose bisphosphate carboxylase. For short periods of time (< 1 h), the amount of P65 formed increased with the duration of the exposure to the UV radiation and with the energy of the radiation applied. Prolonged exposure to UV radiation (1-6 h) resulted in the formation of high-molecular-mass aggregates of ribulose bisphosphate carboxylase. Formation of P65 was shown to depend on the native state of the protein, was stimulated by inhibitors of enzyme activity, and was inhibited by activators of enzyme activity. A UV-independent accumulation of P65 was also achieved by the in vitro incubation of plant crude extracts. However, the UV-dependent and the UV-independent formation of P65 seemed to occur by distinct molecular mechanisms. The UV-dependent accumulation of P65 was immunologically detected in all species examined, including Lemna minor, Arum italicum, Brassica oleracea, Triticum aestivum, Zea mays, Pisum sativum and Phaseolus vulgaris, suggesting that it may constitute a universal response to UV radiation, common to all photo-synthetic tissues.

  3. Evaluation of human antibody responses to diphtheria toxin subunits A and B in various age groups.

    PubMed

    Karakus, R; Caglar, K; Aybay, C

    2007-11-01

    This study aimed to evaluate human antibody responses to diphtheria toxin subunits in various age groups. Antibodies against the intact diphtheria toxin and the diphtheria toxin subunits A and B were evaluated in 1319 individuals using a double-antigen ELISA. Although high levels of protection (83.6%, 95% CI 79.2-87.4) were found in children and adolescents, the middle-aged adult population was less protected (28.8%, 95% CI 24.3-33.6). An increase in age was associated with a decrease in the frequency of protected individuals in the 0-39-year age group (p <0.001). Anti-subunit B levels correlated well (p <0.01) with levels of antibodies against the intact toxin. In children aged < or =16 years, the intervals at which the peaks in geometric mean titres of anti-subunit B antibodies were observed were found to correlate with the ages at which booster doses are administered. Overall, males appeared to be more protected than females (OR 1.67, 95% CI 1.34-2.08, p <0.001). A small group of individuals had antibody levels of > or =0.1 IU/mL against the intact toxin, but did not have protective antibody against subunit B. Determination of anti-subunit B antibody levels should help in evaluating the effectiveness of diphtheria boosters and other aspects of diphtheria immunity.

  4. A translation-like cycle is a quality control checkpoint for maturing 40S ribosome subunits.

    PubMed

    Strunk, Bethany S; Novak, Megan N; Young, Crystal L; Karbstein, Katrin

    2012-07-06

    Assembly factors (AFs) prevent premature translation initiation on small (40S) ribosomal subunit assembly intermediates by blocking ligand binding. However, it is unclear how AFs are displaced from maturing 40S ribosomes, if or how maturing subunits are assessed for fidelity, and what prevents premature translation initiation once AFs dissociate. Here we show that maturation involves a translation-like cycle whereby the translation factor eIF5B, a GTPase, promotes joining of large (60S) subunits with pre-40S subunits to give 80S-like complexes, which are subsequently disassembled by the termination factor Rli1, an ATPase. The AFs Tsr1 and Rio2 block the mRNA channel and initiator tRNA binding site, and therefore 80S-like ribosomes lack mRNA or initiator tRNA. After Tsr1 and Rio2 dissociate from 80S-like complexes Rli1-directed displacement of 60S subunits allows for translation initiation. This cycle thus provides a functional test of 60S subunit binding and the GTPase site before ribosomes enter the translating pool.

  5. Multiple origins of the ascidian-Prochloron symbiosis: molecular phylogeny of photosymbiotic and non-symbiotic colonial ascidians inferred from 18S rDNA sequences.

    PubMed

    Yokobori, Shin-Ichi; Kurabayashi, Atsushi; Neilan, Brett A; Maruyama, Tadashi; Hirose, Euichi

    2006-07-01

    In the tropics, certain didemnid ascidians harbor the prokaryotic photosymbiont Prochloron. To date, this photosymbiosis has been found in four didemnid genera that include non-symbiotic species. Here, we report the molecular phylogeny of symbiotic and non-symbiotic didemnids based on their 18S rDNA sequences. The data cover all four genera containing symbiotic species and one other genus comprised of only non-symbiotic species. Near-complete nucleotide sequences of 18S rDNAs were determined for four non-didemnid species and 52 didemnid samples (five genera), including 48 photosymbiotic samples collected from the Ryukyu Archipelago, the Great Barrier Reef, Hawaii, and Bali. Our phylogenetic trees indicated a monophyletic origin of the family Didemnidae, as well as each of the didemnid genera. The results strongly support the hypothesis that establishment of the ascidian-Prochloron symbiosis occurred independently in the Didemnidae lineage at least once in each of the genera that possess symbiotic species.

  6. 18S rRNA Gene Variation among Common Airborne Fungi, and Development of Specific Oligonucleotide Probes for the Detection of Fungal Isolates

    PubMed Central

    Wu, Zhihong; Tsumura, Yoshihiko; Blomquist, Göran; Wang, Xiao-Ru

    2003-01-01

    In this study, we sequenced 18S rRNA genes (rDNA) from 49 fungal strains representing 31 species from 15 genera. Most of these species are common airborne fungi and pathogens that may cause various public health concerns. Sequence analysis revealed distinct divergence between Zygomycota and Ascomycota. Within Ascomycota, several strongly supported clades were identified that facilitate the taxonomic placement of several little-studied fungi. Wallemia appeared as the group most diverged from all the other Ascomycota species. Based on the 18S rDNA sequence variation, 108 oligonucleotide probes were designed for each genus and species included in this study. After homology searches and DNA hybridization evaluations, 33 probes were verified as genus or species specific. The optimal hybridization temperatures to achieve the best specificity for these 33 probes were determined. These new probes can contribute to the molecular diagnostic research for environmental monitoring. PMID:12957927

  7. Localization of 18S ribosomal genes in suckermouth armoured catfishes Loricariidae (Teleostei, Siluriformes) with discussion on the Ag-NOR evolution

    PubMed Central

    Alves, Anderson Luis; de Borba, Rafael Splendore; Pozzobon, Allan Pierre Bonetti; Oliveira, Claudio; Nirchio, Mauro; Granado, Angel; Foresti, Fausto

    2012-01-01

    Abstract The family Loricariidae with about 690 species divided into six subfamilies, is one of the world’s largest fish families. Cytogenetic studies conducted in the family showed that among 90 species analyzed the diploid number ranges from 2n=38 in Ancistrus sp. to 2n=96 in Hemipsilichthys gobio Luetken, 1874. In the present study, fluorescence in situ hybridization (FISH) was employed to determine the chromosomal localization of the 18S rDNA gene in four suckermouth armoured catfishes: Kronichthys lacerta (Nichols, 1919), Pareiorhaphis splendens (Bizerril, 1995), Liposarcus multiradiatus (Hancock, 1828) and Hypostomus prope plecostomus (Linnaeus, 1758). All species analyzed showed one chromosome pair with 18S rDNA sequences, as observed in the previous Ag-NORs analyses. The presence of size and numerical polymorphism was observed and discussed, with proposing a hypothesis of the Ag-NOR evolution in Loricariidae. PMID:24260671

  8. Fast evolving 18S rRNA sequences from Solenogastres (Mollusca) resist standard PCR amplification and give new insights into mollusk substitution rate heterogeneity

    PubMed Central

    2010-01-01

    Background The 18S rRNA gene is one of the most important molecular markers, used in diverse applications such as molecular phylogenetic analyses and biodiversity screening. The Mollusca is the second largest phylum within the animal kingdom and mollusks show an outstanding high diversity in body plans and ecological adaptations. Although an enormous amount of 18S data is available for higher mollusks, data on some early branching lineages are still limited. Despite of some partial success in obtaining these data from Solenogastres, by some regarded to be the most "basal" mollusks, this taxon still remained problematic due to contamination with food organisms and general amplification difficulties. Results We report here the first authentic 18S genes of three Solenogastres species (Mollusca), each possessing a unique sequence composition with regions conspicuously rich in guanine and cytosine. For these GC-rich regions we calculated strong secondary structures. The observed high intra-molecular forces hamper standard amplification and appear to increase formation of chimerical sequences caused by contaminating foreign DNAs from potential prey organisms. In our analyses, contamination was avoided by using RNA as a template. Indication for contamination of previously published Solenogastres sequences is presented. Detailed phylogenetic analyses were conducted using RNA specific models that account for compensatory substitutions in stem regions. Conclusions The extreme morphological diversity of mollusks is mirrored in the molecular 18S data and shows elevated substitution rates mainly in three higher taxa: true limpets (Patellogastropoda), Cephalopoda and Solenogastres. Our phylogenetic tree based on 123 species, including representatives of all mollusk classes, shows limited resolution at the class level but illustrates the pitfalls of artificial groupings formed due to shared biased sequence composition. PMID:20214780

  9. Karyotypes, male meiosis and comparative FISH mapping of 18S ribosomal DNA and telomeric (TTAGG) n repeat in eight species of true bugs (Hemiptera, Heteroptera)

    PubMed Central

    Grozeva, S.; Kuznetsova, V.G.; Anokhin, B.A.

    2011-01-01

    Abstract Eight species belonging to five true bug families were analyzed using DAPI/CMA3-staining and fluorescence in situ hybridization (FISH) with telomeric (TTAGG)n and 18S rDNA probes. Standard chromosomal complements are reported for the first time for Deraeocoris rutilus (Herrich-Schäffer, 1838) (2n=30+2m+XY) and Deraeocoris ruber(Linnaeus, 1758) (2n=30+2m+XY) from the family Miridae. Using FISH, the location of a 18S rDNA cluster was detected in these species and in five more species: Megaloceroea recticornis (Geoffroy, 1785) (2n=30+XY) from the Miridae; Oxycarenus lavaterae (Fabricius, 1787) (2n=14+2m+XY) from the Lygaeidae s.l.; Pyrrhocoris apterus (Linnaeus, 1758) (2n=22+X) from the Pyrrhocoridae; Eurydema oleracea (Linnaeus, 1758) (2n=12+XY) and Graphosoma lineatum (Linnaeus, 1758) (2n=12+XY) from the Pentatomidae. The species were found to differ with respect to location of a 18S rRNA gene cluster which resides on autosomes in Oxycarenus lavaterae and Pyrrhocoris apterus, whereas it locates on sex chromosomes in other five species. The 18S rDNA location provides the first physical landmark of the genomes of the species studied. The insect consensus telomeric pentanucleotide (TTAGG)n was demonstrated to be absent in all the species studied in this respect, Deraeocoris rutilus, Megaloceroea recticornis, Cimex lectularius Linnaeus, 1758 (Cimicidae), Eurydema oleracea, and Graphosoma lineatum, supporting the hypothesis that this motif was lost in early evolution of the Heteroptera and secondarily replaced with another motif (yet unknown) or the alternative telomerase-independent mechanisms of telomere maintenance. Dot-blot hybridization analysis of the genomic DNA from Cimex lectularius, Nabis sp. and Oxycarenus lavaterae with (TTAGG)n and six other telomeric probes likewise provided a negative result. PMID:24260641

  10. Molecular Analyses of a Three-Subunit Euryarchaeal Clamp Loader Complex from Methanosarcina acetivorans▿ †

    PubMed Central

    Chen, Yi-Hsing; Lin, Yuyen; Yoshinaga, Aya; Chhotani, Benazir; Lorenzini, Jenna L.; Crofts, Alexander A.; Mei, Shou; Mackie, Roderick I.; Ishino, Yoshizumi; Cann, Isaac K. O.

    2009-01-01

    Chromosomal DNA replication is dependent on processive DNA synthesis. Across the three domains of life and in certain viruses, a toroidal sliding clamp confers processivity to replicative DNA polymerases by encircling the DNA and engaging the polymerase in protein/protein interactions. Sliding clamps are ring-shaped; therefore, they have cognate clamp loaders that open and load them onto DNA. Here we use biochemical and mutational analyses to study the structure/function of the Methanosarcina acetivorans clamp loader or replication factor C (RFC) homolog. M. acetivorans RFC (RFCMa), which represents an intermediate between the common archaeal RFC and the eukaryotic RFC, comprises two different small subunits (RFCS1 and RFCS2) and a large subunit (RFCL). Size exclusion chromatography suggested that RFCS1 exists in oligomeric states depending on protein concentration, while RFCS2 exists as a monomer. Protein complexes of RFCS1/RFCS2 formed in solution; however, they failed to stimulate DNA synthesis by a cognate DNA polymerase in the presence of its clamp. Determination of the subunit composition and previous mutational analysis allowed the prediction of the spatial distribution of subunits in this new member of the clamp loader family. Three RFCS1 subunits are flanked by an RFCS2 and an RFCL. The spatial distribution is, therefore, reminiscent of the minimal Escherichia coli clamp loader that exists in space as three γ-subunits (motor) flanked by the δ′ (stator) and the δ (wrench) subunits. Mutational analysis, however, suggested that the similarity between the two clamp loaders does not translate into the complete conservation of the functions of individual subunits within the RFCMa complex. PMID:19717601

  11. Drosophila laminin: sequence of B2 subunit and expression of all three subunits during embryogenesis

    PubMed Central

    1989-01-01

    In a previous study, we described the cloning of the genes encoding the three subunits of Drosophila laminin, a substrate adhesion molecule, and the cDNA sequence of the B1 subunit (Montell and Goodman, 1988). This analysis revealed the similarity of Drosophila laminin with the mouse and human complexes in subunit composition, domain structure, and amino acid sequence. In this paper, we report the deduced amino acid sequence of the B2 subunit. We then describe the expression and tissue distribution of the three subunits of laminin during Drosophila embryogenesis using both in situ hybridization and immunolocalization techniques, with particular emphasis on its expression in and around the developing nervous system. PMID:2808533

  12. Chromosomal localization of 5S and 18S-5.8S-25S ribosomal DNA sites in five Asian pines using fluorescence in situ hybridization.

    PubMed

    Liu, Z-L; Zhang, D; Hong, D-Y; Wang, X-R

    2003-01-01

    Fluorescence in situ hybridization (FISH) was employed on mitotic metaphase chromosome preparations of five Asian Pinus species: Pinus tabuliformis, Pinus yunnanensis, Pinus densata, Pinus massoniana and Pinus merkusii, using simultaneously DNA probes of the 18S rRNA gene and the 5S rRNA gene including the non-transcribed spacer sequences. The number and location of 18S rDNA sites varied markedly (5-10 pairs of strong signals) among the five pines. A maximum of 20 major 18S rDNA sites was observed in the diploid genome (2n = 24) of P. massoniana. The 5S rDNA FISH pattern was less variable, with one major site and one minor site commonly observed in each species. The differentiation of rDNA sites on chromosomes among the five pines correlates well with their phylogenic positions in Pinus as reconstructed from other molecular data. P. densata, a species of hybrid origin, resembles its parents ( P. tabuliformis and P. yunnanensis), including some components characteristic of each parent in its pattern. However, the species is unique, showing new features resulting possibly from recombination and genome reorganization.

  13. Reconstructing the Phylogeny of Capsosiphon fulvescens (Ulotrichales, Chlorophyta) from Korea Based on rbcL and 18S rDNA Sequences.

    PubMed

    Sun, Sang-Mi; Yang, Seung Hwan; Golokhvast, Kirill S; Le, Bao; Chung, Gyuhwa

    2016-01-01

    Capsosiphon fulvescens is a filamentous green algae in the class Ulvophyceae. It has been consumed as food with unique flavor and soft texture to treat stomach disorders and hangovers, and its economic value justifies studying its nutritional and potential therapeutic effects. In contrast to these applications, only a few taxonomic studies have been conducted on C. fulvescens. In particular, classification and phylogenetic relationships of the C. fulvescens below the order level are controversial. To determine its phylogenetic position in the class, we used rbcL and 18S rDNA sequences as molecular markers to construct phylogenetic trees. The amplified rbcL and 18S rDNA sequences from 4 C. fulvescens isolates (Jindo, Jangheung, Wando, and Koheung, Korea) were used for phylogenetic analysis by employing three different phylogenetic methods: neighbor joining (NJ), maximum parsimony (MP), and maximum likelihood (ML). The rbcL phylogenetic tree showed that all taxa in the order Ulvales were clustered as a monophyletic group and resolved the phylogenetic position of C. fulvescens in the order Ulotrichales. The significance of our study is that the 18S rDNA phylogenetic tree shows the detailed taxonomic position of C. fulvescens. In our result, C. fulvescens is inferred as a member of Ulotrichaceae, along with Urospora and Acrosiphonia.

  14. Molecular characterization of Argulus bengalensis and Argulus siamensis (Crustacea: Argulidae) infecting the cultured carps in West Bengal, India using 18S rRNA gene sequences

    PubMed Central

    Patra, Avijit; Mondal, Anjan; Banerjee, Sayani; Adikesavalu, Harresh; Joardar, Siddhartha Narayan; Abraham, Thangapalam Jawahar

    2016-01-01

    The present study characterized Argulus spp. infecting the cultured carps using 18S rRNA gene sequences, estimated the genetic similarity among Argulus spp. and established their phylogenetic relationship. Of the 320 fish samples screened, 34 fish (10.6%) had Argulus infection. The parasitic frequency index (PFI) was observed to be high (20%) in Hypophthalmichthys molitrix and Labeo bata. The frequency of infection was high in September (PFI: 17%) and October (PFI: 12.9%). The 18S rRNA sequences of five A. bengalensis (KF583878, KF192316, KM016968, KM016969, and KM016970) and one A. siamensis (KF583879) of this study showed genetic heterogeneity and exhibited 77-99% homology among the 18S rRNA gene sequences of Argulus spp. of NCBI GenBank database. Among the Indian Argulus spp. the sequence homology was 87–100%. Evolutionary pair-wise distances between Indian Argulus spp. and other Argulus spp. ranged from 0 to 20.20%. In the phylogenetic tree, all the crustaceans were clustered together as a separate clade with two distinct lineages. The lineage-1 comprised exclusive of Branchiura (Argulus spp.). All Argulus bengalensis clustered together and A. siamensis (KF583879) was closely related to Argulus sp. JN558648. The results of the present study provided baseline data for future work on population structure analysis of Indian Argulus species. PMID:28097169

  15. Co-located 18S/5S rDNA arrays: an ancient and unusual chromosomal trait in Julidini species (Labridae, Perciformes)

    PubMed Central

    Amorim, Karlla Danielle Jorge; Cioffi, Marcelo de Bello; Bertollo, Luiz Antonio Carlos; Soares, Rodrigo Xavier; de Souza, Allyson Santos; da Costa, Gideão Wagner Werneck Felix; Molina, Wagner Franco

    2016-01-01

    Abstract Wrasses (Labridae) are extremely diversified marine fishes, whose species exhibit complex interactions with the reef environment. They are widely distributed in the Indian, Pacific and Atlantic oceans. Their species have displayed a number of karyotypic divergent processes, including chromosomal regions with complex structural organization. Current cytogenetic information for this family is phylogenetically and geographically limited and mainly based on conventional cytogenetic techniques. Here, the distribution patterns of heterochromatin, GC-specific chromosome regions and Ag-NORs, and the organization of 18S and 5S rDNA sites of the Atlantic species Thalassoma noronhanum (Boulenger, 1890), Halichoeres poeyi (Steindachner, 1867), Halichoeres radiatus (Linnaeus, 1758), Halichoeres brasiliensis (Bloch, 1791) and Halichoeres penrosei Starks, 1913, belonging to the tribe Julidini were analyzed. All the species exhibited 2n=48 chromosomes with variation in the number of chromosome arms among genera. Thalassoma noronhanum has 2m+46a, while species of the genus Halichoeres Rüppell, 1835 share karyotypes with 48 acrocentric chromosomes. The Halichoeres species exhibit differences in the heterochromatin distribution patterns and in the number and distribution of 18S and 5S rDNA sites. The occurrence of 18S/5S rDNA syntenic arrangements in all the species indicates a functionally stable and adaptive genomic organization. The phylogenetic sharing of this rDNA organization highlights a marked and unusual chromosomal singularity inside the family Labridae. PMID:28123678

  16. Subunit-selective role of the M3 transmembrane domain of the nicotinic acetylcholine receptor in channel gating.

    PubMed

    De Rosa, María José; Corradi, Jeremías; Bouzat, Cecilia

    2008-02-01

    The nicotinic acetylcholine receptor (AChR) can be either hetero-pentameric, composed of alpha and non-alpha subunits, or homo-pentameric, composed of alpha7 subunits. To explore the subunit-selective contributions of transmembrane domains to channel gating we analyzed single-channel activity of chimeric muscle AChRs. We exchanged M3 between alpha1 and epsilon or alpha7 subunits. The replacement of M3 in alpha1 by epsilonM3 significantly alters activation properties. Channel activity appears as bursts of openings whose durations are 20-fold longer than those of wild-type AChRs. In contrast, 7-fold briefer openings are observed in AChRs containing the reverse epsilon chimeric subunit. The duration of the open state decreases with the increase in the number of alpha1M3 segments, indicating additive contributions of M3 of all subunits to channel closing. Each alpha1M3 segment decreases the energy barrier of the closing process by approximately 0.8 kcal/mol. Partial chimeric subunits show that small stretches of the M3 segment contribute additively to the open duration. The replacement of alpha1 sequence by alpha7 in M3 leads to 3-fold briefer openings whereas in M1 it leads to 10-fold prolonged openings, revealing that the subunit-selective role is unique to each transmembrane segment.

  17. Diversity of insect nicotinic acetylcholine receptor subunits.

    PubMed

    Jones, Andrew K; Sattelle, David B

    2010-01-01

    Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that mediate fast synaptic transmission in the insect nervous system and are targets of a major group of insecticides, the neonicotinoids. They consist of five subunits arranged around a central ion channeL Since the subunit composition determines the functional and pharmacological properties of the receptor the presence of nAChR families comprising several subunit-encodinggenes provides a molecular basis for broad functional diversity. Analyses of genome sequences have shown that nAChR gene families remain compact in diverse insect species, when compared to their nematode andvertebrate counterparts. Thus, the fruit fly (Drosophila melanogaster), malaria mosquito (Anopheles gambiae), honey bee (Apis mellifera), silk worm (Bombyx mon) and the red flour beetle (Tribolium castaneum) possess 10-12 nAChR genes while human and the nematode Caenorhabditis elegans have 16 and 29 respectively. Although insect nAChRgene families are amongst the smallest known, receptor diversity can be considerably increased by the posttranscriptional processes alternative splicing and mRNA A-to-I editingwhich can potentially generate protein products which far outnumber the nAChR genes. These two processes can also generate species-specific subunit isoforms. In addition, each insect possesses at least one highly divergent nAChR subunit which may perform species-specific functions. Species-specific subunit diversification may offer promising targets for future rational design of insecticides that target specific pest insects while sparing beneficial species.

  18. Comparison of Voltage Gated K(+) Currents in Arterial Myocytes with Heterologously Expressed K v Subunits.

    PubMed

    Cox, Robert H; Fromme, Samantha

    2016-12-01

    We have shown that three components contribute to functional voltage gated K(+) (K v) currents in rat small mesenteric artery myocytes: (1) Kv1.2 plus Kv1.5 with Kvβ1.2 subunits, (2) Kv2.1 probably associated with Kv9.3 subunits, and (3) Kv7.4 subunits. To confirm and address subunit stoichiometry of the first two, we have compared the biophysical properties of K v currents in small mesenteric artery myocytes with those of Kv subunits heterologously expressed in HEK293 cells using whole cell voltage clamp methods. Selective inhibitors of Kv1 (correolide, COR) and Kv2 (stromatoxin, ScTx) channels were used to separate these K v current components. Conductance-voltage and steady state inactivation data along with time constants of activation, inactivation, and deactivation of native K v components were generally well represented by those of Kv1.2-1.5-β1.2 and Kv2.1-9.3 channels. The slope of the steady state inactivation-voltage curve (availability slope) proved to be the most sensitive measure of accessory subunit presence. The availability slope curves exhibited a single peak for both native K v components. Availability slope curves for Kv1.2-1.5-β1.2 and Kv2.1-9.3 channels expressed in human embryonic kidney cells also exhibited a single peak that shifted to more depolarized voltages with increasing accessory to α subunit transfection ratio. Availability slope curves for SxTc-insensitive currents were similar to those of Kv1.2-1.5 expressed with Kvβ1.2 at a 1:5 molar ratio while curves for COR-insensitive currents closely resembled those of Kv2.1 expressed with Kv9.3 at a 1:1 molar ratio. These results support the suggested Kv subunit combinations in small mesenteric artery, and further suggest that Kv1 α and Kvβ1.2 but not Kv2.1 and Kv9.3 subunits are present in a saturated (4:4) stoichiometry.

  19. Subunit Recombinant Vaccine Protects Against Monkeypox

    DTIC Science & Technology

    2006-05-27

    Subunit Recombinant Vaccine Protects against Monkeypox1 Jean-Michel Heraud,* Yvette Edghill-Smith,*† Victor Ayala,‡ Irene Kalisz,‡ Janie Parrino ...GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Heraud, JM Edghill-Smith, Y Ayala, V Kalisz, I Parrino , J Kalyanaraman, VS Manischewitz, J King

  20. Localization of the human genes encoding the two subunits of general transcription factor TFIIE.

    PubMed

    Purrello, M; Di Pietro, C; Rapisarda, A; Motta, S; Pavone, L; Grzeschik, K H; Sichel, G

    1994-09-01

    TFIIE is a general transcription factor for class II genes composed of two types of subunits, a large one of 56 kDa and a small of 34 kDa. By Southern analysis at high and at low stringency of a panel of mouse/human hybrid cell lines and by in situ chromosomal hybridization, we have demonstrated that both polypeptides are encoded by genes that are single copy in the human genome and are localized at 3q13-q21 and at 8p12, respectively. A TaqI RFLP (heterozygosity index of 0.07) was detected at the locus for the 56-kDa subunit.

  1. Isolation and preliminary characterization of the subunits of the terminal component of naphthalene dioxygenase from Pseudomonas putida NCIB 9816-4.

    PubMed Central

    Suen, W C; Gibson, D T

    1993-01-01

    The terminal oxygenase component (ISPNAP) of naphthalene dioxygenase from Pseudomonas putida NCIB 9816-4 was purified to homogeneity. The protein contained approximately 4 g-atoms each of iron and acid-labile sulfide per mol of ISPNAP, and enzyme activity was stimulated significantly by addition of exogenous iron. The large (alpha) and small (beta) subunits of ISPNAP were isolated by two different procedures. The NH2-terminal amino acid sequences of the alpha and beta subunits were identical to the deduced amino acid sequences reported for the ndoB and ndoC genes from P. putida NCIB 9816 and almost identical to the NH2-terminal amino acid sequences determined for the large and small subunits of ISPNAP from P. putida G7. Gel filtration in the presence of 6 M urea gave an alpha subunit with an absorption maximum at 325 nm and broad absorption between 420 and 450 nm. The alpha subunit contained approximately 2 g-atoms each of iron and acid-labile sulfide per mol of the subunit. The beta subunit did not contain iron or acid-labile sulfide. These results, taken in conjunction with the deduced amino acid sequences of the large subunits from several iron-sulfur oxygenases, indicate that each alpha subunit of ISPNAP contains a Rieske [2Fe-2S] center. Images PMID:8376335

  2. Development of a cob-18S rRNA gene real-time PCR assay for quantifying Pfiesteria shumwayae in the natural environment.

    PubMed

    Zhang, Huan; Lin, Senjie

    2005-11-01

    Despite the fact that the heterotrophic dinoflagellate Pfiesteria shumwayae is an organism of high interest due to alleged toxicity, its abundance in natural environments is poorly understood. To address this inadequacy, a real-time quantitative PCR assay based on mitochondrial cytochrome b (cob) and 18S rRNA gene was developed and P. shumwayae abundance was investigated in several geographic locations. First, cob and its 5'-end region were isolated from a P. shumwayae culture, revealing three different copies, each consisting of an identical cob coding region and an unidentified region (X) of variable length and sequence. The unique sequences in cob and the X region were then used to develop a P. shumwayae-specific primer set. This primer set was used with reported P. shumwayae-specific 18S primers in parallel real-time PCRs to investigate P. shumwayae abundance from Maine to North Carolina along the U.S. east coast and along coasts in Chile, Hawaii, and China. Both genes generally gave similar results, indicating that this species was present, but at low abundance (mostly <10 cells x ml(-1)), in all the American coast locations investigated (with the exception of Long Island Sound, where which both genes gave negative results). Genetic variation was detected by use of both genes in most of the locations, and while cob consistently detected P. shumwayae or close genetic variants, some of the 18S PCR products were unrelated to P. shumwayae. We conclude that (i) the real-time PCR assay developed is useful for specific quantification of P. shumwayae, and (ii) P. shumwayae is distributed widely at the American coasts, but normally only as a minor component of plankton even in high-risk estuaries (Neuse River and the Chesapeake Bay).

  3. Molecular phylogeny and barcoding of Caulerpa (Bryopsidales) based on the tufA, rbcL, 18S rDNA and ITS rDNA genes.

    PubMed

    Kazi, Mudassar Anisoddin; Reddy, C R K; Jha, Bhavanath

    2013-01-01

    The biodiversity assessment of different taxa of the genus Caulerpa is of interest from the context of morphological plasticity, invasive potential of some species and biotechnological and pharmacological applications. The present study investigated the identification and molecular phylogeny of different species of Caulerpa occurring along the Indian coast inferred from tufA, rbcL, 18S rDNA and ITS rDNA nucleotide sequences. Molecular data confirmed the identification of 10 distinct Caulerpa species: C. veravalensis, C. verticillata, C. racemosa, C. microphysa, C. taxifolia, C. sertularioides, C. scalpelliformis, C. serrulata, C. peltata and C. mexicana. All datasets significantly supported the sister relationship between C. veravalensis and C. racemosa var. cylindracea. It was also concluded from the results that the specimen identified previously as C. microphysa and C. lentillifera could not be considered as separate species. The molecular data revealed the presence of multiple lineages for C. racemosa which can be resolved into separate species. All four markers were used to ascertain their utility for DNA barcoding. The tufA gene proved a better marker with monophyletic association as the main criteria for identification at the species level. The results also support the use of 18S rDNA insertion sequences to delineate the Caulerpa species through character-based barcoding. The ITS rDNA (5.8S-ITS2) phylogenetic analysis also served as another supporting tool. Further, more sequences from additional Caulerpa specimens will need to be analysed in order to support the role of these two markers (ITS rDNA and 18S insertion sequence) in identification of Caulerpa species. The present study revealed the phylogeny of Caulerpa as complete as possible using the currently available data, which is the first comprehensive report illustrating the molecular phylogeny and barcoding of the genus Caulerpa from Indian waters.

  4. Molecular Phylogeny and Barcoding of Caulerpa (Bryopsidales) Based on the tufA, rbcL, 18S rDNA and ITS rDNA Genes

    PubMed Central

    Kazi, Mudassar Anisoddin; Reddy, C. R. K.; Jha, Bhavanath

    2013-01-01

    The biodiversity assessment of different taxa of the genus Caulerpa is of interest from the context of morphological plasticity, invasive potential of some species and biotechnological and pharmacological applications. The present study investigated the identification and molecular phylogeny of different species of Caulerpa occurring along the Indian coast inferred from tufA, rbcL, 18S rDNA and ITS rDNA nucleotide sequences. Molecular data confirmed the identification of 10 distinct Caulerpa species: C. veravalensis, C. verticillata, C. racemosa, C. microphysa, C. taxifolia, C. sertularioides, C. scalpelliformis, C. serrulata, C. peltata and C. mexicana. All datasets significantly supported the sister relationship between C. veravalensis and C. racemosa var. cylindracea. It was also concluded from the results that the specimen identified previously as C. microphysa and C. lentillifera could not be considered as separate species. The molecular data revealed the presence of multiple lineages for C. racemosa which can be resolved into separate species. All four markers were used to ascertain their utility for DNA barcoding. The tufA gene proved a better marker with monophyletic association as the main criteria for identification at the species level. The results also support the use of 18S rDNA insertion sequences to delineate the Caulerpa species through character-based barcoding. The ITS rDNA (5.8S-ITS2) phylogenetic analysis also served as another supporting tool. Further, more sequences from additional Caulerpa specimens will need to be analysed in order to support the role of these two markers (ITS rDNA and 18S insertion sequence) in identification of Caulerpa species. The present study revealed the phylogeny of Caulerpa as complete as possible using the currently available data, which is the first comprehensive report illustrating the molecular phylogeny and barcoding of the genus Caulerpa from Indian waters. PMID:24340028

  5. Use of Subgenic 18S Ribosomal DNA PCR and Sequencing for Genus and Genotype Identification of Acanthamoebae from Humans with Keratitis and from Sewage Sludge

    PubMed Central

    Schroeder, Jill M.; Booton, Gregory C.; Hay, John; Niszl, Ingrid A.; Seal, David V.; Markus, Miles B.; Fuerst, Paul A.; Byers, Thomas J.

    2001-01-01

    This study identified subgenic PCR amplimers from 18S rDNA that were (i) highly specific for the genus Acanthamoeba, (ii) obtainable from all known genotypes, and (iii) useful for identification of individual genotypes. A 423- to 551-bp Acanthamoeba-specific amplimer ASA.S1 obtained with primers JDP1 and JDP2 was the most reliable for purposes i and ii. A variable region within this amplimer also identified genotype clusters, but purpose iii was best achieved with sequencing of the genotype-specific amplimer GTSA.B1. Because this amplimer could be obtained from any eukaryote, axenic Acanthamoeba cultures were required for its study. GTSA.B1, produced with primers CRN5 and 1137, extended between reference bp 1 and 1475. Genotypic identification relied on three segments: bp 178 to 355, 705 to 926, and 1175 to 1379. ASA.S1 was obtained from single amoeba, from cultures of all known 18S rDNA genotypes, and from corneal scrapings of Scottish patients with suspected Acanthamoeba keratitis (AK). The AK PCR findings were consistent with culture results for 11 of 15 culture-positive specimens and detected Acanthamoeba in one of nine culture-negative specimens. ASA.S1 sequences were examined for 6 of the 11 culture-positive isolates and were most closely associated with genotypic cluster T3-T4-T11. A similar distance analysis using GTSA.B1 sequences identified nine South African AK-associated isolates as genotype T4 and three isolates from sewage sludge as genotype T5. Our results demonstrate the usefulness of 18S ribosomal DNA PCR amplimers ASA.S1 and GTSA.B1 for Acanthamoeba-specific detection and reliable genotyping, respectively, and provide further evidence that T4 is the predominant genotype in AK. PMID:11326011

  6. Use of subgenic 18S ribosomal DNA PCR and sequencing for genus and genotype identification of acanthamoebae from humans with keratitis and from sewage sludge.

    PubMed

    Schroeder, J M; Booton, G C; Hay, J; Niszl, I A; Seal, D V; Markus, M B; Fuerst, P A; Byers, T J

    2001-05-01

    This study identified subgenic PCR amplimers from 18S rDNA that were (i) highly specific for the genus Acanthamoeba, (ii) obtainable from all known genotypes, and (iii) useful for identification of individual genotypes. A 423- to 551-bp Acanthamoeba-specific amplimer ASA.S1 obtained with primers JDP1 and JDP2 was the most reliable for purposes i and ii. A variable region within this amplimer also identified genotype clusters, but purpose iii was best achieved with sequencing of the genotype-specific amplimer GTSA.B1. Because this amplimer could be obtained from any eukaryote, axenic Acanthamoeba cultures were required for its study. GTSA.B1, produced with primers CRN5 and 1137, extended between reference bp 1 and 1475. Genotypic identification relied on three segments: bp 178 to 355, 705 to 926, and 1175 to 1379. ASA.S1 was obtained from single amoeba, from cultures of all known 18S rDNA genotypes, and from corneal scrapings of Scottish patients with suspected Acanthamoeba keratitis (AK). The AK PCR findings were consistent with culture results for 11 of 15 culture-positive specimens and detected Acanthamoeba in one of nine culture-negative specimens. ASA.S1 sequences were examined for 6 of the 11 culture-positive isolates and were most closely associated with genotypic cluster T3-T4-T11. A similar distance analysis using GTSA.B1 sequences identified nine South African AK-associated isolates as genotype T4 and three isolates from sewage sludge as genotype T5. Our results demonstrate the usefulness of 18S ribosomal DNA PCR amplimers ASA.S1 and GTSA.B1 for Acanthamoeba-specific detection and reliable genotyping, respectively, and provide further evidence that T4 is the predominant genotype in AK.

  7. Development of a cob-18S rRNA Gene Real-Time PCR Assay for Quantifying Pfiesteria shumwayae in the Natural Environment†

    PubMed Central

    Zhang, Huan; Lin, Senjie

    2005-01-01

    Despite the fact that the heterotrophic dinoflagellate Pfiesteria shumwayae is an organism of high interest due to alleged toxicity, its abundance in natural environments is poorly understood. To address this inadequacy, a real-time quantitative PCR assay based on mitochondrial cytochrome b (cob) and18S rRNA gene was developed and P. shumwayae abundance was investigated in several geographic locations. First, cob and its 5′-end region were isolated from a P. shumwayae culture, revealing three different copies, each consisting of an identical cob coding region and an unidentified region (X) of variable length and sequence. The unique sequences in cob and the X region were then used to develop a P. shumwayae-specific primer set. This primer set was used with reported P. shumwayae-specific 18S primers in parallel real-time PCRs to investigate P. shumwayae abundance from Maine to North Carolina along the U.S. east coast and along coasts in Chile, Hawaii, and China. Both genes generally gave similar results, indicating that this species was present, but at low abundance (mostly <10 cells · ml−1), in all the American coast locations investigated (with the exception of Long Island Sound, where which both genes gave negative results). Genetic variation was detected by use of both genes in most of the locations, and while cob consistently detected P. shumwayae or close genetic variants, some of the 18S PCR products were unrelated to P. shumwayae. We conclude that (i) the real-time PCR assay developed is useful for specific quantification of P. shumwayae, and (ii) P. shumwayae is distributed widely at the American coasts, but normally only as a minor component of plankton even in high-risk estuaries (Neuse River and the Chesapeake Bay). PMID:16269741

  8. Determination of phylogenetic relationships among Eimeria species, which parasitize cattle, on the basis of nuclear 18S rDNA sequence.

    PubMed

    Kokuzawa, Takuya; Ichikawa-Seki, Madoka; Itagaki, Tadashi

    2013-11-01

    We analyzed almost complete 18S rDNA sequences of 10 bovine Eimeria species, namely Eimeria alabamensis, E. auburnensis, E. bovis, E. bukidnonensis, E. canadensis, E. cylindrica, E. ellipsoidalis, E. subspherica, E. wyomingensis and E. zuernii. Although these sequences showed intraspecific variation in 8 species, the sequences of each species were clustered in monophyletic groups in all species, except E. auburnensis. The sequences constituted 3 distinct clusters in a phylogenetic tree with relatively high bootstrap values; however, the members including each cluster shared no similarities in oocyst morphology.

  9. Identification and characterization of high-molecular-weight glutenin subunits from Agropyron intermedium.

    PubMed

    Cao, Shuanghe; Li, Zhixin; Gong, Caiyan; Xu, Hong; Yang, Ran; Hao, Shanting; Wang, Xianping; Wang, Daowen; Zhang, Xiangqi

    2014-01-01

    High-molecular-weight glutenin subunit (HMW-GS) is a primary determinant of processing quality of wheat. Considerable progress has been made in understanding the structure, function and genetic regulation of HMW-GS in wheat and some of its related species, but less is known about their orthologs in Agropyron intermedium, a useful related species for wheat improvement. Here seven HMW-GSs in Ag. intermedium were identified using SDS-PAGE and Western blotting experiments. Subsequently, the seven genes (Glu-1Aix1 ∼ 4 and Glu-1Aiy1 ∼ 3) encoding the seven HMW-GSs were isolated using PCR technique with degenerate primers, and confirmed by bacterial expression and Western blotting. Sequence analysis indicated that the seven Ag. intermedium HMW-GSs shared high similarity in primary structure to those of wheat, but four of the seven subunits were unusually small compared to the representatives of HMW-GS from wheat and two of them possessed extra cysteine residues. The alignment and clustering analysis of deduced amino acid sequences revealed that 1Aix1 and 1Aiy1 subunits had special molecular structure, belonging to the hybrid type compounding between typical x- and y-type subunit. The xy-type subunit 1Aix1 is composed of the N-terminal of x-type and C-terminal of y-type, whereas yx-type subunit 1Aiy1 comprises the N-terminal of y-type and C-terminal of x-type. This result strongly supported the hypothesis of unequal crossover mechanism that might generate the novel coding sequence for the hybrid type of HMW-GSs. In addition to the aforementioned, the other novel characteristics of the seven subunits were also discussed. Finally, phylogenetic analysis based on HMW-GS genes was carried out and provided new insights into the evolutionary biology of Ag. intermedium.

  10. Relationship between auxiliary gamma subunits and mallotoxin on BK channel modulation

    PubMed Central

    Guan, Xin; Li, Qin; Yan, Jiusheng

    2017-01-01

    The large-conductance, calcium- and voltage-activated K+(BK) channel consists of the pore-forming α subunits (BKα) and auxiliary subunits. The auxiliary γ1-3 subunits potently modulate the BK channel by shifting its voltage-dependence of channel activation toward the hyperpolarizing direction by approximately 145 mV (γ1), 100 mV (γ2), and 50 mV (γ3). Mallotoxin is a potent small-molecule BK channel activator. We analyzed the relationship between mallotoxin and the γ subunits in their BK channel-activating effects in membrane patches excised from HEK-293 cells. We found that mallotoxin, when applied extracellularly, shifted the half-activation voltage (V1/2) of BKα channels by −72 mV. The channel-activating effect of mallotoxin was greatly attenuated in the presence of the γ1, γ2, or γ3 subunit, with resultant ΔV1/2 (+/− mallotoxin) values of −9, −28, or −15 mV, respectively. Most examined γ1 mutant subunits antagonized mallotoxin’s channel-activating effect in a manner that was largely dependent on its own modulatory function. However, mallotoxin caused an irreversible functional and structural disengagement of the γ1-F273S mutant from BK channels. We infer that the auxiliary γ subunit effectively interferes with mallotoxin on BK channel modulation via either a direct steric competition or an indirect allosteric influence on mallotoxin’s binding and action on BKα. PMID:28165042

  11. Thermostable cross-protective subunit vaccine against Brucella species.

    PubMed

    Cherwonogrodzky, John W; Barabé, Nicole D; Grigat, Michelle L; Lee, William E; Poirier, Robert T; Jager, Scott J; Berger, Bradley J

    2014-12-01

    A subunit vaccine candidate was produced from Brucella suis 145 (biovar 4; expressing both the A antigen of Brucella abortus and the M antigen of Brucella melitensis). The preparation consisted mostly of polysaccharide (PS; >90% [wt/wt]; both cell-associated PS and exo-PS were combined) and a small amount of protein (1 to 3%) with no apparent nucleic acids. Vaccinated mice were protected (these had a statistically significant reduction in bacterial colonization compared to that of unvaccinated controls) when challenged with representative strains of three Brucella species most pathogenic for humans, i.e., B. abortus, B. melitensis, and B. suis. As little as 1 ng of the vaccine, without added adjuvant, protected mice against B. suis 145 infection (5 × 10(5) CFU), and a single injection of 1 μg of this subunit vaccine protected mice from B. suis 145 challenge for at least 14 months. A single immunization induced a serum IgG response to Brucella antigens that remained elevated for up to 9 weeks. The use of heat (i.e., boiling-water bath, autoclaving) in the vaccine preparation showed that it was thermostable. This method also ensured safety and security. The vaccine produced was immunogenic and highly protective against multiple strains of Brucella and represents a promising candidate for further evaluation.

  12. Deciphering the subunit composition of multimeric proteins by counting photobleaching steps.

    PubMed

    Arant, Ryan J; Ulbrich, Maximilian H

    2014-03-17

    The limit of subdiffraction imaging with fluorescent proteins currently lies at 20 nm, and therefore most protein complexes are too small (2-5 nm) to spatially resolve their individual subunits by optical means. However, the number and stoichiometry of subunits within an immobilized protein complex can be resolved by the observation of photobleaching steps of individual fluorophores or co-localization of single-molecule fluorescence emission in multiple colors. We give an overview of the proteins that have been investigated by this approach and the different techniques that can be used to immobilize and label the proteins. This minireview should serve as a guideline for scientists who want to employ single-molecule subunit counting for their research.

  13. Mutations in RNAs of both ribosomal subunits cause defects in translation termination.

    PubMed Central

    Arkov, A L; Freistroffer, D V; Ehrenberg, M; Murgola, E J

    1998-01-01

    Mutations in RNAs of both subunits of the Escherichia coli ribosome caused defects in catalysis of peptidyl-tRNA hydrolysis in a realistic in vitro termination system. Assaying the two codon-dependent cytoplasmic proteins that drive termination, RF1 and RF2, we observed large defects with RF2 but not with RF1, a result consistent with the in vivo properties of the mutants. Our study presents the first direct in vitro evidence demonstrating the involvement of RNAs from both the large and the small ribosomal subunits in catalysis of peptidyl-tRNA hydrolysis during termination of protein biosynthesis. The results and conclusions are of general significance since the rRNA nucleotides studied have been virtually universally conserved throughout evolution. Our findings suggest a novel role for rRNAs of both subunits as molecular transmitters of a signal for termination. PMID:9482747

  14. Embryonic Expression of the Putative γ Subunit of the Sodium Pump Is Required for Acquisition of Fluid Transport Capacity during Mouse Blastocyst Development

    PubMed Central

    Jones, D. Holstead; Davies, Tyler C.; Kidder, Gerald M.

    1997-01-01

    The sodium/potassium pump, Na+,K+-ATPase, is generally understood to function as a heterodimer of two subunits, a catalytic α subunit and a noncatalytic, glycosylated β subunit. Recently, a putative third subunit, the γ subunit, was cloned. This small protein (6.5 kD) coimmunoprecipitates with the α and β subunits and is closely associated with the ouabain binding site on the holoenzyme, but its function is unknown. We have investigated the expression of the γ subunit in preimplantation mouse development, where Na+,K+-ATPase plays a critical role as the driving force for blastocoel formation (cavitation). Using reverse transcriptase-polymerase chain reaction, we demonstrated that the γ subunit mRNA accumulates continuously from the eight-cell stage onward and that it cosediments with polyribosomes from its time of first appearance. Confocal immunofluorescence microscopy revealed that the γ subunit itself accumulates and is localized at the blastomere surfaces up to the blastocyst stage. In contrast with the α and β subunits, the γ subunit is not concentrated in the basolateral surface of the polarized trophectoderm layer, but is strongly expressed at the apical surface as well. When embryos were treated with antisense oligodeoxynucleotide complementary to the γ subunit mRNA, ouabain-sensitive K+ transport (as indicated by 86Rb+ uptake) was reduced and cavitation delayed. However, Na+,K+-ATPase enzymatic activity was unaffected as determined by a direct phosphorylation assay (“back door” phosphorylation) applied to plasma membrane preparations. These results indicate that the γ subunit, although not an integral component of Na+,K+-ATPase, is an important determinant of active cation transport and that, as such, its embryonic expression is essential for blastocoel formation in the mouse. PMID:9396759

  15. Subunit-selective proteasome activity profiling uncovers uncoupled proteasome subunit activities during bacterial infections.

    PubMed

    Misas-Villamil, Johana C; van der Burgh, Aranka M; Grosse-Holz, Friederike; Bach-Pages, Marcel; Kovács, Judit; Kaschani, Farnusch; Schilasky, Sören; Emon, Asif Emran Khan; Ruben, Mark; Kaiser, Markus; Overkleeft, Hermen S; van der Hoorn, Renier A L

    2017-01-24

    The proteasome is a nuclear - cytoplasmic proteolytic complex involved in nearly all regulatory pathways in plant cells. The three different catalytic activities of the proteasome can have different functions but tools to monitor and control these subunits selectively are not yet available in plant science. Here, we introduce subunit-selective inhibitors and dual-color fluorescent activity-based probes for studying two of the three active catalytic subunits of the plant proteasome. We validate these tools in two model plants and use this to study the proteasome during plant-microbe interactions. Our data reveals that Nicotiana benthamiana incorporates two different paralogs of each catalytic subunit into active proteasomes. Interestingly, both β1 and β5 activities are significantly increased upon infection with pathogenic Pseudomonas syringae pv. tomato DC3000 lacking hopQ1-1 (PtoDC3000(ΔhQ)) whilst the activity profile of the β1 subunit changes. Infection with wild-type PtoDC3000 causes proteasome activities that range from strongly induced β1 and β5 activities to strongly suppressed β5 activities, revealing that β1 and β5 activities can be uncoupled during bacterial infection. These selective probes and inhibitors are now available to the plant science community and can be widely and easily applied to study the activity and role of the different catalytic subunits of the proteasome in different plant species. This article is protected by copyright. All rights reserved.

  16. Nucleotide sequence of the genetic loci encoding subunits of Bradyrhizobium japonicum uptake hydrogenase.

    PubMed Central

    Sayavedra-Soto, L A; Powell, G K; Evans, H J; Morris, R O

    1988-01-01

    An indispensable part of the hydrogen-recycling system in Bradyrhizobium japonicum is the uptake hydrogenase, which is composed of 34.5- and 65.9-kDa subunits. The gene encoding the large subunit is located on a 5.9-kilobase fragment of the H2-uptake-complementing cosmid pHU52 [Zuber, M., Harker, A.R., Sultana, M.A. & Evans, H.J. (1986) Proc. Natl. Acad. Sci. USA 83, 7668-7672]. We have now determined that the structural genes for both subunits are present on this fragment. Two open reading frames are present that correspond in size and deduced amino acid sequence to the hydrogenase subunits, except that the small-subunit coding region contains a leader peptide of 46 amino acids. The two genes are separated by a 32-nucleotide intergenic region and likely constitute an operon. Comparison of the deduced amino acid sequences of the B. japonicum genes with those from Desulfovibrio gigas, Desulfovibrio baculatus, and Rhodobacter capsulatus indicates significant sequence identity. Images PMID:3054886

  17. Localization of the regulatory particle subunit Sem1 in the 26S proteasome

    SciTech Connect

    Bohn, Stefan; Sakata, Eri; Beck, Florian; Pathare, Ganesh R.; Schnitger, Jérôme; Nágy, Istvan; Baumeister, Wolfgang Förster, Friedrich

    2013-05-31

    Highlights: •26S proteasome subunit Sem1 was mapped using cryo-EM and cross-linking data. •C-terminal helix of Sem1 located near winged helix motif of Rpn7. •N-terminal part of Sem1 tethers Rpn7, Rpn3 and lid helical bundle. •Sem1 binds differently to PCI-domains of proteasome subunit Rpn7 and TREX-2 subunit Thp1. -- Abstract: The ubiquitin–proteasome system is responsible for regulated protein degradation in the cell with the 26S proteasome acting as its executive arm. The molecular architecture of this 2.5 MDa complex has been established recently, with the notable exception of the small acidic subunit Sem1. Here, we localize the C-terminal helix of Sem1 binding to the PCI domain of the subunit Rpn7 using cryo-electron microscopy single particle reconstruction of proteasomes purified from yeast cells with sem1 deletion. The approximate position of the N-terminal region of Sem1 bridging the cleft between Rpn7 and Rpn3 was inferred based on site-specific cross-linking data of the 26S proteasome. Our structural studies indicate that Sem1 can assume different conformations in different contexts, which supports the idea that Sem1 functions as a molecular glue stabilizing the Rpn3/Rpn7 heterodimer.

  18. Fungal community analysis in the deep-sea sediments of the Pacific Ocean assessed by comparison of ITS, 18S and 28S ribosomal DNA regions

    NASA Astrophysics Data System (ADS)

    Xu, Wei; Luo, Zhu-Hua; Guo, Shuangshuang; Pang, Ka-Lai

    2016-03-01

    We investigated the diversity of fungal communities in 6 different deep-sea sediment samples of the Pacific Ocean based on three different types of clone libraries, including internal transcribed spacer (ITS), 18S rDNA, and 28S rDNA regions. A total of 1978 clones were generated from 18 environmental clone libraries, resulting in 140 fungal operational taxonomic units (OTUs), including 18 OTUs from ITS, 44 OTUs from 18S rDNA, and 78 OTUs from 28S rDNA gene primer sets. The majority of the recovered sequences belonged to diverse phylotypes of the Ascomycota and Basidiomycota. Additionally, our study revealed a total of 46 novel fungal phylotypes, which showed low similarities (<97%) with available fungal sequences in the GenBank, including a novel Zygomycete lineage, suggesting possible new fungal taxa occurring in the deep-sea sediments. The results suggested that 28S rDNA is an efficient target gene to describe fungal community in deep-sea environment.

  19. Molecular phylogenetic analysis of the coccidian cephalopod parasites Aggregata octopiana and Aggregata eberthi (Apicomplexa: Aggregatidae) from the NE Atlantic coast using 18S rRNA sequences.

    PubMed

    Castellanos-Martínez, Sheila; Pérez-Losada, Marcos; Gestal, Camino

    2013-08-01

    The coccidia genus Aggregata is responsible for intestinal coccidiosis in wild and cultivated cephalopods. Two coccidia species, Aggregata octopiana, (infecting the common octopus Octopus vulgaris), and A. eberthi, (infecting the cuttlefish Sepia officinalis), are identified in European waters. Extensive investigation of their morphology resulted in a redescription of A. octopiana in octopuses from the NE Atlantic Coast (NW Spain) thus clarifying confusing descriptions recorded in the past. The present study sequenced the 18S rRNA gene in A. octopiana and A. eberthi from the NE Atlantic coast in order to assess their taxonomic and phylogenetic status. Phylogenetic analyses revealed conspecific genetic differences (2.5%) in 18S rRNA sequences between A. eberthi from the Ria of Vigo (NW Spain) and the Adriatic Sea. Larger congeneric differences (15.9%) were observed between A. octopiana samples from the same two areas, which suggest the existence of two species. Based on previous morphological evidence, host specificity data, and new molecular phylogenetic analyses, we suggest that A. octopiana from the Ria of Vigo is the valid type species.

  20. Recent Advances in Subunit Vaccine Carriers

    PubMed Central

    Vartak, Abhishek; Sucheck, Steven J.

    2016-01-01

    The lower immunogenicity of synthetic subunit antigens, compared to live attenuated vaccines, is being addressed with improved vaccine carriers. Recent reports indicate that the physio-chemical properties of these carriers can be altered to achieve optimal antigen presentation, endosomal escape, particle bio-distribution, and cellular trafficking. The carriers can be modified with various antigens and ligands for dendritic cells targeting. They can also be modified with adjuvants, either covalently or entrapped in the matrix, to improve cellular and humoral immune responses against the antigen. As a result, these multi-functional carrier systems are being explored for use in active immunotherapy against cancer and infectious diseases. Advancing technology, improved analytical methods, and use of computational methodology have also contributed to the development of subunit vaccine carriers. This review details recent breakthroughs in the design of nano-particulate vaccine carriers, including liposomes, polymeric nanoparticles, and inorganic nanoparticles. PMID:27104575

  1. Cloning and characterization of subunit genes of ribonucleotide reductase, a cell-cycle-regulated enzyme, from Plasmodium falciparum.

    PubMed Central

    Chakrabarti, D; Schuster, S M; Chakrabarti, R

    1993-01-01

    Ribonucleotide reductase (EC 1.17.4.1; RNR), a cell-cycle-regulated enzyme, catalyzes the rate-limiting step in the de novo synthesis of deoxyribonucleotides by the reduction of the corresponding ribonucleotides. The important role of the RNR in DNA synthesis and cell division makes this enzyme an excellent target for chemotherapy. However, nothing is known about this enzyme from the malaria parasite Plasmodium falciparum. We have isolated cDNA clones encoding both the large and small RNR subunits. The sequences of full-length clones of the large and small RNR subunits revealed an open reading frame encoding 806 and 349 amino acids, respectively, and showed significant identity with other RNR sequences in the data base. RNA blot analysis showed that the size of the large and small RNR subunit transcripts are 5.4 kb and 2.2 kb, respectively. Both the RNR subunit transcripts fluctuate in level during the cell cycle, reaching a peak preceding maximal DNA synthesis activity. An oligodeoxynucleotide phosphorothioate that is complementary to sequences around the translational initiation codon of the small RNR subunit showed significant inhibition of growth, as measured by the inhibition in DNA synthesis. Images Fig. 2 PMID:8265664

  2. Subunit organization in cytoplasmic dynein subcomplexes

    PubMed Central

    King, Stephen J.; Bonilla, Myriam; Rodgers, Michael E.; Schroer, Trina A.

    2002-01-01

    Because cytoplasmic dynein plays numerous critical roles in eukaryotic cells, determining the subunit composition and the organization and functions of the subunits within dynein are important goals. This has been difficult partly because of accessory polypeptide heterogeneity of dynein populations. The motor domain containing heavy chains of cytoplasmic dynein are associated with multiple intermediate, light intermediate, and light chain accessory polypeptides. We examined the organization of these subunits within cytoplasmic dynein by separating the molecule into two distinct subcomplexes. These subcomplexes were competent to reassemble into a molecule with dynein-like properties. One subcomplex was composed of the dynein heavy and light intermediate chains whereas the other subcomplex was composed of the intermediate and light chains. The intermediate and light chain subcomplex could be further separated into two pools, only one of which contained dynein light chains. The two pools had distinct intermediate chain compositions, suggesting that intermediate chain isoforms have different light chain–binding properties. When the two intermediate chain pools were characterized by analytical velocity sedimentation, at least four molecular components were seen: intermediate chain monomers, intermediate chain dimers, intermediate chain monomers with bound light chains, and a mixture of intermediate chain dimers with assorted bound light chains. These data provide new insights into the compositional heterogeneity and assembly of the cytoplasmic dynein complex and suggest that individual dynein molecules have distinct molecular compositions in vivo. PMID:11967380

  3. 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

  4. DNA sequences, recombinant DNA molecules and processes for producing the A and B subunits of cholera toxin and preparations containing so-obtained subunit or subunits

    SciTech Connect

    Harford, N.; De Wilde, M.

    1987-05-19

    A recombinant DNA molecule is described comprising at least a portion coding for subunits A and B of cholera toxin, or a fragment or derivative of the portion wherein the fragment or derivative codes for a polypeptide have an activity which can induce an immune response to subunit A; can induce an immune response to subunit A and cause epithelial cell penetration and the enzymatic effect leading to net loss of fluid into the gut lumen; can bind to the membrane receptor for the B subunit of cholera toxin; can induce an immune response to subunit B; can induce an immune response to subunit B and bind to the membrane receptor; or has a combination of the activities.

  5. Carbohydrate binding specificities and crystal structure of the cholera toxin-like B-subunit from Citrobacter freundii.

    PubMed

    Jansson, Lena; Angström, Jonas; Lebens, Michael; Imberty, Anne; Varrot, Annabelle; Teneberg, Susann

    2010-05-01

    Enterotoxigenic Escherichia coli and Vibrio cholerae are well known causative agents of severe diarrheal diseases. Both pathogens produce AB(5) toxins, with one enzymatically active A-subunit and a pentamer of receptor-binding B-subunits. The primary receptor for both B-subunits is the GM1 ganglioside (Galbeta3GalNAcbeta4(NeuAcalpha3)Galbeta4GlcbetaCer), but the B-subunits from porcine isolates of E. coli also bind neolacto-(Galbeta4GlcNAcbeta-)terminated glycoconjugates and the B-subunits from human isolates of E. coli (hLTB) have affinity for blood group A type 2-(GalNAcalpha3(Fucalpha2)Galbeta4GlcNAcbeta-)terminated glycoconjugates. A B-subunit with 73% sequence identity to the B-subunits of cholera toxin and the heat-labile toxin of E. coli is produced by certain strains of enteropathogenic E. coli and by Citrobacter freundii. This C. freundii B-subunit (CFXB) has now been expressed in V. cholerae, and isolated in high yields. Glycosphingolipid binding studies show that CFXB binds to the GM1 ganglioside with high affinity. In addition, CFXB has high affinity for both neolacto-terminated and blood group A type 2-terminated glycoconjugates. The crystal structure of the pentameric arrangement of C. freundii B-subunits display high structural similarity with related proteins from E. coli and V. cholerae and oligosaccharide binding sites can be identified on the protein surface. Small changes in the 88-95 loop connecting the GM1 and blood group A binding sites explains the minor changes in affinity seen for these two ligands. However, the enhanced affinity of CFXB for neolacto-terminated structures can be sought in the Lys34Tyr substitution affording additional hydrogen bond interactions between the tyrosyl side chain and the GlcNAcbeta3Galb4Glcbeta1 segment of neolactotetraosylceramide via bridging water molecules.

  6. The β-conglycinin deficiency in wild soybean is associated with the tail-to-tail inverted repeat of the α-subunit genes.

    PubMed

    Tsubokura, Yasutaka; Hajika, Makita; Kanamori, Hiroyuki; Xia, Zhengjun; Watanabe, Satoshi; Kaga, Akito; Katayose, Yuichi; Ishimoto, Masao; Harada, Kyuya

    2012-02-01

    β-conglycinin, a major seed protein in soybean, is composed of α, α', and β subunits sharing a high homology among them. Despite its many health benefits, β-conglycinin has a lower amino acid score and lower functional gelling properties compared to glycinin, another major soybean seed protein. In addition, the α, α', and β subunits also contain major allergens. A wild soybean (Glycine soja Sieb et Zucc.) line, 'QT2', lacks all of the β-conglycinin subunits, and the deficiency is controlled by a single dominant gene, Scg-1 (Suppressor of β-conglycinin). This gene was characterized using a soybean cultivar 'Fukuyutaka', 'QY7-25', (its near-isogenic line carrying the Scg-1 gene), and the F₂ population derived from them. The physical map of the Scg-1 region covered by lambda phage genomic clones revealed that the two α-subunit genes, a β-subunit gene, and a pseudo α-subunit gene were closely organized. The two α-subunit genes were arranged in a tail-to-tail orientation, and the genes were separated by 197 bp in Scg-1 compared to 3.3 kb in the normal allele (scg-1). In addition, small RNA was detected in immature seeds of the mutants by northern blot analysis using an RNA probe of the α subunit. These results strongly suggest that β-conglycinin deficiency in QT2 is controlled by post-transcriptional gene silencing through the inverted repeat of the α subunits.

  7. 18S rDNA analysis of alkenone-producing haptophyte(s) preserved in surface sediments of Lake Toyoni, Japan

    NASA Astrophysics Data System (ADS)

    McColl, J. L.; Couto, J.; Bendle, J. A.; Henderson, A. C.; Seki, O.; Phoenix, V. R.; Toney, J. L.

    2013-12-01

    Alkenones (long chain ketones) are readily preserved in sedimentary archives and have the potential to provide quantitative reconstructions of past water temperature. Alkenones are produced by a limited number of haptophyte algae in the marine and also some lacustrine systems. However, lakes are heterogeneous: an individual lake will have a unique combination of ecological conditions, haptophyte species and seasonal alkenone production that contributes to the sedimentary record. Haptophyte algae species have different sensitivities to temperature; therefore identifying the alkenone producer(s) prior to down-core temperature reconstructions is critical before selecting the most appropriate temperature calibration. We present a study from Lake Toyoni, a freshwater lake in Hokkaido, Japan that has alkenones preserved in surface sediments. The aim of this study is to identify the alkenone producer(s) within the lake using 18S rDNA analyses. Preserved rDNA of planktonic phototrophic algae was extracted from surface sediments of Lake Toyoni and phylogenetic analyses of the rDNA sequences suggest alkenones are produced by a single haptophyte within the class Prymnesiophyceae (order Isochrysidales). The Lake Toyoni alkenone-producer shares a distinct phylotype with a haptophyte reported from water filter samples collected in Lake BrayaSø, Greenland (D'Andrea et al., 2006). Similarity between the 18S rDNA sequences from Lake Toyoni and Lake BrayaSø provides a basis for applying (and updating) the Greenland lake temperature calibration. Applying this temperature calibration (T°C = 40.8 [UK37] + 31.8, R2=0.96; n=34) to the surface sediment alkenone unsaturation index from Lake Toyoni gives an estimated lake surface temperature (LST) of 8°C. This is in line with observed LST at Lake Toyoni, which ranges between 7 - 22°C (Apr 2011 to Nov 2011). The occurrence and identification of a single alkenone producer in Lake Toyoni means problems posed by a mixture of haptophytes in

  8. Composition of the summer photosynthetic pico and nanoplankton communities in the Beaufort Sea assessed by T-RFLP and sequences of the 18S rRNA gene from flow cytometry sorted samples

    PubMed Central

    Balzano, Sergio; Marie, Dominique; Gourvil, Priscillia; Vaulot, Daniel

    2012-01-01

    The composition of photosynthetic pico and nanoeukaryotes was investigated in the North East Pacific and the Arctic Ocean with special emphasis on the Beaufort Sea during the MALINA cruise in summer 2009. Photosynthetic populations were sorted using flow cytometry based on their size and pigment fluorescence. Diversity of the sorted photosynthetic eukaryotes was determined using terminal-restriction fragment length polymorphism analysis and cloning/sequencing of the 18S ribosomal RNA gene. Picoplankton was dominated by Mamiellophyceae, a class of small green algae previously included in the prasinophytes: in the North East Pacific, the contribution of an Arctic Micromonas ecotype increased steadily northward becoming the only taxon occurring at most stations throughout the Beaufort Sea. In contrast, nanoplankton was more diverse: North Pacific stations were dominated by Pseudo-nitzschia sp. whereas those in the Beaufort Sea were dominated by two distinct Chaetoceros species as well as by Chrysophyceae, Pelagophyceae and Chrysochromulina spp.. This study confirms the importance of Arctic Micromonas within picoplankton throughout the Beaufort Sea and demonstrates that the photosynthetic picoeukaryote community in the Arctic is much less diverse than at lower latitudes. Moreover, in contrast to what occurs in warmer waters, most of the key pico- and nanoplankton species found in the Beaufort Sea could be successfully established in culture. PMID:22278671

  9. Prefoldin Subunits Are Protected from Ubiquitin-Proteasome System-mediated Degradation by Forming Complex with Other Constituent Subunits*

    PubMed Central

    Miyazawa, Makoto; Tashiro, Erika; Kitaura, Hirotake; Maita, Hiroshi; Suto, Hiroo; Iguchi-Ariga, Sanae M. M.; Ariga, Hiroyoshi

    2011-01-01

    The molecular chaperone prefoldin (PFD) is a complex comprised of six different subunits, PFD1-PFD6, and delivers newly synthesized unfolded proteins to cytosolic chaperonin TRiC/CCT to facilitate the folding of proteins. PFD subunits also have functions different from the function of the PFD complex. We previously identified MM-1α/PFD5 as a novel c-Myc-binding protein and found that MM-1α suppresses transformation activity of c-Myc. However, it remains unclear how cells regulate protein levels of individual subunits and what mechanisms alter the ratio of their activities between subunits and their complex. In this study, we found that knockdown of one subunit decreased protein levels of other subunits and that transfection of five subunits other than MM-1α into cells increased the level of endogenous MM-1α. We also found that treatment of cells with MG132, a proteasome inhibitor, increased the level of transfected/overexpressed MM-1α but not that of endogenous MM-1α, indicating that overexpressed MM-1α, but not endogenous MM-1α, was degraded by the ubiquitin proteasome system (UPS). Experiments using other PFD subunits showed that the UPS degraded a monomer of PFD subunits, though extents of degradation varied among subunits. Furthermore, the level of one subunit was increased after co-transfection with the respective subunit, indicating that there are specific combinations between subunits to be stabilized. These results suggest mutual regulation of protein levels among PFD subunits and show how individual subunits form the PFD complex without degradation. PMID:21478150

  10. Prefoldin subunits are protected from ubiquitin-proteasome system-mediated degradation by forming complex with other constituent subunits.

    PubMed

    Miyazawa, Makoto; Tashiro, Erika; Kitaura, Hirotake; Maita, Hiroshi; Suto, Hiroo; Iguchi-Ariga, Sanae M M; Ariga, Hiroyoshi

    2011-06-03

    The molecular chaperone prefoldin (PFD) is a complex comprised of six different subunits, PFD1-PFD6, and delivers newly synthesized unfolded proteins to cytosolic chaperonin TRiC/CCT to facilitate the folding of proteins. PFD subunits also have functions different from the function of the PFD complex. We previously identified MM-1α/PFD5 as a novel c-Myc-binding protein and found that MM-1α suppresses transformation activity of c-Myc. However, it remains unclear how cells regulate protein levels of individual subunits and what mechanisms alter the ratio of their activities between subunits and their complex. In this study, we found that knockdown of one subunit decreased protein levels of other subunits and that transfection of five subunits other than MM-1α into cells increased the level of endogenous MM-1α. We also found that treatment of cells with MG132, a proteasome inhibitor, increased the level of transfected/overexpressed MM-1α but not that of endogenous MM-1α, indicating that overexpressed MM-1α, but not endogenous MM-1α, was degraded by the ubiquitin proteasome system (UPS). Experiments using other PFD subunits showed that the UPS degraded a monomer of PFD subunits, though extents of degradation varied among subunits. Furthermore, the level of one subunit was increased after co-transfection with the respective subunit, indicating that there are specific combinations between subunits to be stabilized. These results suggest mutual regulation of protein levels among PFD subunits and show how individual subunits form the PFD complex without degradation.

  11. Phylogeny of freshwater parasitic copepods in the Ergasilidae (Copepoda: Poecilostomatoida) based on 18S and 28S rDNA sequences.

    PubMed

    Song, Y; Wang, G T; Yao, W J; Gao, Q; Nie, P

    2008-01-01

    The phylogenetic relationships among the Ergasilidae genera are poorly understood. In this study, 14 species from four genera in the Ergasilidae including Sinergasilus, Ergasilus, Pseudergasilus, and Paraergasilus were collected in China, and their phylogenetic relationships were examined using neighbor-joining, maximum parsimony, maximum likelihood, and Bayesian inference methods based on partial sequences of 18S and 28S ribosomal deoxyribonucleic acid, respectively. All the analyses suggest that the Sinergasilus and Paraergasilus are both monophyletic, but the Ergasilus is polyphyletic rather than monophyletic. Considering the relationships among the four genera, the phylogenetic analyses and subsequent hypothesis tests all suggest that Pseudergasilus clustered with some Ergasilus species may have a closer relationship with Sinergasilus rather than with Paraergasilus. It is proposed that the Sinergasilus and the Pseudergasilus species might have evolved from Ergasilus species.

  12. Slow molecular evolution in 18S rDNA, rbcL and nad5 genes of mosses compared with higher plants.

    PubMed

    Stenøien, H K

    2008-03-01

    The evolutionary potential of bryophytes (mosses, liverworts and hornworts) has been debated for decades. Fossil record and biogeographical distribution patterns suggest very slow morphological evolution and the retainment of several ancient traits since the split with vascular plants some 450 million years ago. Many have argued that bryophytes may evolve as rapidly as higher plants on the molecular level, but this hypothesis has not been tested so far. Here, it is shown that mosses have experienced significantly lower rates of molecular evolution than higher plants within 18S rDNA (nuclear), rbcL (chloroplast) and nad5 (mitochondrial) genes. Mosses are on an average evolving 2-3 times slower than ferns, gymnosperms and angiosperms; and also green algae seem to be evolving faster than nonvascular plants. These results support the observation of a general correlation between morphological and molecular evolutionary rates in plants and also show that mosses are 'evolutionary sphinxes' regarding both morphological and molecular evolutionary potential.

  13. Molecular characterization and phylogeny of Linguatula serrata (Pentastomida: Linguatulidae) based on the nuclear 18S rDNA and mitochondrial cytochrome c oxidase I gene

    PubMed Central

    MOHANTA, Uday Kumar; ITAGAKI, Tadashi

    2016-01-01

    Linguatula serrata, a cosmopolitan parasite, is commonly known as tongue worm belonging to the subclass Pentastomida.We collected the nymphal stage of the worm from mesenteric lymph nodes of cattle and identified these as L. serrata based on morphology and morphometry. The 18S rDNA sequences showed no intraspecific variation, although cox1 sequences showed 99.7–99.9% homology. In the phylogenies inferred from both gene loci, members of the genus Linguatula (order Porocephalida) were closer to those of the order Cephalobaenida than to those of Porocephalida, reflecting a mismatch with the corresponding morphology-based taxonomy. Accordingly, analyses of additional gene loci using a larger number of taxa across the Pentastomida should be undertaken to determine an accurate phylogenetic position within the Arthropoda. PMID:27941305

  14. Internal phylogeny of the Chilopoda (Myriapoda, Arthropoda) using complete 18S rDNA and partial 28S rDNA sequences.

    PubMed Central

    Giribet, G; Carranza, S; Riutort, M; Baguñà, J; Ribera, C

    1999-01-01

    The internal phylogeny of the 'myriapod' class Chilopoda is evaluated for 12 species belonging to the five extant centipede orders, using 18S rDNA complete gene sequence and 28S rDNA partial gene sequence data. Equally and differentially weighted parsimony, neighbour-joining and maximum-likelihood were used for phylogenetic reconstruction, and bootstrapping and branch support analyses were performed to evaluate tree topology stability. The results show that the Chilopoda constitute a monophyletic group that is divided into two lines, Notostigmophora (= Scutigeromorpha) and Pleurostigmophora, as found in previous morphological analyses. The Notostigmophora are markedly modified for their epigenic mode of life. The first offshoot of the Pleurostigmophora are the Lithobiomorpha, followed by the Craterostigmomorpha and by the Epimorpha s. str. (= Scolopendromorpha + Geophilomorpha), although strong support for the monophyly of the Epimorpha s. lat. (= Craterostigmomorpha + Epimorpha s. str.) is only found in the differentially weighted parsimony analysis. PMID:10087567

  15. Protist 18S rRNA gene Sequence Analysis Reveals Multiple Sources of Organic Matter Contributing to Turbidity Maxima of the Columbia River Estuary

    SciTech Connect

    Herfort, Lydie; Peterson, Tawnya D.; McCue, Lee Ann; Zuber, Peter A.

    2011-10-05

    The Columbia River estuary is traditionally considered a detritus-based ecosystem fueled in summer by organic matter (OM) from expired freshwater diatoms. Since Estuarine Turbidity Maxima (ETM) are sites of accumulation and transformation of this phytoplankton-derived OM, to further characterize the ETM protist assemblage, we collected in August 2007 bottom waters throughout an ETM event, as well as surface water during the peak of bottom turbidity, and performed biogeochemical, microscopic and molecular (18S rRNA gene clone libraries) analyses. These data confirmed that the majority of the particulate OM in ETMs is derived from chlorophyll a-poor particulate organic carbon tagged by DNA too damaged to be detected by molecular analysis.

  16. Triploblastic relationships with emphasis on the acoelomates and the position of Gnathostomulida, Cycliophora, Plathelminthes, and Chaetognatha: a combined approach of 18S rDNA sequences and morphology.

    PubMed

    Giribet, G; Distel, D L; Polz, M; Sterrer, W; Wheeler, W C

    2000-09-01

    Triploblastic relationships were examined in the light of molecular and morphological evidence. Representatives for all triploblastic "phyla" (except Loricifera) were represented by both sources of phylogenetic data. The 18S ribosomal (rDNA) sequence data for 145 terminal taxa and 276 morphological characters coded for 36 supraspecific taxa were combined in a total evidence regime to determine the most consistent picture of triploblastic relationships for these data. Only triploblastic taxa are used to avoid rooting with distant outgroups, which seems to happen because of the extreme distance that separates diploblastic from triploblastic taxa according to the 18S rDNA data. Multiple phylogenetic analyses performed with variable analysis parameters yield largely inconsistent results for certain groups such as Chaetognatha, Acoela, and Nemertodermatida. A normalized incongruence length metric is used to assay the relative merit of the multiple analyses. The combined analysis having the least character incongruence yields the following scheme of relationships of four main clades: (1) Deuterostomia [((Echinodermata + Enteropneusta) (Cephalochordata (Urochordata + Vertebrata)))]; (2) Ecdysozoa [(((Priapulida + Kinorhyncha) (Nematoda + Nematomorpha)) ((Onychophora + Tardigrada) Arthropoda))]; (3) Trochozoa [((Phoronida + Brachiopoda) (Entoprocta (Nemertea (Sipuncula (Mollusca (Pogonophora (Echiura + Annelida)))))))]; and (4) Platyzoa [((Gnathostomulida (Cycliophora + Syndermata)) (Gastrotricha + Plathelminthes))]. Chaetognatha, Nemertodermatida, and Bryozoa cannot be assigned to any one of these four groups. For the first time, a data analysis recognizes a clade of acoelomates, the Platyzoa (sensu Cavalier-Smith, Biol. Rev. 73:203-266, 1998). Other relationships that corroborate some morphological analyses are the existence of a clade that groups Gnathostomulida + Syndermata (= Gnathifera), which is expanded to include the enigmatic phylum Cycliophora, as sister group

  17. Inherent conformational flexibility of F1-ATPase α-subunit.

    PubMed

    Hahn-Herrera, Otto; Salcedo, Guillermo; Barril, Xavier; García-Hernández, Enrique

    2016-09-01

    The core of F1-ATPase consists of three catalytic (β) and three noncatalytic (α) subunits, forming a hexameric ring in alternating positions. A wealth of experimental and theoretical data has provided a detailed picture of the complex role played by catalytic subunits. Although major conformational changes have only been seen in β-subunits, it is clear that α-subunits have to respond to these changes in order to be able to transmit information during the rotary mechanism. However, the conformational behavior of α-subunits has not been explored in detail. Here, we have combined unbiased molecular dynamics (MD) simulations and calorimetrically measured thermodynamic signatures to investigate the conformational flexibility of isolated α-subunits, as a step toward deepening our understanding of its function inside the α3β3 ring. The simulations indicate that the open-to-closed conformational transition of the α-subunit is essentially barrierless, which is ideal to accompany and transmit the movement of the catalytic subunits. Calorimetric measurements of the recombinant α-subunit from Geobacillus kaustophilus indicate that the isolated subunit undergoes no significant conformational changes upon nucleotide binding. Simulations confirm that the nucleotide-free and nucleotide-bound subunits show average conformations similar to that observed in the F1 crystal structure, but they reveal an increased conformational flexibility of the isolated α-subunit upon MgATP binding, which might explain the evolutionary conserved capacity of α-subunits to recognize nucleotides with considerable strength. Furthermore, we elucidate the different dependencies that α- and β-subunits show on Mg(II) for recognizing ATP.

  18. Dimeric human sulfotransferase 1B1 displays cofactor-dependent subunit communication

    PubMed Central

    Tibbs, Zachary E; Falany, Charles N

    2015-01-01

    The cytosolic sulfotransferases (SULTs) are dimeric enzymes that catalyze the transformation of hydrophobic drugs and hormones into hydrophilic sulfate esters thereby providing the body with an important pathway for regulating small molecule activity and excretion. While SULT dimerization is highly conserved, the necessity for the interaction has not been established. To perform its function, a SULT must efficiently bind the universal sulfate donor, 3′-phosphoadenosine-5′-phosphosulfate (PAPS), and release the byproduct, 3′, 5′-diphosphoadenosine (PAP), following catalysis. We hypothesize this efficient binding and release of PAPS/PAP may be connected to SULT dimerization. To allow for the visualization of dynamic protein interactions critical for addressing this hypothesis and to generate kinetically testable hypotheses, molecular dynamic simulations (MDS) of hSULT1B1 were performed with PAPS and PAP bound to each dimer subunit in various combinations. The results suggest the dimer subunits may possess the capability of communicating with one another in a manner dependent on the presence of the cofactor. PAP or PAPS binding to a single side of the dimer results in decreased backbone flexibility of both the bound and unbound subunits, implying the dimer subunits may not act independently. Further, binding of PAP to one subunit of the dimer and PAPS to the other caused increased flexibility in the subunit bound to the inactive cofactor (PAP). These results suggest SULT dimerization may be important in maintaining cofactor binding/release properties of SULTs and provide hypothetical explanations for SULT half-site reactivity and substrate inhibition, which can be analyzed in vitro. PMID:26236487

  19. Interaction between Subunits of Heterodimeric Splicing Factor U2AF Is Essential In Vivo

    PubMed Central

    Rudner, David Z.; Kanaar, Roland; Breger, Kevin S.; Rio, Donald C.

    1998-01-01

    The heterodimeric pre-mRNA splicing factor, U2AF (U2 snRNP auxiliary factor), plays a critical role in 3′ splice site selection. Although the U2AF subunits associate in a tight complex, biochemical experiments designed to address the requirement for both subunits in splicing have yielded conflicting results. We have taken a genetic approach to assess the requirement for the Drosophila U2AF heterodimer in vivo. We developed a novel Escherichia coli copurification assay to map the domain on the Drosophila U2AF large subunit (dU2AF50) that interacts with the Drosophila small subunit (dU2AF38). A 28-amino-acid fragment on dU2AF50 that is both necessary and sufficient for interaction with dU2AF38 was identified. Using the copurification assay, we scanned this 28-amino-acid interaction domain for mutations that abrogate heterodimer formation. A collection of these dU2AF50 point mutants was then tested in vivo for genetic complementation of a recessive lethal dU2AF50 allele. A mutation that completely abolished interaction with dU2AF38 was incapable of complementation, whereas dU2AF50 mutations that did not effect heterodimer formation rescued the recessive lethal dU2AF50 allele. Analysis of heterodimer formation in embryo extracts derived from these interaction mutant lines revealed a perfect correlation between the efficiency of subunit association and the ability to complement the dU2AF50 recessive lethal allele. These data indicate that Drosophila U2AF heterodimer formation is essential for viability in vivo, consistent with a requirement for both subunits in splicing in vitro. PMID:9528748

  20. Chloroplast ATP synthase contains one single copy of subunit delta that is indispensable for photophosphorylation.

    PubMed

    Engelbrecht, S; Schürmann, K; Junge, W

    1989-01-15

    F0F1 ATP synthases synthesize ATP in their F1 portion at the expense of free energy supplied by proton flow which enters the enzyme through their channel portion F0. The smaller subunits of F1, especially subunit delta, may act as energy transducers between these rather distant functional units. We have previously shown that chloroplast delta, when added to thylakoids partially depleted of the coupling factor CF1, can reconstitute photophosphorylation by inhibiting proton leakage through exposed coupling factor CF0. In view of controversies in the literature, we reinvestigated two further aspects related to subunit delta, namely (a) its stoichiometry in CF0CF1 and (b) whether or not delta is required for photophosphorylation. By rocket immunoelectrophoresis of thylakoid membranes and calibration against purified delta, we confirmed a stoichiometry of one delta per CF0CF1. In CF1-depleted thylakoids photophosphorylation could be reconstituted not only by adding CF1 and subunit delta but, surprisingly, also by CF1 (-delta). We found that the latter was attributable to a contamination of CF1 (-delta) preparations with integral CF1. To lesser extent CF1 (-delta) acted by complementary rebinding to CF0 channels that were closed because they contained delta [CF0(+delta)]. This added catalytic capacity to proton-tight thylakoid vesicles. The ability of subunit delta to control proton flow through CF0 and the absolute requirement for delta in restoration of photophosphorylation suggest an essential role of this small subunit at the interface between the large portions of ATP synthase: delta may be part of the coupling site between electrochemical, conformational and chemical events in this enzyme.

  1. Evolutionary and structural analyses of heterodimeric proteins composed of subunits with same fold.

    PubMed

    Sudha, Govindarajan; Naveenkumar, Nagarajan; Srinivasan, Narayanaswamy

    2015-10-01

    Heterodimeric proteins with homologous subunits of same fold are involved in various biological processes. The objective of this study is to understand the evolution of structural and functional features of such heterodimers. Using a non-redundant dataset of 70 such heterodimers of known 3D structure and an independent dataset of 173 heterodimers from yeast, we note that the mean sequence identity between interacting homologous subunits is only 23-24% suggesting that, generally, highly diverged paralogues assemble to form such a heterodimer. We also note that the functional roles of interacting subunits/domains are generally quite different. This suggests that, though the interacting subunits/domains are homologous, the high evolutionary divergence characterize their high functional divergence which contributes to a gross function for the heterodimer considered as a whole. The inverse relationship between sequence identity and RMSD of interacting homologues in heterodimers is not followed. We also addressed the question of formation of homodimers of the subunits of heterodimers by generating models of fictitious homodimers on the basis of the 3D structures of the heterodimers. Interaction energies associated with these homodimers suggests that, in overwhelming majority of the cases, such homodimers are unlikely to be stable. Majority of the homologues of heterodimers of known structures form heterodimers (51.8%) and a small proportion (14.6%) form homodimers. Comparison of 3D structures of heterodimers with homologous homodimers suggests that interfacial nature of residues is not well conserved. In over 90% of the cases we note that the interacting subunits of heterodimers are co-localized in the cell.

  2. [Nose surgical anatomy in six aesthetic subunits].

    PubMed

    Chaput, B; Lauwers, F; Lopez, R; Saboye, J; André, A; Grolleau, J-L; Chavoin, J-P

    2013-04-01

    The nose is a complex entity, combining aesthetic and functional roles. Descriptive anatomy is a fundamental science that it can be difficult to relate directly to our daily surgical activity. Reasoning in terms of aesthetic subunits to decide on his actions appeared to us so obvious. The aim of this paper is to resume the anatomical bases relevant to our daily practice in order to fully apprehend the restorative or cosmetic procedures. We discuss the limits of the systematization of these principles in nasal oncology.

  3. Reporter mutation studies show that nicotinic acetylcholine receptor (nAChR) α5 Subunits and/or variants modulate function of α6*-nAChR.

    PubMed

    Dash, Bhagirathi; Chang, Yongchang; Lukas, Ronald J

    2011-11-04

    To further the understanding of functional α6α5*-nicotinic acetylcholine receptors (nAChR; the asterisk (*) indicates known or possible presence of other subunits), we have heterologously expressed in oocytes different, mouse or human, nAChR subunit combinations. Coexpression with wild-type α5 subunits or chimeric α5/β3 subunits (in which the human α5 subunit N-terminal, extracellular domain is linked to the remaining domains of the human β3 subunit) almost completely abolishes the very small amount of function seen for α6β4*-nAChR and does not induce function of α6β2*-nAChR. Coexpression with human α5(V9)'(S) subunits bearing a valine 290 to serine mutation in the 9' position of the second transmembrane domain does not rescue the function of α6β4*-nAChR or induce function of α6β2*-nAChR. However, coexpression with mutant chimeric α5/β3(V9)'(S) subunits has a gain-of-function effect (higher functional expression and agonist sensitivity and spontaneous opening inhibited by mecamylamine) on α6β4*-nAChR. Moreover, N143D + M145V mutations in the α6 subunit N-terminal domain enable α5/β3(V9)'(S) subunits to have a gain-of-function effect on α6β2*-nAChR. nAChR containing chimeric α6/α3 subunits plus either β2 or β4 subunits have some function that is modulated in the presence of α5 or α5/β3 subunits. Coexpression with α5/β3(V9)'(S) subunits has a gain-of-function effect more pronounced than that in the presence of α5(V9)'(S) subunits. Gain-of-function effects are dependent, sometimes subtly, on the nature and apparently the extracellular, cytoplasmic, and/or transmembrane domain topology of partner subunits. These studies yield insight into assembly of functional α6α5*-nAChR and provide tools for development of α6*-nAChR-selective ligands that could be important in the treatment of nicotine dependence, and perhaps other neurological diseases.

  4. Nuclear omnipotent suppressors of premature termination codons in mitochondrial genes affect the 37S mitoribosomal subunit.

    PubMed

    Boguta, M; Mieszczak, M; Zagórski, W

    1988-02-01

    nam3 and R705, yeast nuclear omnipotent suppressors of mitochondrial mit- mutations, reverse the superimposed spectrum of trans-recessive splicing defects by affecting the protein composition of the small mitoribosomal subunit. Analysis of the suppressor's interaction suggests that suppression results from mutations in the mitoribosomal polypeptides. These data indicate an obligatory connection between mitoribosome function and splicing of introns bI2, bI4 and aI1 in yeast mitochondria.

  5. Analysis of conformational changes in 16 S rRNA during the course of 30 S subunit assembly.

    PubMed

    Holmes, Kristi L; Culver, Gloria M

    2005-11-25

    Ribosome biogenesis involves an integrated series of binding events coupled with conformational changes that ultimately result in the formation of a functional macromolecular complex. In vitro, Escherichia coli 30 S subunit assembly occurs in a cooperative manner with the ordered addition of 20 ribosomal proteins (r-proteins) with 16 S rRNA. The assembly pathway for 30 S subunits has been dissected in vitro into three steps, where specific r-proteins associate with 16 S rRNA early in 30 S subunit assembly, followed by a mid-assembly conformational rearrangement of the complex that then enables the remaining r-proteins to associate in the final step. Although the three steps of 30 S subunit assembly have been known for some time, few details have been elucidated about changes that occur as a result of these three specific stages. Here, we present a detailed analysis of the concerted early and late stages of small ribosomal subunit assembly. Conformational changes, roles for base-pairing and r-proteins at specific stages of assembly, and a polar nature to the assembly process have been revealed. This work has allowed a more comprehensive and global view of E.coli 30 S ribosomal subunit assembly to be obtained.

  6. The ω Subunit Governs RNA Polymerase Stability and Transcriptional Specificity in Staphylococcus aureus.

    PubMed

    Weiss, Andy; Moore, Brittney D; Tremblay, Miguel H J; Chaput, Dale; Kremer, Astrid; Shaw, Lindsey N

    2017-01-15

    Staphylococcus aureus is a major human pathogen that causes infection in a wide variety of sites within the human body. Its ability to adapt to the human host and to produce a successful infection requires precise orchestration of gene expression. While DNA-dependent RNA polymerase (RNAP) is generally well characterized, the roles of several small accessory subunits within the complex have yet to be fully explored. This is particularly true for the omega (ω or RpoZ) subunit, which has been extensively studied in Gram-negative bacteria but largely neglected in Gram-positive counterparts. In Escherichia coli, it has been shown that ppGpp binding, and thus control of the stringent response, is facilitated by ω. Interestingly, key residues that facilitate ppGpp binding by ω are not conserved in S. aureus, and consequently, survival under starvation conditions is unaffected by rpoZ deletion. Further to this, ω-lacking strains of S. aureus display structural changes in the RNAP complex, which result from increased degradation and misfolding of the β' subunit, alterations in δ and σ factor abundance, and a general dissociation of RNAP in the absence of ω. Through RNA sequencing analysis we detected a variety of transcriptional changes in the rpoZ-deficient strain, presumably as a response to the negative effects of ω depletion on the transcription machinery. These transcriptional changes translated to an impaired ability of the rpoZ mutant to resist stress and to fully form a biofilm. Collectively, our data underline, for the first time, the importance of ω for RNAP stability, function, and cellular physiology in S. aureus IMPORTANCE: In order for bacteria to adjust to changing environments, such as within the host, the transcriptional process must be tightly controlled. Transcription is carried out by DNA-dependent RNA polymerase (RNAP). In addition to its major subunits (α2ββ') a fifth, smaller subunit, ω, is present in all forms of life. Although this

  7. Unequal synthesis and differential degradation of propionyl CoA carboxylase subunits in cells from normal and propionic acidemia patients.

    PubMed Central

    Ohura, T; Kraus, J P; Rosenberg, L E

    1989-01-01

    We have characterized further the molecular basis of human inherited propionyl CoA carboxylase deficiency by measuring steady state levels of the mRNAs coding for the enzyme's two protein subunits (alpha and beta) and by estimating initial synthesis and steady state levels of the protein subunits in skin fibroblasts from controls and affected patients. We studied cell lines from both major complementation groups (pccA and pccBC) corresponding, respectively, to defects in the carboxylase's alpha and beta subunits. Analysis of pccA lines revealed the absence of alpha chain mRNA in three and an abnormally small alpha-mRNA in a fourth. Despite the presence of normal beta-mRNA in each of these pccA lines, there was complete absence of both alpha and beta protein subunits under steady state conditions, even though new synthesis and mitochondrial import of beta precursors was normal. Results in nine pccBC lines revealed normal alpha mRNA in each, while the amounts of beta-mRNA were distinctly reduced in every case. Correspondingly, alpha protein subunits were present in normal amounts at steady-state, but beta subunits were uniformly decreased. In addition, in six of the nine beta deficient cell lines, partially degraded beta-subunits were observed. To help interpret these results, synthesis and stability of carboxylase subunits were studied in intact HeLa cells using a pulse-chase protocol. Whereas alpha chains were stable over the four hour interval studied, beta chains--initially synthesized in large excess over alpha chains--were degraded rapidly reaching equivalence with alpha chains after two hours.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:2741949

  8. Multi-site Phosphorylation of Voltage-Gated Sodium Channel α Subunits from Rat Brain

    PubMed Central

    Berendt, Frank J.; Park, Kang-Sik; Trimmer, James S.

    2010-01-01

    Reversible phosphorylation of ion channels underlies cellular plasticity in mammalian neurons. Voltage-gated sodium or Nav channels underlie action potential initiation and propagation, dendritic excitability, and many other aspects of neuronal excitability. Various protein kinases have been suggested to phosphorylate the primary α subunit of Nav channels, affecting diverse aspects of channel function. Previous studies of Nav α subunit phosphorylation have led to the identification of a small set of phosphorylation sites important in meditating aspects of Nav channel function. Here we use nanoflow liquid chromatography tandem mass spectrometry (nano-LC MS/MS) on Nav α subunits affinity-purified from rat brain with two distinct monoclonal antibodies to identify 15 phosphorylation sites on Nav1.2, 12 of which have not been previously reported. We also found 3 novel phosphorylation sites on Nav1.1. In general, commonly used phosphorylation site prediction algorithms did not accurately predict these novel in vivo phosphorylation sites. Our results demonstrate that specific Nav α subunits isolated from rat brain are highly phosphorylated, and suggest extensive modulation of Nav channel activity in mammalian brain. Identification of phosphorylation sites using monoclonal antibody-based immunopurification and mass spectrometry is an effective approach to define the phosphorylation status of Nav channels and important membrane proteins in mammalian brain. PMID:20131913

  9. Proteolysis of the proofreading subunit controls the assembly of Escherichia coli DNA polymerase III catalytic core.

    PubMed

    Bressanin, Daniela; Stefan, Alessandra; Piaz, Fabrizio Dal; Cianchetta, Stefano; Reggiani, Luca; Hochkoeppler, Alejandro

    2009-11-01

    The C-terminal region of the proofreading subunit (epsilon) of Escherichia coli DNA polymerase III is shown here to be labile and to contain the residues (identified between F187 and R213) responsible for association with the polymerase subunit (alpha). We also identify two alpha-helices of the polymerase subunit (comprising the residues E311-M335 and G339-D353, respectively) as the determinants of binding to epsilon. The C-terminal region of epsilon is degraded by the ClpP protease assisted by the GroL molecular chaperone, while other factors control the overall concentration in vivo of epsilon. Among these factors, the chaperone DnaK is of primary importance for preserving the integrity of epsilon. Remarkably, inactivation of DnaK confers to Escherichia coli inviable phenotype at 42 degrees C, and viability can be restored over-expressing epsilon. Altogether, our observations indicate that the association between epsilon and alpha subunits of DNA polymerase III depends on small portions of both proteins, the association of which is controlled by proteolysis of epsilon. Accordingly, the factors catalysing (ClpP, GroL) or preventing (DnaK) this proteolysis exert a crucial checkpoint of the assembly of Escherichia coli DNA polymerase III core.

  10. Understanding Molecular Recognition by G protein βγ Subunits on the Path to Pharmacological Targeting

    PubMed Central

    Lin, Yuan

    2011-01-01

    Heterotrimeric G proteins, composed of Gα and Gβγ subunits, transduce extracellular signals via G-protein-coupled receptors to modulate many important intracellular responses. The Gβγ subunits hold a central position in this signaling system and have been implicated in multiple aspects of physiology and the pathophysiology of disease. The Gβ subunit belongs to a large family of WD40 repeat proteins with a circular β-bladed propeller structure. This structure allows Gβγ to interact with a broad range of proteins to play diverse roles. How Gβγ interacts with and regulates such a wide variety of partners yet maintains specificity is an interesting problem in protein-protein molecular recognition in signal transduction, where signal transfer by proteins is often driven by modular conserved recognition motifs. Evidence has accumulated that one mechanism for Gβγ multitarget recognition is through an intrinsically flexible protein surface or “hot spot” that accommodates multiple modes of binding. Because each target has a unique recognition mode for Gβγ subunits, it suggests that these interactions could be selectively manipulated with small molecules, which could have significant therapeutic potential. PMID:21737569

  11. Chemically related 4,5-linked aminoglycoside antibiotics drive subunit rotation in opposite directions

    PubMed Central

    Wasserman, Michael R.; Pulk, Arto; Zhou, Zhou; Altman, Roger B.; Zinder, John C.; Green, Keith D.; Garneau-Tsodikova, Sylvie; Doudna Cate, Jamie H.; Blanchard, Scott C.

    2015-01-01

    Dynamic remodelling of intersubunit bridge B2, a conserved RNA domain of the bacterial ribosome connecting helices 44 (h44) and 69 (H69) of the small and large subunit, respectively, impacts translation by controlling intersubunit rotation. Here we show that aminoglycosides chemically related to neomycin—paromomycin, ribostamycin and neamine—each bind to sites within h44 and H69 to perturb bridge B2 and affect subunit rotation. Neomycin and paromomycin, which only differ by their ring-I 6′-polar group, drive subunit rotation in opposite directions. This suggests that their distinct actions hinge on the 6′-substituent and the drug's net positive charge. By solving the crystal structure of the paromomycin–ribosome complex, we observe specific contacts between the apical tip of H69 and the 6′-hydroxyl on paromomycin from within the drug's canonical h44-binding site. These results indicate that aminoglycoside actions must be framed in the context of bridge B2 and their regulation of subunit rotation. PMID:26224058

  12. Complementation of subunits from different bacterial luciferases. Evidence for the role of the. beta. subunit in the bioluminescent mechanism

    SciTech Connect

    Meighen, E.A.; Bartlet, I.

    1980-12-10

    Complementation of the nonidentical subunits (..cap alpha.. and ..beta..) of luciferases isolated from two different bioluminescent strains, Beneckea harveyi and Photobacterium phosphoreum, has resulted in the formation of a functional hybrid luciferase (..cap alpha../sub h/..beta../sub p/) containing the ..cap alpha.. subunit from B. harveyi luciferase (..cap alpha../sub h/) and the ..beta.. subunit from P. phosphoreum luciferase (..beta../sub p/). The complementation was unidirectional; activity could not be restored by complementing the ..cap alpha.. subunit of P. phosphoreum luciferase with the ..beta.. subunit of B. harveyi luciferase, showing that the subunits from these luciferases were not identical. Kinetic parameters of the hybrid luciferase reflecting the intermediate and later steps of the bioluminescent reaction as well as the overall activity and specificity were essentially identical to the same kinetic parameters for B. harveyi luciferase, the source of the ..cap alpha.. subunit, and quite distinct from those of P. phosphoreum luciferase. However, kinetic parameters that reflected the initial step in the reaction involving interaction of FMNH/sub 2/ and luciferase were altered in the hybrid luciferase compared to both the parental luciferases, the K/sub d/ for FMNH/sub 2/ actually being closer to that observed for the P. phosphoreum luciferase (the source of the ..beta.. subunit). These results provide direct evidence that modification or alteration of the ..beta.. subunit in a dimeric luciferase molecule can affect the kinetic properties and indicates that the ..beta.. subunit plays a functional role in the bioluminescent mechanism. It is proposed that both the ..cap alpha.. and ..beta.. subunits are involved with the initial interaction with FMNH/sub 2/, whereas subsequent steps in the mechanism are dictated exclusively by the ..cap alpha.. subunit and are unaffected by alterations in the ..beta.. subunit.

  13. Fal1p is an essential DEAD-box protein involved in 40S-ribosomal-subunit biogenesis in Saccharomyces cerevisiae.

    PubMed Central

    Kressler, D; de la Cruz, J; Rojo, M; Linder, P

    1997-01-01

    A previously uncharacterized Saccharomyces cerevisiae gene, FAL1, was found by sequence comparison as a homolog of the eukaryotic translation initiation factor 4A (eIF4A). Fal1p has 55% identity and 73% similarity on the amino acid level to yeast eIF4A, the prototype of ATP-dependent RNA helicases of the DEAD-box protein family. Although clearly grouped in the eIF4A subfamily, the essential Fal1p displays a different subcellular function and localization. An HA epitope-tagged Fal1p is localized predominantly in the nucleolus. Polysome analyses in a temperature-sensitive fal1-1 mutant and a Fal1p-depleted strain reveal a decrease in the number of 40S ribosomal subunits. Furthermore, these strains are hypersensitive to the aminoglycoside antibiotics paromomycin and neomycin. Pulse-chase labeling of pre-rRNA and steady-state-level analysis of pre-rRNAs and mature rRNAs by Northern hybridization and primer extension in the Fal1p-depleted strain show that Fal1p is required for pre-rRNA processing at sites A0, A1, and A2. Consequently, depletion of Fal1p leads to decreased 18S rRNA levels and to an overall deficit in 40S ribosomal subunits. Together, these results implicate Fal1p in the 18S rRNA maturation pathway rather than in translation initiation. PMID:9372960

  14. Formation of active bacterial luciferase between interspecific subunits in vivo.

    PubMed

    Almashanu, S; Tuby, A; Hadar, R; Einy, R; Kuhn, J

    1995-01-01

    Interspecific complementation between luxAs and luxBs from Vibrio harveyi, Vibrio fischeri, Photobacterium leiognathi and Xenorhabdus luminescens was examined in vivo. The individual genes from these species were cloned on different compatible plasmids or amplified by PCR and brought together to yield cis combinations without extraneous DNA. The beta subunits from V. harveyi and X. luminescens form active enzyme only with alpha subunits from one of these species. All other combinations yield active enzymes. The lack of activity of the V. harveyi and X. luminescens beta subunits with the alpha subunits from V. fischeri and P. leiognathi results from a lack of association. This was shown by in vivo competition in which these beta subunits were overproduced in comparison with the beta and alpha of V. fischeri. No reduction in light was found. Overall, the in vivo results parallel those found in vitro using isolated denatured subunits and renaturation by removal of the denaturant.

  15. Sodium channel β subunits: emerging targets in channelopathies

    PubMed Central

    O’Malley, Heather A.; Isom, Lori L.

    2016-01-01

    Voltage-gated sodium channels (VGSCs) are responsible for initiation and propagation of action potentials in excitable cells. VGSCs in mammalian brain are heterotrimeric complexes of α and β subunits. Originally called “auxiliary,” we now know that β subunit proteins are multifunctional signaling molecules that play roles in both excitable and non-excitable cell types, and with or without the pore-forming α subunit present. β subunits function in VGSC and potassium channel modulation, cell adhesion, and gene regulation, with particularly important roles in brain development. Mutations in the genes encoding β subunits are linked to a number of diseases, including epilepsy, sudden death syndromes like SUDEP and SIDS, and cardiac arrhythmia. While VGSC β subunit-specific drugs have not yet been developed, this protein family is an emerging therapeutic target. PMID:25668026

  16. Design and Evaluation of Illumina MiSeq-Compatible, 18S rRNA Gene-Specific Primers for Improved Characterization of Mixed Phototrophic Communities

    PubMed Central

    2016-01-01

    ABSTRACT The use of high-throughput sequencing technologies with the 16S rRNA gene for characterization of bacterial and archaeal communities has become routine. However, the adoption of sequencing methods for eukaryotes has been slow, despite their significance to natural and engineered systems. There are large variations among the target genes used for amplicon sequencing, and for the 18S rRNA gene, there is no consensus on which hypervariable region provides the most suitable representation of diversity. Additionally, it is unclear how much PCR/sequencing bias affects the depiction of community structure using current primers. The present study amplified the V4 and V8-V9 regions from seven microalgal mock communities as well as eukaryotic communities from freshwater, coastal, and wastewater samples to examine the effect of PCR/sequencing bias on community structure and membership. We found that degeneracies on the 3′ end of the current V4-specific primers impact read length and mean relative abundance. Furthermore, the PCR/sequencing error is markedly higher for GC-rich members than for communities with balanced GC content. Importantly, the V4 region failed to reliably capture 2 of the 12 mock community members, and the V8-V9 hypervariable region more accurately represents mean relative abundance and alpha and beta diversity. Overall, the V4 and V8-V9 regions show similar community representations over freshwater, coastal, and wastewater environments, but specific samples show markedly different communities. These results indicate that multiple primer sets may be advantageous for gaining a more complete understanding of community structure and highlight the importance of including mock communities composed of species of interest. IMPORTANCE The quantification of error associated with community representation by amplicon sequencing is a critical challenge that is often ignored. When target genes are amplified using currently available primers, differential

  17. Molecular organization of the 25S-18S rDNA IGS of Fagus sylvatica and Quercus suber: a comparative analysis.

    PubMed

    Inácio, Vera; Rocheta, Margarida; Morais-Cecílio, Leonor

    2014-01-01

    The 35S ribosomal DNA (rDNA) units, repeated in tandem at one or more chromosomal loci, are separated by an intergenic spacer (IGS) containing functional elements involved in the regulation of transcription of downstream rRNA genes. In the present work, we have compared the IGS molecular organizations in two divergent species of Fagaceae, Fagus sylvatica and Quercus suber, aiming to comprehend the evolution of the IGS sequences within the family. Self- and cross-hybridization FISH was done on representative species of the Fagaceae. The IGS length variability and the methylation level of 18 and 25S rRNA genes were assessed in representatives of three genera of this family: Fagus, Quercus and Castanea. The intergenic spacers in Beech and Cork Oak showed similar overall organizations comprising putative functional elements needed for rRNA gene activity and containing a non-transcribed spacer (NTS), a promoter region, and a 5'-external transcribed spacer. In the NTS: the sub-repeats structure in Beech is more organized than in Cork Oak, sharing some short motifs which results in the lowest sequence similarity of the entire IGS; the AT-rich region differed in both spacers by a GC-rich block inserted in Cork Oak. The 5'-ETS is the region with the higher similarity, having nonetheless different lengths. FISH with the NTS-5'-ETS revealed fainter signals in cross-hybridization in agreement with the divergence between genera. The diversity of IGS lengths revealed variants from ∼ 2 kb in Fagus, and Quercus up to 5.3 kb in Castanea, and a lack of correlation between the number of variants and the number of rDNA loci in several species. Methylation of 25S Bam HI site was confirmed in all species and detected for the first time in the 18S of Q. suber and Q. faginea. These results provide important clues for the evolutionary trends of the rDNA 25S-18S IGS in the Fagaceae family.

  18. Quantifying the cooperative subunit action in a multimeric membrane receptor

    PubMed Central

    Wongsamitkul, Nisa; Nache, Vasilica; Eick, Thomas; Hummert, Sabine; Schulz, Eckhard; Schmauder, Ralf; Schirmeyer, Jana; Zimmer, Thomas; Benndorf, Klaus

    2016-01-01

    In multimeric membrane receptors the cooperative action of the subunits prevents exact knowledge about the operation and the interaction of the individual subunits. We propose a method that permits quantification of ligand binding to and activation effects of the individual binding sites in a multimeric membrane receptor. The power of this method is demonstrated by gaining detailed insight into the subunit action in olfactory cyclic nucleotide-gated CNGA2 ion channels. PMID:26858151

  19. Genetic analysis of neuronal ionotropic glutamate receptor subunits.

    PubMed

    Granger, Adam J; Gray, John A; Lu, Wei; Nicoll, Roger A

    2011-09-01

    In the brain, fast, excitatory synaptic transmission occurs primarily through AMPA- and NMDA-type ionotropic glutamate receptors. These receptors are composed of subunit proteins that determine their biophysical properties and trafficking behaviour. Therefore, determining the function of these subunits and receptor subunit composition is essential for understanding the physiological properties of synaptic transmission. Here, we discuss and evaluate various genetic approaches that have been used to study AMPA and NMDA receptor subunits. These approaches have demonstrated that the GluA1 AMPA receptor subunit is required for activity-dependent trafficking and contributes to basal synaptic transmission, while the GluA2 subunit regulates Ca(2+) permeability, homeostasis and trafficking to the synapse under basal conditions. In contrast, the GluN2A and GluN2B NMDA receptor subunits regulate synaptic AMPA receptor content, both during synaptic development and plasticity. Ongoing research in this field is focusing on the molecular interactions and mechanisms that control these functions. To accomplish this, molecular replacement techniques are being used, where native subunits are replaced with receptors containing targeted mutations. In this review, we discuss a single-cell molecular replacement approach which should arguably advance our physiological understanding of ionotropic glutamate receptor subunits, but is generally applicable to study of any neuronal protein.

  20. Expression of GABA receptor rho subunits in rat brain.

    PubMed

    Boue-Grabot, E; Roudbaraki, M; Bascles, L; Tramu, G; Bloch, B; Garret, M

    1998-03-01

    The GABA receptor rho1, rho2, and rho3 subunits are expressed in the retina where they form bicuculline-insensitive GABA(C) receptors. We used northern blot, in situ hybridization, and RT-PCR analysis to study the expression of rho subunits in rat brains. In situ hybridization allowed us to detect rho-subunit expression in the superficial gray layer of the superior colliculus and in the cerebellar Purkinje cells. RT-PCR experiments indicated that (a) in retina and in domains that may contain functional GABA(C) receptors, rho2 and rho1 subunits are expressed at similar levels; and (b) in domains and in tissues that are unlikely to contain GABA(C) receptors, rho2 mRNA is enriched relative to rho1 mRNA. These results suggest that both rho1 and rho2 subunits are necessary to form a functional GABA(C) receptor. The use of RT-PCR also showed that, except in the superior colliculus, rho3 is expressed along with rho1 and rho2 subunits. We also raised an antibody against a peptide sequence unique to the rho1 subunit. The use of this antibody on cerebellum revealed the rat rho1 subunit in the soma and dendrites of Purkinje neurons. The allocation of GABA(C) receptor subunits to identified neurons paves the way for future electrophysiological studies.

  1. Virus-induced gene silencing of RPC5-like subunit of RNA polymerase III caused pleiotropic effects in Nicotiana benthamiana

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In eukaryotic cells, RNA polymerase III is highly conserved, contains 17 subunits and transcribes housekeeping genes such as ribosomal 50S rRNA, tRNA and other small RNAs. Functional roles of the RPC5 are poorly characterized in the literature. In this work, we report that virus-induced gene silenci...

  2. Suppression of type 1 pilus assembly in uropathogenic Escherichia coli by chemical inhibition of subunit polymerization

    PubMed Central

    Lo, Alvin W. H.; Van de Water, Karen; Gane, Paul J.; Chan, A.W. Edith; Steadman, David; Stevens, Kiri; Selwood, David L.; Waksman, Gabriel; Remaut, Han

    2014-01-01

    Objectives To identify and to characterize small-molecule inhibitors that target the subunit polymerization of the type 1 pilus assembly in uropathogenic Escherichia coli (UPEC). Methods Using an SDS–PAGE-based assay, in silico pre-filtered small-molecule compounds were screened for specific inhibitory activity against the critical subunit polymerization step of the chaperone–usher pathway during pilus biogenesis. The biological activity of one of the compounds was validated in assays monitoring UPEC type 1 pilus biogenesis, type 1 pilus-dependent biofilm formation and adherence to human bladder epithelial cells. The time dependence of the in vivo inhibitory activity and the overall effect of the compound on UPEC growth were determined. Results N-(4-chloro-phenyl)-2-{5-[4-(pyrrolidine-1-sulfonyl)-phenyl]-[1,3,4]oxadiazol-2-yl sulfanyl}-acetamide (AL1) inhibited in vitro pilus subunit polymerization. In bacterial cultures, AL1 disrupted UPEC type 1 pilus biogenesis and pilus-dependent biofilm formation, and resulted in the reduction of bacterial adherence to human bladder epithelial cells, without affecting bacterial cell growth. Bacterial exposure to the inhibitor led to an almost instantaneous loss of type 1 pili. Conclusions We have identified and characterized a small molecule that interferes with the assembly of type 1 pili. The molecule targets the polymerization step during the subunit incorporation cycle of the chaperone–usher pathway. Our discovery provides new insight into the design and development of novel anti-virulence therapies targeting key virulence factors of bacterial pathogens. PMID:24324225

  3. Phylogeny of coral-inhabiting barnacles (Cirripedia; Thoracica; Pyrgomatidae) based on 12S, 16S and 18S rDNA analysis.

    PubMed

    Simon-Blecher, N; Huchon, D; Achituv, Y

    2007-09-01

    The traditional phylogeny of the coral-inhabiting barnacles, the Pyrgomatidae, is based on morphological characteristics, mainly of the hard parts. It has been difficult to establish the phylogenetic relationships among Pyrgomatidae because of the apparent convergence of morphological characteristics, and due to the use of non-cladistic systematics, which emphasize ancestor-descendant relationships rather than sister-clade relationships. We used partial sequences of two mithochondrial genes, 12S rDNA and 16S rDNA, and a nuclear gene, 18S rDNA, to infer the molecular phylogeny of the pyrgomatids. Our phylogenetic results allowed us to reject previous classifications of Pyrgomatidae based on morphological characteristics. Our results also suggested the possibility of paraphyly of the Pyrgomatidae. The hydrocoral barnacle Wanella is not found on the same clade as the other pyrgomatids, but rather, with the free-living balanids. The basal position of Megatrema and Ceratoconcha is supported. The archeaobalanid Armatobalanus is grouped with Cantellius at the base of the Indo-Pacific pyrgomatines. Fusion of the shell plate and modification of the opercular valves are homoplasious features that occurred more than three times on different clades. The monophyly of the "Savignium" group, comprising four nominal genera, is also not supported, and the different taxa are placed on different clades.

  4. Prevalence of infection and 18S rRNA gene sequences of Cytauxzoon species in Iberian lynx (Lynx pardinus) in Spain.

    PubMed

    Millán, J; Naranjo, V; Rodríguez, A; de la Lastra, J M Pérez; Mangold, A J; de la Fuente, J

    2007-07-01

    The Iberian lynx (Lynx pardinus) is the most endangered felid in the world. Only about 160 individuals remain in 2 separate metapopulations in Southern Spain (Sierra Morena and Doñana). We obtained blood samples of 20 lynxes captured from 2004 to 2006, and determined the prevalence of infection and genetic diversity of Cytauxzoon spp. using 18S rRNA PCR and sequence analysis. Prevalence of infection was 15% (3 of 20). Cytauxzoon sp. was only detected in Sierra Morena. For phylogenetic analysis, we used the sequences reported in the present study and those characterized in different domestic and wild felids and ticks from North and South America, Asia and Europe. Three different Cytauxzoon sp. sequences were obtained. They were closely related to that obtained from a Spanish cat, but diverged in up to 1.0% with respect to the only previously reported sequence from an Iberian lynx. Conversely, the latter sequence clustered together with C. manul sequences obtained from Pallas cats (Otocolobus manul) in Mongolia. Our analysis yields a separate cluster of C. felis sequences from cats, wild felids and ticks in the United States and Brazil. These results suggest that at least 2 different Cytauxzoon spp. may be present in Iberian lynx. The apparent absence in one of the areas, together with the possibility of fatal cytauxzoonosis in lynxes makes necessary disease risks to be taken into account in management conservation strategies, such as translocations and re-introductions.

  5. Morphology and 18S rDNA sequencing identifies Henneguya visibilis n. sp., a parasite of Leporinus obtusidens from Mogi Guaçu River, Brazil.

    PubMed

    Moreira, Gabriel S A; Adriano, Edson A; Silva, Marcia R M; Ceccarelli, Paulo S; Maia, Antônio A M

    2014-01-01

    During a survey of myxozoan parasites of freshwater fish from the Mogi Guaçu River in São Paulo State, Brazil, plasmodia of Henneguya visibilis n. sp. were found on the fins of Leporinus obtusidens (Characiformes: Anostomidae). The plasmodia, which were observed on five out of eight (62.5%) L. obtusidens examined, were 400-1,000 μm long. Mature spores were elongated with a spore body 10.8 ± 0.6 μm long and 3.9 ± 0.2 μm wide, a caudal process 18 ± 1.2 μm long, and a total spore length of 26.8 ± 1.1 μm. Polar capsules were elongated 4.9 ± 0.3 μm long and 1.4 ± 0.1 μm wide. Histological examination indicated that the plasmodia developed in the connective tissue, and no inflammatory infiltrate was observed at the infection site. Ultrastructural analysis showed a plasmodium wall with a single membrane and several pinocytotic canals. Sporogenesis occurred from the periphery to the center of the plasmodia. Phylogenetic analysis of the 18S rDNA sequence using maximum likelihood and maximum parsimony methods showed H. visibilis n. sp. positioned in a sub-clade composed of Henneguya/Myxobolus parasites of several freshwater fish families.

  6. The phylogenetic position of Myxobolus carnaticus (Myxozoa, Myxosporea, Bivalvulida) infecting gill lamellae of Cirrhinus mrigala (Hamilton, 1822) based on 18S rRNA sequence analysis

    PubMed Central

    Banerjee, Sayani; Patra, Avijit; Adikesavalu, Harresh; Mondal, Anjan; Jawahar Abraham, Thangapalam

    2015-01-01

    Myxozoans are an economically important group of microscopic parasites best known for the diseases they cause in commercially important fish hosts. The classification of myxosporeans is generally based on the morphology of their myxospores. Without molecular data, it is very difficult to identify new or existing species. DNA sequence information is therefore, a prerequisite to taxonomic and phylogenic studies of myxosporeans. In the present study, a myxozoan parasite, Myxobolus carnaticus, infecting the gill lamellae of mrigal carp, Cirrhinus mrigala, was characterized by the 18S rRNA gene sequence. The DNA sequence of M. carnaticus clustered phylogenetically with other gill infecting Myxobolus spp. of freshwater clades, forming a dichotomy with closely related M. pavlovskii (HM991164) that infects the gill lamellae epithelium of silver carp, Hypophthalmichthys molitrix with 95% similarity. Evolutionary pair-wise distances among M. carnaticus and other species of myxosporeans indicated high genetic diversity among myxosporeans. The present study demonstrated that tissue tropism, host specificity and habitat play important roles in phylogenetic relationships among myxozoan species. PMID:27844004

  7. Gregarine site-heterogeneous 18S rDNA trees, revision of gregarine higher classification, and the evolutionary diversification of Sporozoa.

    PubMed

    Cavalier-Smith, Thomas

    2014-10-01

    Gregarine 18S ribosomal DNA trees are hard to resolve because they exhibit the most disparate rates of rDNA evolution of any eukaryote group. As site-heterogeneous tree-reconstruction algorithms can give more accurate trees, especially for technically unusually challenging groups, I present the first site-heterogeneous rDNA trees for 122 gregarines and an extensive set of 452 appropriate outgroups. While some features remain poorly resolved, these trees fit morphological diversity better than most previous, evolutionarily less realistic, maximum likelihood trees. Gregarines are probably polyphyletic, with some 'eugregarines' and all 'neogregarines' (both abandoned as taxa) being more closely related to Cryptosporidium and Rhytidocystidae than to archigregarines. I establish a new subclass Orthogregarinia (new orders Vermigregarida, Arthrogregarida) for gregarines most closely related to Cryptosporidium and group Orthogregarinia, Cryptosporidiidae, and Rhytidocystidae as revised class Gregarinomorphea. Archigregarines are excluded from Gregarinomorphea and grouped with new orders Velocida (Urosporoidea superfam. n. and Veloxidium) and Stenophorida as a new sporozoan class Paragregarea. Platyproteum and Filipodium never group with Orthogregarinia or Paragregarea and are sufficiently different morphologically to merit a new order Squirmida. I revise gregarine higher-level classification generally in the light of site-heterogeneous-model trees, discuss their evolution, and also sporozoan cell structure and life-history evolution, correcting widespread misinterpretations.

  8. Molecular Characterization of Cryptosporidium spp. in Wild Rodents of Southwestern Iran Using 18s rRNA Gene Nested-PCR-RFLP and Sequencing Techniques

    PubMed Central

    Saki, Jasem; Asadpouri, Reza

    2016-01-01

    Background. Rodents could act as reservoir for Cryptosporidium spp. specially C. parvum, a zoonotic agent responsible for human infections. Since there is no information about Cryptosporidium infection in rodents of Ahvaz city, southwest of Iran, hence, this survey was performed to determine the prevalence and molecular characterization of Cryptosporidium spp. in this region. Materials and Methods. One hundred rodents were trapped from different regions of Ahvaz city. Intestine contents and fecal specimens of rodents were studied using both microscopy examination to identify oocyst and nested-polymerase chain reaction (PCR) technique for 18s rRNA gene detection. Eventually restriction fragment length polymorphism (RFLP) method using SspI and VspI restriction enzymes was carried out to genotype the species and then obtained results were sequenced. Results. Three out of 100 samples were diagnosed as positive and overall prevalence of Cryptosporidium spp. was 3% using both modified Ziehl-Neelsen staining under light microscope and nested-PCR (830 bp) methods. Afterwards, PCR-RFLP was performed on positive samples and C. parvum pattern was identified. Finally PCR-RFLP findings were sequenced and presence of C. parvum was confirmed again. Conclusions. Our study showed rodents could be potential reservoir for C. parvum. So an integrated program for control and combat with them should be adopted and continued. PMID:27956905

  9. Nanoscale copper sulfide hollow spheres with phase-engineered composition: covellite (CuS), digenite (Cu1.8S), chalcocite (Cu2S).

    PubMed

    Leidinger, Peter; Popescu, Radian; Gerthsen, Dagmar; Lünsdorf, Heinrich; Feldmann, Claus

    2011-06-01

    Covellite (CuS), digenite (Cu(1.8)S) and chalcocite (Cu(2)S) are prepared as nanoscaled hollow spheres by reaction at the liquid-to-liquid phase boundary of a w/o-microemulsion. According to electron microscopy (SEM, STEM, TEM, HRTEM) the hollow spheres exhibit an outer diameter of 32-36 nm, a wall thickness of 8-12 nm and an inner cavity of 8-16 nm in diameter. The phase composition is determined based on HRTEM, electron-energy loss spectroscopy, X-ray powder diffraction and thermal analysis. In face of the advanced morphology of the hollow spheres, precise control of its phase composition is nevertheless possible by adjusting the experimental conditions (i.e. type and concentration of the copper precursor, concentration of ammonia inside of the micelle). Such phase-engineering of nanoscale hollow spheres is firstly observed and might allow adjusting even further compositions/structures as well as tailoring of phase-specific properties in the future.

  10. Phylogenetic position of phylum Nemertini, inferred from 18S rRNA sequences: molecular data as a test of morphological character homology.

    PubMed

    Turbeville, J M; Field, K G; Raff, R A

    1992-03-01

    Partial 18S rRNA sequence of the nemertine Cerebratulus lacteus was obtained and compared with those of coelomate metazoans and acoelomate platyhelminths to test whether nemertines share a most recent common ancestor with the platyhelminths, as traditionally has been implied, or whether nemertines lie within a protostome coelomate clade, as suggested by more recent morphological analyses. Maximum-parsimony analysis supports the inclusion of the nemertine within a protostome-coelomate clade that falls within a more inclusive coelomate clade. Bootstrap analysis indicates strong support for a monophyletic Coelomata composed of a deuterostome and protostome-coelomate clade. Support for a monophyletic protostome Coelomata is weak. Inference by distance analysis is consistent with that of maximum parsimony. Analysis of down-weighted paired sites by maximum parsimony reveals variation in topology only within the protostome-coelomate clade. The relationships among the protostome coelomates cannot be reliably inferred from the partial sequences, suggesting that coelomate protostomes diversified rapidly. Results with evolutionary parsimony are consistent with the inclusion of the nemertine in a coelomate clade. The molecular inference corroborates recent morphological character analyses that reveal no synapomorphies of nemertines and flatworms but instead suggest that the circulatory system and rhynchocoel of nemertines are homologous to coelomic cavities of protostome coelomates, thus supporting the corresponding hypothesis that nemertines belong within a protostome-coelomate clade. The sequence data provide an independent test of morphological character homology.

  11. Detection of Kudoa septempunctata 18S ribosomal DNA in patient fecal samples from novel food-borne outbreaks caused by consumption of raw olive flounder (Paralichthys olivaceus).

    PubMed

    Harada, Tetsuya; Kawai, Takao; Jinnai, Michio; Ohnishi, Takahiro; Sugita-Konishi, Yoshiko; Kumeda, Yuko

    2012-09-01

    Kudoa septempunctata is a newly identified myxosporean parasite of olive flounder (Paralichthys olivaceus) and a suspected causative agent of several food-borne gastroenteritis outbreaks in Japan. Here, we report the detection of K. septempunctata 18S ribosomal DNA in fecal samples of outbreak patients using an efficient method based on real-time PCR. We first performed a spiking experiment to assess whether our previously developed real-time PCR assay was applicable to detect K. septempunctata in feces. Simultaneously, we compared the relative extraction efficacy of K. septempunctata DNA using three commercial kits. Finally, our detection method was validated by testing 45 clinical samples obtained from 13 food-borne outbreaks associated with the consumption of raw flounder and 41 fecal samples from diarrhea patients epidemiologically unrelated to the ingestion of raw fish. We found that the FastDNA Spin Kit for Soil (MP Biomedicals) was the most efficient method for extracting K. septempunctata DNA from fecal samples. Using this kit, the detection limit of our real-time PCR assay was 1.6 × 10(1) spores per g of feces, and positive results were obtained for 21 fecal and 2 vomitus samples obtained from the food-borne outbreaks. To our knowledge, this is the first report to describe the detection of K. septempunctata DNA in patient fecal samples. We anticipate that our detection method will be useful for confirming food-borne diseases caused by K. septempunctata in laboratory investigations.

  12. Design and validation of four new primers for next-generation sequencing to target the 18S rRNA genes of gastrointestinal ciliate protozoa.

    PubMed

    Ishaq, Suzanne L; Wright, André-Denis G

    2014-09-01

    Four new primers and one published primer were used to PCR amplify hypervariable regions within the protozoal 18S rRNA gene to determine which primer pair provided the best identification and statistical analysis. PCR amplicons of 394 to 498 bases were generated from three primer sets, sequenced using Roche 454 pyrosequencing with Titanium, and analyzed using the BLAST database (NCBI) and MOTHUR version 1.29. The protozoal diversity of rumen contents from moose in Alaska was assessed. In the present study, primer set 1, P-SSU-316F and GIC758R (amplicon of 482 bases), gave the best representation of diversity using BLAST classification, and the set amplified Entodinium simplex and Ostracodinium spp., which were not amplified by the other two primer sets. Primer set 2, GIC1080F and GIC1578R (amplicon of 498 bases), had similar BLAST results and a slightly higher percentage of sequences that were identified with a higher sequence identity. Primer sets 1 and 2 are recommended for use in ruminants. However, primer set 1 may be inadequate to determine protozoal diversity in nonruminants. The amplicons created by primer set 1 were indistinguishable for certain species within the genera Bandia, Blepharocorys, Polycosta, and Tetratoxum and between Hemiprorodon gymnoprosthium and Prorodonopsis coli, none of which are normally found in the rumen.

  13. Dynamic changes in the distribution of a satellite homologous to intergenic 26-18S rDNA spacer in the evolution of Nicotiana.

    PubMed Central

    Lim, K Y; Skalicka, K; Koukalova, B; Volkov, R A; Matyasek, R; Hemleben, V; Leitch, A R; Kovarik, A

    2004-01-01

    An approximately 135-bp sequence called the A1/A2 repeat was isolated from the transcribed region of the 26-18S rDNA intergenic spacer (IGS) of Nicotiana tomentosiformis. Fluorescence in situ hybridization (FISH) and Southern blot analysis revealed its occurrence as an independent satellite (termed an A1/A2 satellite) outside of rDNA loci in species of Nicotiana section Tomentosae. The chromosomal location, patterns of genomic dispersion, and copy numbers of its tandemly arranged units varied between the species. In more distantly related Nicotiana species the A1/A2 repeats were found only at the nucleolar organizer regions (NOR). There was a trend toward the elimination of the A1/A2 satellite in N. tabacum (tobacco), an allotetraploid with parents closely related to the diploids N. sylvestris and N. tomentosiformis. This process may have already commenced in an S(3) generation of synthetic tobacco. Cytosine residues in the IGS were significantly hypomethylated compared with the A1/A2 satellite. There was no clear separation between the IGS and satellite fractions in sequence analysis of individual clones and we found no evidence for CG suppression. Taken together the data indicate a dynamic nature of the A1/A2 repeats in Nicotiana genomes, with evidence for recurrent integration, copy number expansions, and contractions. PMID:15126410

  14. Design and Validation of Four New Primers for Next-Generation Sequencing To Target the 18S rRNA Genes of Gastrointestinal Ciliate Protozoa

    PubMed Central

    Wright, André-Denis G.

    2014-01-01

    Four new primers and one published primer were used to PCR amplify hypervariable regions within the protozoal 18S rRNA gene to determine which primer pair provided the best identification and statistical analysis. PCR amplicons of 394 to 498 bases were generated from three primer sets, sequenced using Roche 454 pyrosequencing with Titanium, and analyzed using the BLAST database (NCBI) and MOTHUR version 1.29. The protozoal diversity of rumen contents from moose in Alaska was assessed. In the present study, primer set 1, P-SSU-316F and GIC758R (amplicon of 482 bases), gave the best representation of diversity using BLAST classification, and the set amplified Entodinium simplex and Ostracodinium spp., which were not amplified by the other two primer sets. Primer set 2, GIC1080F and GIC1578R (amplicon of 498 bases), had similar BLAST results and a slightly higher percentage of sequences that were identified with a higher sequence identity. Primer sets 1 and 2 are recommended for use in ruminants. However, primer set 1 may be inadequate to determine protozoal diversity in nonruminants. The amplicons created by primer set 1 were indistinguishable for certain species within the genera Bandia, Blepharocorys, Polycosta, and Tetratoxum and between Hemiprorodon gymnoprosthium and Prorodonopsis coli, none of which are normally found in the rumen. PMID:24973070

  15. When molecules support morphology: Phylogenetic reconstruction of the family Onuphidae (Eunicida, Annelida) based on 16S rDNA and 18S rDNA.

    PubMed

    Budaeva, Nataliya; Schepetov, Dmitry; Zanol, Joana; Neretina, Tatiana; Willassen, Endre

    2016-01-01

    Onuphid polychaetes are tubicolous marine worms commonly reported worldwide from intertidal areas to hadal depths. They often dominate in benthic communities and have economic importance in aquaculture and recreational fishing. Here we report the phylogeny of the family Onuphidae based on the combined analyses of nuclear (18S rDNA) and mitochondrial (16S rDNA) genes. Results of Bayesian and Maximum Likelihood analyses supported the monophyly of Onuphidae and its traditional subdivision into two monophyletic subfamilies: Onuphinae and Hyalinoeciinae. Ten of 22 recognized genera were monophyletic with strong node support; four more genera included in this study were either monotypic or represented by a single species. None of the genera appeared para- or polyphyletic and this indicates a strong congruence between the traditional morphology-based systematics of the family and the newly obtained molecular-based phylogenetic reconstructions. Intergeneric relationships within Hyalinoeciinae were not resolved. Two strongly supported monophyletic groups of genera were recovered within Onuphinae: ((Onuphis, Aponuphis), Diopatra, Paradiopatra) and (Hirsutonuphis, (Paxtonia, (Kinbergonuphis, Mooreonuphis))). A previously accepted hypothesis on the subdivision of Onuphinae into the Onuphis group of genera and the Diopatra group of genera was largely rejected.

  16. Subunit vaccine efficacy against Botulinum neurotoxin subtypes.

    PubMed

    Henkel, James S; Tepp, William H; Przedpelski, Amanda; Fritz, Robert B; Johnson, Eric A; Barbieri, Joseph T

    2011-10-13

    Botulinum neurotoxins (BoNT) are classified into 7 serotypes (A-G) based upon neutralization by serotype-specific anti-sera. Several recombinant serotype-specific subunit BoNT vaccines have been developed, including a subunit vaccine comprising the receptor binding domain (HCR) of the BoNTs. Sequencing of the genes encoding BoNTs has identified variants (subtypes) that possess up to 32% primary amino acid variation among different BoNT serotypes. Studies were conducted to characterize the ability of the HCR of BoNT/A to protect against challenge by heterologous BoNT/A subtypes (A1-A3). High dose vaccination with HCR/A subtypes A1-A4 protected mice from challenge by heterologous BoNT/A subtype A1-A3, while low dose HCR vaccination yielded partial protection to heterologous BoNT/A subtype challenge. Absolute IgG titers to HCRs correlated to the dose of HCR used for vaccination, where HCR/A1 elicited an A1 subtype-specific IgG response, which was not observed with HCR/A2 vaccination. Survival of mice challenged to heterologous BoNT/A2 following low dose HCR/A1 vaccination correlated with elevated IgG titers directed to the denatured C-terminal sub-domain of HCR/A2, while survival of mice to heterologous BoNT/A1 following low dose HCR/A2 vaccination correlated to elevated IgG titers directed to native HCRc/A1. This implies that low dose vaccinations with HCR/A subtypes elicit unique IgG responses, and provides a basis to define how the host develops a neutralizing immune response to BoNT intoxication. These results may provide a reference for the development of pan-BoNT vaccines.

  17. Conformational studies on the beta subunits of human hemoglobin and their arginyl-COOH peptides.

    PubMed

    Bucci, C F; Bucci, E

    1975-10-07

    The beta subunits of hemoglobin upon alkylation of the cysteinyl residues with iodoacetamide showed a sedimentation velocity with an S20w, near 1.8 as for monomeric subunits. They reacted with alpha chains to give a tetrameric hemoglobin with a sedimentation constant near 4.4. Their CD spectrum was indistinguishable from that of untreated beta chains below 270 nm, otherwise they showed some deviation that became pronounced in the Soret region, where the optical activity of the alkylated subunits was definitely lower than that of the native subunits. Upon removal of the heme the apo-beta subunits showed a decreased optical activity in the far-uv region of the spectrum indicating a substantial loss of helical content. Their sedimentation behavior was consistent with the presence of large aggregates, which dissociates into monomers upon reconstitution with cyanoheme. The apo-beta subunits could be renatured from 6 M guanidine hydrochloride. They showed a stoichiometric reaction with heme in the molar ratio 1:1. Upon reconstitution with the heme their optical activity became similar to that of the native beta chains in the far-uv region of the spectrum, but remained lower in the near-uv and Soret regions. After acylation of the lysyl residues with citraconic anhydride the apo-beta subunits were digested with trypsin and the arginyl-COOH peptides beta(1-30), beta(31-40), beta(41-104), and beta(105-146) were separated by gel chromatography. With the exception of the peptide beta/105-146), which was insoluble at neutral pH, the sedimentation behavior of the other peptides showed the presence of small polymers. The sedimentation behavior of the peptide beta(31-40) was not tested. The percentage of alpha helix, beta conformation, and of random coil (or unordered structure) of the various proteins and peptides was measured fitting their CD spectra in the far-uv region with the parameter published by Y.H. Chen et al. ((1974), Biochemistry 13, 3350) and by N. Greenfield and G

  18. Novel structure of an N-terminal domain that is crucial for the dimeric assembly and DNA-binding of an archaeal DNA polymerase D large subunit from Pyrococcus horikoshii.

    PubMed

    Matsui, Ikuo; Urushibata, Yuji; Shen, Yulong; Matsui, Eriko; Yokoyama, Hideshi

    2011-02-04

    Archaea-specific D-family DNA polymerase forms a heterotetramer consisting of two large polymerase subunits and two small exonuclease subunits. The N-terminal (1-300) domain structure of the large subunit was determined by X-ray crystallography, although ∼50 N-terminal residues were disordered. The determined structure consists of nine alpha helices and three beta strands. We also identified the DNA-binding ability of the domain by SPR measurement. The N-terminal (1-100) region plays crucial roles in the folding of the large subunit dimer by connecting the ∼50 N-terminal residues with their own catalytic region (792-1163).

  19. Subunit Interfaces Contribute Differently to Activation and Allosteric Modulation of Neuronal Nicotinic Acetylcholine Receptors

    PubMed Central

    Short, Caitlin A.; Cao, Angela T.; Wingfield, Molly A.; Doers, Matthew E.; Jobe, Emily M.; Wang, Nan; Levandoski, Mark M.

    2015-01-01

    Neuronal nicotinic acetylcholine receptors (nAChRs) are widely distributed in the nervous system and are implicated in many normal and pathological processes. The structural determinants of allostery in nAChRs are not well understood. One class of nAChR allosteric modulators, including the small molecule morantel (Mor), acts from a site that is structurally homologous to the canonical agonist site but exists in the β(+)/α(–) subunit interface. We hypothesized that all nAChR subunits move with respect to each other during channel activation and allosteric modulation. We therefore studied five pairs of residues predicted to span the interfaces of α3β2 receptors, one at the agonist interface and four at the modulator interface. Substituting cysteines in these positions, we used disulfide trapping to perturb receptor function. The pair α3Y168-β2D190, involving the C loop region of the β2 subunit, mediates modulation and agonist activation, because evoked currents were reduced up to 50% following oxidation (H2O2) treatment. The pair α3S125-β2Q39, below the canonical site, is also involved in channel activation, in accord with previous studies of the muscle-type receptor; however, the pair is differentially sensitive to ACh activation and Mor modulation (currents decreased 60% and 80%, respectively). The pairs α3Q37-β2A127 and α3E173-β2R46, both in the non-canonical interface, showed increased currents following oxidation, suggesting that subunit movements are not symmetrical. Together, our results from disulfide trapping and further mutation analysis indicate that subunit interface movement is important for allosteric modulation of nAChRs, but that the two types of interfaces contribute unequally to receptor activation. PMID:25486620

  20. Structural model of the 50S subunit of E.Coli ribosomes from solution scattering

    SciTech Connect

    Svergun, D.I.; Koch, M.H.J.; Pedersen, J.S.; Serdyuk, I.N.

    1994-12-31

    The application of new methods of small-angle scattering data interpretation to a contrast variation study of the 50S ribosomal subunit of Escherichia coli in solution is described. The X-ray data from contrast variation with sucrose are analyzed in terms of the basic scattering curves from the volume inaccessible to sucrose and from the regions inside this volume occupied mainly by RNA and by proteins. From these curves models of the shape of the 50S and its RNA-rich core are evaluated and positioned so that their difference produces a scattering curve which is in good agreement with the scattering from the protein moiety. Basing on this preliminary model, the X-ray and neutron contrast variation data of the 50S subunit in aqueous solutions are interpreted in the frame of the advanced two-phase model described by the shapes of the 50S subunit and its RNA-rich core taking into account density fluctuations inside the RNA and the protein moiety. The shape of the envelope of the 50S subunit and of the RNA-rich core are evaluated with a resolution of about 40A. The shape of the envelope is in good agreement with the models of the 50S subunit obtained from electron microscopy on isolated particles. The shape of the RNA-rich core correlates well with the model of the entire particle determined by the image reconstruction from ordered sheets indicating that the latter model which is based on the subjective contouring of density maps is heavily biased towards the RNA.

  1. Liposome-Based Adjuvants for Subunit Vaccines: Formulation Strategies for Subunit Antigens and Immunostimulators

    PubMed Central

    Tandrup Schmidt, Signe; Foged, Camilla; Smith Korsholm, Karen; Rades, Thomas; Christensen, Dennis

    2016-01-01

    The development of subunit vaccines has become very attractive in recent years due to their superior safety profiles as compared to traditional vaccines based on live attenuated or whole inactivated pathogens, and there is an unmet medical need for improved vaccines and vaccines against pathogens for which no effective vaccines exist. The subunit vaccine technology exploits pathogen subunits as antigens, e.g., recombinant proteins or synthetic peptides, allowing for highly specific immune responses against the pathogens. However, such antigens are usually not sufficiently immunogenic to induce protective immunity, and they are often combined with adjuvants to ensure robust immune responses. Adjuvants are capable of enhancing and/or modulating immune responses by exposing antigens to antigen-presenting cells (APCs) concomitantly with conferring immune activation signals. Few adjuvant systems have been licensed for use in human vaccines, and they mainly stimulate humoral immunity. Thus, there is an unmet demand for the development of safe and efficient adjuvant systems that can also stimulate cell-mediated immunity (CMI). Adjuvants constitute a heterogeneous group of compounds, which can broadly be classified into delivery systems or immunostimulators. Liposomes are versatile delivery systems for antigens, and they can carefully be customized towards desired immune profiles by combining them with immunostimulators and optimizing their composition, physicochemical properties and antigen-loading mode. Immunostimulators represent highly diverse classes of molecules, e.g., lipids, nucleic acids, proteins and peptides, and they are ligands for pattern-recognition receptors (PRRs), which are differentially expressed on APC subsets. Different formulation strategies might thus be required for incorporation of immunostimulators and antigens, respectively, into liposomes, and the choice of immunostimulator should ideally be based on knowledge regarding the specific PRR

  2. Divergent Evolution of Nuclear Localization Signal Sequences in Herpesvirus Terminase Subunits.

    PubMed

    Sankhala, Rajeshwer S; Lokareddy, Ravi K; Cingolani, Gino

    2016-05-20

    The tripartite terminase complex of herpesviruses assembles in the cytoplasm of infected cells and exploits the host nuclear import machinery to gain access to the nucleus, where capsid assembly and genome-packaging occur. Here we analyzed the structure and conservation of nuclear localization signal (NLS) sequences previously identified in herpes simplex virus 1 (HSV-1) large terminase and human cytomegalovirus (HCMV) small terminase. We found a monopartite NLS at the N terminus of large terminase, flanking the ATPase domain, that is conserved only in α-herpesviruses. In contrast, small terminase exposes a classical NLS at the far C terminus of its helical structure that is conserved only in two genera of the β-subfamily and absent in α- and γ-herpesviruses. In addition, we predicted a classical NLS in the third terminase subunit that is partially conserved among herpesviruses. Bioinformatic analysis revealed that both location and potency of NLSs in terminase subunits evolved more rapidly than the rest of the amino acid sequence despite the selective pressure to keep terminase gene products active and localized in the nucleus. We propose that swapping NLSs among terminase subunits is a regulatory mechanism that allows different herpesviruses to regulate the kinetics of terminase nuclear import, reflecting a mechanism of virus:host adaptation.

  3. Crystal Structure of the 25 kDa Subunit of Human Cleavage Factor I{m}

    SciTech Connect

    Coseno,M.; Martin, G.; Berger, C.; Gilmartin, G.; Keller, W.; Doublie, S.

    2008-01-01

    Cleavage factor Im is an essential component of the pre-messenger RNA 3'-end processing machinery in higher eukaryotes, participating in both the polyadenylation and cleavage steps. Cleavage factor Im is an oligomer composed of a small 25 kDa subunit (CF Im25) and a variable larger subunit of either 59, 68 or 72 kDa. The small subunit also interacts with RNA, poly(A) polymerase, and the nuclear poly(A)-binding protein. These protein-protein interactions are thought to be facilitated by the Nudix domain of CF Im25, a hydrolase motif with a characteristic {alpha}/{beta}/{alpha} fold and a conserved catalytic sequence or Nudix box. We present here the crystal structures of human CF Im25 in its free and diadenosine tetraphosphate (Ap4A) bound forms at 1.85 and 1.80 Angstroms, respectively. CF Im25 crystallizes as a dimer and presents the classical Nudix fold. Results from crystallographic and biochemical experiments suggest that CF Im25 makes use of its Nudix fold to bind but not hydrolyze ATP and Ap4A. The complex and apo protein structures provide insight into the active oligomeric state of CF Im and suggest a possible role of nucleotide binding in either the polyadenylation and/or cleavage steps of pre-messenger RNA 3'-end processing.

  4. Crystal structure of Sec10, a subunit of the exocyst complex

    PubMed Central

    Chen, Jianxing; Yamagata, Atsushi; Kubota, Keiko; Sato, Yusuke; Goto-Ito, Sakurako; Fukai, Shuya

    2017-01-01

    The exocyst complex is a heterooctameric protein complex composed of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84. This complex plays an essential role in trafficking secretory vesicles to the plasma membrane through its interaction with phosphatidylinositol 4,5-bisphosphate and small GTPases. To date, the near-full-length structural information of each subunit has been limited to Exo70, although the C-terminal half structures of Sec6, Sec15 and Exo84 and the structures of the small GTPase-binding domains of Sec3, Sec5 and Exo84 have been reported. Here, we report the crystal structure of the near-full-length zebrafish Sec10 (zSec10) at 2.73 Å resolution. The structure of zSec10 consists of tandem antiparallel helix bundles that form a straight rod, like helical core regions of other exocyst subunits. This structure provides the first atomic details of Sec10, which may be useful for future functional and structural studies of this subunit and the exocyst complex. PMID:28098232

  5. Epitopes from two soybean glycinin subunits antigenic in pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Glycinin is a seed storage protein in soybean (Glycine max) that is allergenic in pigs. Glycinin is a hexamer composed of subunits consisting of a basic and acidic portion joined by disulfide bridges. There are 5 glycinin subunits designated Gy1-Gy5. Results: Twenty seven out of 30 pi...

  6. Specific Roles of NMDA Receptor Subunits in Mental Disorders

    PubMed Central

    Yamamoto, H.; Hagino, Y.; Kasai, S.; Ikeda, K.

    2015-01-01

    N-methyl-D-aspartate (NMDA) receptor plays important roles in learning and memory. NMDA receptors are a tetramer that consists of two glycine-binding subunits GluN1, two glutamate-binding subunits (i.e., GluN2A, GluN2B, GluN2C, and GluN2D), a combination of a GluN2 subunit and glycine-binding GluN3 subunit (i.e., GluN3A or GluN3B), or two GluN3 subunits. Recent studies revealed that the specific expression and distribution of each subunit are deeply involved in neural excitability, plasticity, and synaptic deficits. The present article summarizes reports on the dysfunction of NMDA receptors and responsible subunits in various neurological and psychiatric disorders, including schizophrenia, autoimmune-induced glutamatergic receptor dysfunction, mood disorders, and autism. A key role for the GluN2D subunit in NMDA receptor antagonist-induced psychosis has been recently revealed. PMID:25817860

  7. 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…

  8. Time-series of water column alkenones and 18S rRNA confirm that Uk'37 is a viable SST proxy in Narragansett Bay, RI

    NASA Astrophysics Data System (ADS)

    Salacup, J.; Theroux, S.; Herbert, T.; Prell, W. L.

    2011-12-01

    Alkenones, produced in the sunlit mixed layer by specific Haptophyte algae, are a well-established and widely-applied proxy for sea surface temperature (SST) in the world's open-oceans. However, the proxy's utility in estuarine environments remains largely untested. A reliable SST proxy is needed to identify the estuary's sensitivity and response to past and present global change because SST can exert strong control on stratification and circulation patterns, and thus oxygenation and ecosystem health, in these shallow basins. Knowing the estuaries response should help local managers and policy-makers plan mitigation and adaptation strategies. Additionally, the rapid deposition of both marine and terrestrial organic and inorganic material in estuarine systems makes them potential archives of high-resolution paleo-environmental information. A previous investigation of estuarine alkenones suggested that the Uk'37 proxy may be sensitive to the composition of the alkenone-producing Haptophyte population, which may be affected by local nutrient and fresh water fluxes. In particular, low-salinity coastal Haptophytes such as Isochrysis galbana may have a different relationship to SST than higher-salinity open-ocean Haptophytes and their presence may complicate interpretations of the Uk'37 proxy in estuaries. To better understand how the alkenone-based Uk'37 SST proxy is produced in estuarine systems, we present a two-year time-series (monthly-to-thrice-weekly resolution) of alkenone concentrations in particulate organic matter from Narragansett Bay. Alkenone concentrations are coupled with 18S ribosomal RNA (rRNA) measurements to identify the alkenone-producing population. Highest concentrations of alkenones are detected at different times in the upper and lower Bay such that the highest alkenone concentrations occur in the winter-spring (upper Bay) and summer/fall (lower Bay). This result is consistent with the established seasonal blooms and seasonal changes in nutrient

  9. Free-Living Protozoa in Two Unchlorinated Drinking Water Supplies, Identified by Phylogenic Analysis of 18S rRNA Gene Sequences▿ †

    PubMed Central

    Valster, Rinske M.; Wullings, Bart A.; Bakker, Geo; Smidt, Hauke; van der Kooij, Dick

    2009-01-01

    Free-living protozoan communities in water supplies may include hosts for Legionella pneumophila and other undesired bacteria, as well as pathogens. This study aimed at identifying free-living protozoa in two unchlorinated groundwater supplies, using cultivation-independent molecular approaches. For this purpose, samples (<20°C) of treated water, distributed water, and distribution system biofilms were collected from supply A, with a low concentration of natural organic matter (NOM) (<0.5 ppm of C), and from supply B, with a high NOM concentration (7.9 ppm of C). Eukaryotic communities were studied using terminal restriction fragment length polymorphism and clone library analyses of partial 18S rRNA gene fragments and a Hartmannella vermiformis-specific quantitative PCR (qPCR). In both supplies, highly diverse eukaryotic communities were observed, including free-living protozoa, fungi, and metazoa. Sequences of protozoa clustered with Amoebozoa (10 operational taxonomic units [OTUs]), Cercozoa (39 OTUs), Choanozoa (26 OTUs), Ciliophora (29 OTUs), Euglenozoa (13 OTUs), Myzozoa (5 OTUs), and Stramenopiles (5 OTUs). A large variety of protozoa were present in both supplies, but the estimated values for protozoan richness did not differ significantly. H. vermiformis was observed in both supplies but was not a predominant protozoan. One OTU with the highest similarity to Acanthamoeba polyphaga, an opportunistic human pathogen and a host for undesired bacteria, was observed in supply A. The high level of NOM in supply B corresponded with an elevated level of active biomass and with elevated concentrations of H. vermiformis in distributed water. Hence, the application of qPCR may be promising in elucidating the relationship between drinking water quality and the presence of specific protozoa. PMID:19465529

  10. Identification of Habitat-Specific Biomes of Aquatic Fungal Communities Using a Comprehensive Nearly Full-Length 18S rRNA Dataset Enriched with Contextual Data.

    PubMed

    Panzer, Katrin; Yilmaz, Pelin; Weiß, Michael; Reich, Lothar; Richter, Michael; Wiese, Jutta; Schmaljohann, Rolf; Labes, Antje; Imhoff, Johannes F; Glöckner, Frank Oliver; Reich, Marlis

    2015-01-01

    Molecular diversity surveys have demonstrated that aquatic fungi are highly diverse, and that they play fundamental ecological roles in aquatic systems. Unfortunately, comparative studies of aquatic fungal communities are few and far between, due to the scarcity of adequate datasets. We combined all publicly available fungal 18S ribosomal RNA (rRNA) gene sequences with new sequence data from a marine fungi culture collection. We further enriched this dataset by adding validated contextual data. Specifically, we included data on the habitat type of the samples assigning fungal taxa to ten different habitat categories. This dataset has been created with the intention to serve as a valuable reference dataset for aquatic fungi including a phylogenetic reference tree. The combined data enabled us to infer fungal community patterns in aquatic systems. Pairwise habitat comparisons showed significant phylogenetic differences, indicating that habitat strongly affects fungal community structure. Fungal taxonomic composition differed considerably even on phylum and class level. Freshwater fungal assemblage was most different from all other habitat types and was dominated by basal fungal lineages. For most communities, phylogenetic signals indicated clustering of sequences suggesting that environmental factors were the main drivers of fungal community structure, rather than species competition. Thus, the diversification process of aquatic fungi must be highly clade specific in some cases.The combined data enabled us to infer fungal community patterns in aquatic systems. Pairwise habitat comparisons showed significant phylogenetic differences, indicating that habitat strongly affects fungal community structure. Fungal taxonomic composition differed considerably even on phylum and class level. Freshwater fungal assemblage was most different from all other habitat types and was dominated by basal fungal lineages. For most communities, phylogenetic signals indicated clustering of

  11. Use of 18S rRNA gene-based PCR assay for diagnosis of acanthamoeba keratitis in non-contact lens wearers in India.

    PubMed

    Pasricha, Gunisha; Sharma, Savitri; Garg, Prashant; Aggarwal, Ramesh K

    2003-07-01

    Identification of Acanthamoeba cysts and trophozoites in ocular tissues requires considerable expertise and is often time-consuming. An 18S rRNA gene-based PCR test, highly specific for the genus Acanthamoeba, has recently been reported in the molecular diagnosis of Acanthamoeba keratitis. This PCR assay was compared with conventional microbiological tests for the diagnosis of Acanthamoeba keratitis. In a pilot study, the PCR conditions with modifications were first tested on corneal scrapings from patients with culture-proven non-contact lens-related Acanthamoeba, bacterial, and fungal keratitis. This was followed by testing of corneal scrapings from 53 consecutive cases of microbial keratitis to determine sensitivity, specificity, and predictive values of the assay. All corneal scrapings from patients with proven Acanthamoeba keratitis showed a 463-bp amplicon, while no amplicon was obtained from patients with bacterial or fungal keratitis. Some of these amplified products were sequenced and compared with EMBL database reference sequences to validate these to be of Acanthamoeba origin. Out of 53 consecutive cases of microbial keratitis included for evaluating the PCR, 10 (18.9%) cases were diagnosed as Acanthamoeba keratitis on the basis of combined results of culture, smear, and PCR of corneal scrapings. Based on culture results as the "gold standard," the sensitivity of PCR was the same as that of the smear (87.5%); however, the specificity and the positive and negative predictive values of PCR were marginally higher than the smear examination (97.8 versus 95.6%, 87.5 versus 77.8%, and 97.8 versus 97.7%) although the difference was not significant. This study confirms the efficacy of the PCR assay and is the first study to evaluate a PCR-based assay against conventional methods of diagnosis in a clinical setting.

  12. Use of 18S rRNA Gene-Based PCR Assay for Diagnosis of Acanthamoeba Keratitis in Non-Contact Lens Wearers in India

    PubMed Central

    Pasricha, Gunisha; Sharma, Savitri; Garg, Prashant; Aggarwal, Ramesh K.

    2003-01-01

    Identification of Acanthamoeba cysts and trophozoites in ocular tissues requires considerable expertise and is often time-consuming. An 18S rRNA gene-based PCR test, highly specific for the genus Acanthamoeba, has recently been reported in the molecular diagnosis of Acanthamoeba keratitis. This PCR assay was compared with conventional microbiological tests for the diagnosis of Acanthamoeba keratitis. In a pilot study, the PCR conditions with modifications were first tested on corneal scrapings from patients with culture-proven non-contact lens-related Acanthamoeba, bacterial, and fungal keratitis. This was followed by testing of corneal scrapings from 53 consecutive cases of microbial keratitis to determine sensitivity, specificity, and predictive values of the assay. All corneal scrapings from patients with proven Acanthamoeba keratitis showed a 463-bp amplicon, while no amplicon was obtained from patients with bacterial or fungal keratitis. Some of these amplified products were sequenced and compared with EMBL database reference sequences to validate these to be of Acanthamoeba origin. Out of 53 consecutive cases of microbial keratitis included for evaluating the PCR, 10 (18.9%) cases were diagnosed as Acanthamoeba keratitis on the basis of combined results of culture, smear, and PCR of corneal scrapings. Based on culture results as the “gold standard,” the sensitivity of PCR was the same as that of the smear (87.5%); however, the specificity and the positive and negative predictive values of PCR were marginally higher than the smear examination (97.8 versus 95.6%, 87.5 versus 77.8%, and 97.8 versus 97.7%) although the difference was not significant. This study confirms the efficacy of the PCR assay and is the first study to evaluate a PCR-based assay against conventional methods of diagnosis in a clinical setting. PMID:12843065

  13. Identification of Habitat-Specific Biomes of Aquatic Fungal Communities Using a Comprehensive Nearly Full-Length 18S rRNA Dataset Enriched with Contextual Data

    PubMed Central

    Panzer, Katrin; Yilmaz, Pelin; Weiß, Michael; Reich, Lothar; Richter, Michael; Wiese, Jutta; Schmaljohann, Rolf; Labes, Antje; Imhoff, Johannes F.; Glöckner, Frank Oliver; Reich, Marlis

    2015-01-01

    Molecular diversity surveys have demonstrated that aquatic fungi are highly diverse, and that they play fundamental ecological roles in aquatic systems. Unfortunately, comparative studies of aquatic fungal communities are few and far between, due to the scarcity of adequate datasets. We combined all publicly available fungal 18S ribosomal RNA (rRNA) gene sequences with new sequence data from a marine fungi culture collection. We further enriched this dataset by adding validated contextual data. Specifically, we included data on the habitat type of the samples assigning fungal taxa to ten different habitat categories. This dataset has been created with the intention to serve as a valuable reference dataset for aquatic fungi including a phylogenetic reference tree. The combined data enabled us to infer fungal community patterns in aquatic systems. Pairwise habitat comparisons showed significant phylogenetic differences, indicating that habitat strongly affects fungal community structure. Fungal taxonomic composition differed considerably even on phylum and class level. Freshwater fungal assemblage was most different from all other habitat types and was dominated by basal fungal lineages. For most communities, phylogenetic signals indicated clustering of sequences suggesting that environmental factors were the main drivers of fungal community structure, rather than species competition. Thus, the diversification process of aquatic fungi must be highly clade specific in some cases.The combined data enabled us to infer fungal community patterns in aquatic systems. Pairwise habitat comparisons showed significant phylogenetic differences, indicating that habitat strongly affects fungal community structure. Fungal taxonomic composition differed considerably even on phylum and class level. Freshwater fungal assemblage was most different from all other habitat types and was dominated by basal fungal lineages. For most communities, phylogenetic signals indicated clustering of

  14. Localization of two major GABA(A) receptor subunits in the dentate gyrus of the rat and cell type-specific up-regulation following entorhinal cortex lesion.

    PubMed

    Simbürger, E; Plaschke, M; Fritschy, J M; Nitsch, R

    2001-01-01

    GABA(A) receptor subunits show a specific regional distribution in the CNS during development and in the adult animal. In the hippocampal formation, individual subsets of GABAergic interneurons are highly immunoreactive for the alpha1-subunit, whereas granule and pyramidal cells show a strong expression of the alpha2-subunit. Using confocal microscopy and digital image analysis, we demonstrate that in the dentate gyrus the alpha1-subunit immunolabeling appears in differently sized clusters. The large clusters, which are confined to dendrites of interneurons, show no alpha2 labeling, whereas the smaller ones coincide with alpha2-subunit-positive clusters. In the molecular layer, the clusters of both alpha-subunits co-localize with the anchoring protein gephyrin. In the granule cell layer and hilus, we found alpha1- and alpha2-subunit-positive clusters which were devoid of gephyrin labeling. Lesions of the medial entorhinal cortex led to the deafferentation of dendrites in the middle molecular layer of the dentate gyrus. This resulted in a significantly increased concentration of alpha2-subunit-positive clusters. We also observed an increase of alpha1-subunit immunolabeling in the deafferented area. We found no change in the co-localization between alpha1 and alpha2, and no significant change in the number of large alpha1-positive clusters along individual dendritic segments of interneurons. In a previous study, we demonstrated that calbindin-immunoreactive dendrites of granule cells revealed a significant increase in gephyrin immunoreactivity following lesion, whereas parvalbumin-positive dendrites showed no such alterations. The predominant localization of small gephyrin clusters in dendrites of granule cells, which was also described in this study, leads to the conclusion that the increase of the alpha2-subunit-positive clusters, demonstrated in the present study, indicates that, following entorhinal cortex lesion, new GABAergic synapses may be formed and that

  15. 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 <