The restriction-modification genes of Escherichia coli K-12 may not be selfish: they do not resist loss and are readily replaced by alleles conferring different specificities.

PubMed

O'Neill, M; Chen, A; Murray, N E

1997-12-23

Type II restriction and modification (R-M) genes have been described as selfish because they have been shown to impose selection for the maintenance of the plasmid that encodes them. In our experiments, the type I R-M system EcoKI does not behave in the same way. The genes specifying EcoKI are, however, normally residents of the chromosome and therefore our analyses were extended to monitor the deletion of chromosomal genes rather than loss of plasmid vector. If EcoKI were to behave in the same way as the plasmid-encoded type II R-M systems, the loss of the relevant chromosomal genes by mutation or recombination should lead to cell death because the cell would become deficient in modification enzyme and the bacterial chromosome would be vulnerable to the restriction endonuclease. Our data contradict this prediction; they reveal that functional type I R-M genes in the chromosome are readily replaced by mutant alleles and by alleles encoding a type I R-M system of different specificity. The acquisition of allelic genes conferring a new sequence specificity, but not the loss of the resident genes, is dependent on the product of an unlinked gene, one predicted [Prakash-Cheng, A., Chung, S. S. & Ryu, J. (1993) Mol. Gen. Genet. 241, 491-496] to be relevant to control of expression of the genes that encode EcoKI. Our evidence suggests that not all R-M systems are evolving as "selfish" units; rather, the diversity and distribution of the family of type I enzymes we have investigated require an alternative selective pressure.

A young root-specific gene (ArMY2) from horseradish encoding a MYR II myrosinase with kinetic preference for the root-specific glucosinolate gluconasturtiin.

PubMed

Loebers, Andreas; Müller-Uri, Frieder; Kreis, Wolfgang

2014-03-01

The pungent taste of horseradish is caused by isothiocyanates which are released from glucosinolates by myrosinases. These enzymes are encoded by genes belonging to one of two subfamilies, termed MYR I and MYR II, respectively. A MYR II-type myrosinase gene was identified for the first time in horseradish. The gene termed ArMY2 was only expressed in young roots. A full-length cDNA encoding a myrosinase termed ArMy2 was isolated and heterologously expressed in Pichia pastoris. The recombinant His-tagged enzyme was characterized biochemically. Substrate affinity was 5 times higher towards gluconasturtiin than towards sinigrin. Gluconasturtiin was found to be the most abundant glucosinolate in young horseradish roots while sinigrin dominated in storage roots and leaves. This indicates that a specialized glucosinolate-myrosinase defense system might be active in young roots. Copyright © 2013 Elsevier Ltd. All rights reserved.

Role of Modular Polyketide Synthases in the Production of Polyether Ladder Compounds in Ciguatoxin-Producing Gambierdiscus polynesiensis and G. excentricus (Dinophyceae).

PubMed

Kohli, Gurjeet S; Campbell, Katrina; John, Uwe; Smith, Kirsty F; Fraga, Santiago; Rhodes, Lesley L; Murray, Shauna A

2017-09-01

Gambierdiscus, a benthic dinoflagellate, produces ciguatoxins that cause the human illness Ciguatera. Ciguatoxins are polyether ladder compounds that have a polyketide origin, indicating that polyketide synthases (PKS) are involved in their production. We sequenced transcriptomes of Gambierdiscus excentricus and Gambierdiscus polynesiensis and found 264 contigs encoding single domain ketoacyl synthases (KS; G. excentricus: 106, G. polynesiensis: 143) and ketoreductases (KR; G. excentricus: 7, G. polynesiensis: 8) with sequence similarity to type I PKSs, as reported in other dinoflagellates. In addition, 24 contigs (G. excentricus: 3, G. polynesiensis: 21) encoding multiple PKS domains (forming typical type I PKSs modules) were found. The proposed structure produced by one of these megasynthases resembles a partial carbon backbone of a polyether ladder compound. Seventeen contigs encoding single domain KS, KR, s-malonyltransacylase, dehydratase and enoyl reductase with sequence similarity to type II fatty acid synthases (FAS) in plants were found. Type I PKS and type II FAS genes were distinguished based on the arrangement of domains on the contigs and their sequence similarity and phylogenetic clustering with known PKS/FAS genes in other organisms. This differentiation of PKS and FAS pathways in Gambierdiscus is important, as it will facilitate approaches to investigating toxin biosynthesis pathways in dinoflagellates. © 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists.

Stoichiometry of DNA binding by the bacteriophage SP01-encoded type II DNA-binding protein TF1.

PubMed

Schneider, G J; Geiduschek, E P

1990-06-25

The stoichiometry of DNA binding by the bacteriophage SP01-encoded type II DNA-binding protein TF1 has been determined. 3H-Labeled TF1 was allowed to bind to a 32P-labeled DNA fragment containing a TF1 binding site. Multiple TF1-DNA complexes were resolved from each other and from unbound DNA by native gel electrophoresis. DNA-protein complexes were cut from polyacrylamide gels, and the amounts of 3H and 32P contained in each slice were measured. A ratio of 1.12 +/- 0.06 TF1 dimer/DNA molecule was calculated for the fastest-migrating TF1-DNA complex. We conclude that TF1 has a DNA-binding unit of one dimer. More slowly migrating complexes are apparently formed by serial addition of single TF1 dimers.

Cloning and characterization of the gene encoding IMP dehydrogenase from Arabidopsis thaliana.

PubMed

Collart, F R; Osipiuk, J; Trent, J; Olsen, G J; Huberman, E

1996-10-03

We have cloned and characterized the gene encoding inosine monophosphate dehydrogenase (IMPDH) from Arabidopsis thaliana (At). The transcription unit of the At gene spans approximately 1900 bp and specifies a protein of 503 amino acids with a calculated relative molecular mass (M(r)) of 54,190. The gene is comprised of a minimum of four introns and five exons with all donor and acceptor splice sequences conforming to previously proposed consensus sequences. The deduced IMPDH amino-acid sequence from At shows a remarkable similarity to other eukaryotic IMPDH sequences, with a 48% identity to human Type II enzyme. Allowing for conservative substitutions, the enzyme is 69% similar to human Type II IMPDH. The putative active-site sequence of At IMPDH conforms to the IMP dehydrogenase/guanosine monophosphate reductase motif and contains an essential active-site cysteine residue.

Methyltransferases acquired by lactococcal 936-type phage provide protection against restriction endonuclease activity.

PubMed

Murphy, James; Klumpp, Jochen; Mahony, Jennifer; O'Connell-Motherway, Mary; Nauta, Arjen; van Sinderen, Douwe

2014-10-01

So-called 936-type phages are among the most frequently isolated phages in dairy facilities utilising Lactococcus lactis starter cultures. Despite extensive efforts to control phage proliferation and decades of research, these phages continue to negatively impact cheese production in terms of the final product quality and consequently, monetary return. Whole genome sequencing and in silico analysis of three 936-type phage genomes identified several putative (orphan) methyltransferase (MTase)-encoding genes located within the packaging and replication regions of the genome. Utilising SMRT sequencing, methylome analysis was performed on all three phages, allowing the identification of adenine modifications consistent with N-6 methyladenine sequence methylation, which in some cases could be attributed to these phage-encoded MTases. Heterologous gene expression revealed that M.Phi145I/M.Phi93I and M.Phi93DAM, encoded by genes located within the packaging module, provide protection against the restriction enzymes HphI and DpnII, respectively, representing the first functional MTases identified in members of 936-type phages. SMRT sequencing technology enabled the identification of the target motifs of MTases encoded by the genomes of three lytic 936-type phages and these MTases represent the first functional MTases identified in this species of phage. The presence of these MTase-encoding genes on 936-type phage genomes is assumed to represent an adaptive response to circumvent host encoded restriction-modification systems thereby increasing the fitness of the phages in a dynamic dairy environment.

Feasibility of Screening for Antibiotic Resistance - Part I

DTIC Science & Technology

2005-09-01

Klebsiella pneumonia (AF052258), Providencia stuartii (AF052259), Serratia marcescens (AF052260). After alignment of the sequences of the various...intracellular targets of fluoroquinolones , the type II DNA topoisomerases gyrase and topoisomerase IV [Balas et al., 1998; Weigel et al., 1998]. Mutations in...the quinolones resistant determining region (QRDR) of the parC gene, which encodes the A subunit of topoisomerase , and the gyrA gene, which encodes

Evolution of the P-type II ATPase gene family in the fungi and presence of structural genomic changes among isolates of Glomus intraradices.

PubMed

Corradi, Nicolas; Sanders, Ian R

2006-03-10

The P-type II ATPase gene family encodes proteins with an important role in adaptation of the cell to variation in external K+, Ca2+ and Na2+ concentrations. The presence of P-type II gene subfamilies that are specific for certain kingdoms has been reported but was sometimes contradicted by discovery of previously unknown homologous sequences in newly sequenced genomes. Members of this gene family have been sampled in all of the fungal phyla except the arbuscular mycorrhizal fungi (AMF; phylum Glomeromycota), which are known to play a key-role in terrestrial ecosystems and to be genetically highly variable within populations. Here we used highly degenerate primers on AMF genomic DNA to increase the sampling of fungal P-Type II ATPases and to test previous predictions about their evolution. In parallel, homologous sequences of the P-type II ATPases have been used to determine the nature and amount of polymorphism that is present at these loci among isolates of Glomus intraradices harvested from the same field. In this study, four P-type II ATPase sub-families have been isolated from three AMF species. We show that, contrary to previous predictions, P-type IIC ATPases are present in all basal fungal taxa. Additionally, P-Type IIE ATPases should no longer be considered as exclusive to the Ascomycota and the Basidiomycota, since we also demonstrate their presence in the Zygomycota. Finally, a comparison of homologous sequences encoding P-type IID ATPases showed unexpectedly that indel mutations among coding regions, as well as specific gene duplications occur among AMF individuals within the same field. On the basis of these results we suggest that the diversification of P-Type IIC and E ATPases followed the diversification of the extant fungal phyla with independent events of gene gains and losses. Consistent with recent findings on the human genome, but at a much smaller geographic scale, we provided evidence that structural genomic changes, such as exonic indel mutations and gene duplications are less rare than previously thought and that these also occur within fungal populations.

Dispersion of the RmInt1 group II intron in the Sinorhizobium meliloti genome upon acquisition by conjugative transfer

PubMed Central

Nisa-Martínez, Rafael; Jiménez-Zurdo, José I.; Martínez-Abarca, Francisco; Muñoz-Adelantado, Estefanía; Toro, Nicolás

2007-01-01

RmInt1 is a self-splicing and mobile group II intron initially identified in the bacterium Sinorhizobium meliloti, which encodes a reverse transcriptase–maturase (Intron Encoded Protein, IEP) lacking the C-terminal DNA binding (D) and DNA endonuclease domains (En). RmInt1 invades cognate intronless homing sites (ISRm2011-2) by a mechanism known as retrohoming. This work describes how the RmInt1 intron spreads in the S.meliloti genome upon acquisition by conjugation. This process was revealed by using the wild-type intron RmInt1 and engineered intron-donor constructs based on ribozyme coding sequence (ΔORF)-derivatives with higher homing efficiency than the wild-type intron. The data demonstrate that RmInt1 propagates into the S.meliloti genome primarily by retrohoming with a strand bias related to replication of the chromosome and symbiotic megaplasmids. Moreover, we show that when expressed in trans from a separate plasmid, the IEP is able to mobilize genomic ΔORF ribozymes that afterward displayed wild-type levels of retrohoming. Our results contribute to get further understanding of how group II introns spread into bacterial genomes in nature. PMID:17158161

Dispersion of the RmInt1 group II intron in the Sinorhizobium meliloti genome upon acquisition by conjugative transfer.

PubMed

Nisa-Martínez, Rafael; Jiménez-Zurdo, José I; Martínez-Abarca, Francisco; Muñoz-Adelantado, Estefanía; Toro, Nicolás

2007-01-01

RmInt1 is a self-splicing and mobile group II intron initially identified in the bacterium Sinorhizobium meliloti, which encodes a reverse transcriptase-maturase (Intron Encoded Protein, IEP) lacking the C-terminal DNA binding (D) and DNA endonuclease domains (En). RmInt1 invades cognate intronless homing sites (ISRm2011-2) by a mechanism known as retrohoming. This work describes how the RmInt1 intron spreads in the S.meliloti genome upon acquisition by conjugation. This process was revealed by using the wild-type intron RmInt1 and engineered intron-donor constructs based on ribozyme coding sequence (DeltaORF)-derivatives with higher homing efficiency than the wild-type intron. The data demonstrate that RmInt1 propagates into the S.meliloti genome primarily by retrohoming with a strand bias related to replication of the chromosome and symbiotic megaplasmids. Moreover, we show that when expressed in trans from a separate plasmid, the IEP is able to mobilize genomic DeltaORF ribozymes that afterward displayed wild-type levels of retrohoming. Our results contribute to get further understanding of how group II introns spread into bacterial genomes in nature.

Single mage gene in the chicken genome encodes CMage, a protein with functional similarities to mammalian type II Mage proteins.

PubMed

López-Sánchez, Noelia; González-Fernández, Zaira; Niinobe, Michio; Yoshikawa, Kazuaki; Frade, José María

2007-07-18

In mammals, the type II melanoma antigen (Mage) protein family is constituted by at least 10 closely related members that are expressed in different tissues, including the nervous system. These proteins are believed to regulate cell cycle withdrawal, neuronal differentiation, and apoptosis. However, the analysis of their specific function has been complicated by functional redundancy. In accordance with previous studies in teleosts and Drosophila, we present evidence that only one mage gene exists in genomes from protists, fungi, plants, nematodes, insects, and nonmammalian vertebrates. We have identified the chicken mage gene and cloned the cDNA encoding the chick Mage protein (CMage). CMage shares close homology with the type II Mage protein family, and, as previously shown for the type II Mage proteins Necdin and Mage-G1, it can interact with the transcription factor E2F-1. CMage is expressed in specific regions of the developing nervous system including the retinal ganglion cell layer, the ventral horn of the spinal cord, and the dorsal root ganglia, coinciding with the expression of the neurotrophin receptor p75 (p75(NTR)) in these regions. We show that the intracellular domain of p75(NTR) can interact with both CMage and Necdin, thus preventing the binding of the latter proteins to the transcription factor E2F-1, and facilitating the proapoptotic activity of E2F-1 in N1E-115 differentiating neurons. The presence of a single mage gene in the chicken genome, together with the close functional resemblance between CMage and Necdin, makes this species ideal to further analyze signal transduction through type II Mage proteins.

Large basolateral processes on type II hair cells comprise a novel processing unit in mammalian vestibular organs

PubMed Central

Pujol, Rémy; Pickett, Sarah B.; Nguyen, Tot Bui; Stone, Jennifer S.

2014-01-01

Sensory receptors in the vestibular system (hair cells) encode head movements and drive central motor reflexes that control gaze, body movements, and body orientation. In mammals, type I and II vestibular hair cells are defined by their shape, contacts with vestibular afferent nerves, and membrane conductance. Here, we describe unique morphological features of type II vestibular hair cells in mature rodents (mice and gerbils) and bats. These features are cytoplasmic processes that extend laterally from the hair cell’s base and project under type I hair cells. Closer analysis of adult mouse utricles demonstrated that the basolateral processes of type II hair cells range in shape, size, and branching, with the longest processes extending 3–4 hair cell widths. The hair cell basolateral processes synapse upon vestibular afferent nerves and receive inputs from vestibular efferent nerves. Further, some basolateral processes make physical contacts with the processes of other type II hair cells, forming some sort of network amongst type II hair cells. Basolateral processes are rare in perinatal mice and do not attain their mature form until 3–6 weeks of age. These observations demonstrate that basolateral processes are significant signaling regions of type II vestibular hair cells, and they suggest type II hair cells may directly communicate with each other, which has not been described in vertebrates. PMID:24825750

Large basolateral processes on type II hair cells are novel processing units in mammalian vestibular organs.

PubMed

Pujol, Rémy; Pickett, Sarah B; Nguyen, Tot Bui; Stone, Jennifer S

2014-10-01

Sensory receptors in the vestibular system (hair cells) encode head movements and drive central motor reflexes that control gaze, body movements, and body orientation. In mammals, type I and II vestibular hair cells are defined by their shape, contacts with vestibular afferent nerves, and membrane conductance. Here we describe unique morphological features of type II vestibular hair cells in mature rodents (mice and gerbils) and bats. These features are cytoplasmic processes that extend laterally from the hair cell base and project under type I hair cells. Closer analysis of adult mouse utricles demonstrated that the basolateral processes of type II hair cells vary in shape, size, and branching, with the longest processes extending three to four hair cell widths. The hair cell basolateral processes synapse upon vestibular afferent nerves and receive inputs from vestibular efferent nerves. Furthermore, some basolateral processes make physical contacts with the processes of other type II hair cells, forming some sort of network among type II hair cells. Basolateral processes are rare in perinatal mice and do not attain their mature form until 3-6 weeks of age. These observations demonstrate that basolateral processes are significant signaling regions of type II vestibular hair cells and suggest that type II hair cells may directly communicate with each other, which has not been described in vertebrates. © 2014 Wiley Periodicals, Inc.

A Dentin Sialophosphoprotein Mutation That Partially Disrupts a Splice Acceptor Site Causes Type II Dentin Dysplasia

PubMed Central

Lee, Sook-Kyung; Hu, Jan C.-C.; Lee, Kyung-Eun; Simmer, James P.; Kim, Jung-Wook

2009-01-01

The dentin sialophosphoprotein (DSPP) gene on chromosome 4q21.3 encodes the major noncollagenous protein in tooth dentin. DSPP mutations are the principal cause of dentin dysplasia type II, dentinogenesis imperfecta type II, and dentinogenesis imperfecta type III. We have identified a DSPP splice junction mutation (IVS2-6T>G) in a family with dentin dysplasia type II. The primary dentition is discolored brown with severe attrition. The mildly discolored permanent dentition has thistle-shaped pulp chambers, pulp stones, and eventual pulp obliteration. The mutation is in the sixth nucleotide from the end of intron 2, perfectly segregates with the disease phenotype, and is absent in 200 normal control chromosomes. An in vitro splicing assay shows that pre-mRNA splicing of the mutant allele generates wild-type mRNA and mRNA lacking exon 3 in approximately equal amounts. Skipping exon 3 might interfere with signal peptide cleavage, causing endoplasmic reticulum stress, and also reduce DSPP secretion, leading to haploinsufficiency. PMID:19026876

Supporting cells remove and replace sensory receptor hair cells in a balance organ of adult mice

PubMed Central

Bucks, Stephanie A; Cox, Brandon C; Vlosich, Brittany A; Manning, James P; Nguyen, Tot B; Stone, Jennifer S

2017-01-01

Vestibular hair cells in the inner ear encode head movements and mediate the sense of balance. These cells undergo cell death and replacement (turnover) throughout life in non-mammalian vertebrates. However, there is no definitive evidence that this process occurs in mammals. We used fate-mapping and other methods to demonstrate that utricular type II vestibular hair cells undergo turnover in adult mice under normal conditions. We found that supporting cells phagocytose both type I and II hair cells. Plp1-CreERT2-expressing supporting cells replace type II hair cells. Type I hair cells are not restored by Plp1-CreERT2-expressing supporting cells or by Atoh1-CreERTM-expressing type II hair cells. Destruction of hair cells causes supporting cells to generate 6 times as many type II hair cells compared to normal conditions. These findings expand our understanding of sensorineural plasticity in adult vestibular organs and further elucidate the roles that supporting cells serve during homeostasis and after injury. DOI: http://dx.doi.org/10.7554/eLife.18128.001 PMID:28263708

Generation of Trichoderma atroviride mutants with constitutively activated G protein signaling by using geneticin resistance as selection marker.

PubMed

Gruber, Sabine; Omann, Markus; Rodrìguez, Carolina Escobar; Radebner, Theresa; Zeilinger, Susanne

2012-11-17

Species of the fungal genus Trichoderma are important industrial producers of cellulases and hemicellulases, but also widely used as biocontrol agents (BCAs) in agriculture. In the latter function Trichoderma species stimulate plant growth, induce plant defense and directly antagonize plant pathogenic fungi through their mycoparasitic capabilities. The recent release of the genome sequences of four mycoparasitic Trichoderma species now forms the basis for large-scale genetic manipulations of these important BCAs. Thus far, only a limited number of dominant selection markers, including Hygromycin B resistance (hph) and the acetamidase-encoding amdS gene, have been available for transformation of Trichoderma spp. For more extensive functional genomics studies the utilization of additional dominant markers will be essential. We established the Escherichia coli neomycin phosphotransferase II-encoding nptII gene as a novel selectable marker for the transformation of Trichoderma atroviride conferring geneticin resistance. The nptII marker cassette was stably integrated into the fungal genome and transformants exhibited unaltered phenotypes compared to the wild-type. Co-transformation of T. atroviride with nptII and a constitutively activated version of the Gα subunit-encoding tga3 gene (tga3Q207L) resulted in a high number of mitotically stable, geneticin-resistant transformants. Further analyses revealed a co-transformation frequency of 68% with 15 transformants having additionally integrated tga3Q207L into their genome. Constitutive activation of the Tga3-mediated signaling pathway resulted in increased vegetative growth and an enhanced ability to antagonize plant pathogenic host fungi. The neomycin phosphotransferase II-encoding nptII gene from Escherichia coli proved to be a valuable tool for conferring geneticin resistance to the filamentous fungus T. atroviride thereby contributing to an enhanced genetic tractability of these important BCAs.

Ketide Synthase (KS) Domain Prediction and Analysis of Iterative Type II PKS Gene in Marine Sponge-Associated Actinobacteria Producing Biosurfactants and Antimicrobial Agents

PubMed Central

Selvin, Joseph; Sathiyanarayanan, Ganesan; Lipton, Anuj N.; Al-Dhabi, Naif Abdullah; Valan Arasu, Mariadhas; Kiran, George S.

2016-01-01

The important biological macromolecules, such as lipopeptide and glycolipid biosurfactant producing marine actinobacteria were analyzed and their potential linkage between type II polyketide synthase (PKS) genes was explored. A unique feature of type II PKS genes is their high amino acid (AA) sequence homology and conserved gene organization. These enzymes mediate the biosynthesis of polyketide natural products with enormous structural complexity and chemical nature by combinatorial use of various domains. Therefore, deciphering the order of AA sequence encoded by PKS domains tailored the chemical structure of polyketide analogs still remains a great challenge. The present work deals with an in vitro and in silico analysis of PKS type II genes from five actinobacterial species to correlate KS domain architecture and structural features. Our present analysis reveals the unique protein domain organization of iterative type II PKS and KS domain of marine actinobacteria. The findings of this study would have implications in metabolic pathway reconstruction and design of semi-synthetic genomes to achieve rational design of novel natural products. PMID:26903957

Specific Molecular Signatures for Type II Crustins in Penaeid Shrimp Uncovered by the Identification of Crustin-Like Antimicrobial Peptides in Litopenaeus vannamei

PubMed Central

Barreto, Cairé; Coelho, Jaqueline da Rosa; Yuan, Jianbo; Xiang, Jianhai; Perazzolo, Luciane Maria

2018-01-01

Crustins form a large family of antimicrobial peptides (AMPs) in crustaceans composed of four sub-groups (Types I-IV). Type II crustins (Type IIa or “Crustins” and Type IIb or “Crustin-like”) possess a typical hydrophobic N-terminal region and are by far the most representative sub-group found in penaeid shrimp. To gain insight into the molecular diversity of Type II crustins in penaeids, we identified and characterized a Type IIb crustin in Litopenaeus vannamei (Crustin-like Lv) and compared Type II crustins at both molecular and transcriptional levels. Although L. vannamei Type II crustins (Crustin Lv and Crustin-like Lv) are encoded by separate genes, they showed a similar tissue distribution (hemocytes and gills) and transcriptional response to the shrimp pathogens Vibrio harveyi and White spot syndrome virus (WSSV). As Crustin Lv, Crustin-like Lv transcripts were found to be present early in development, suggesting a maternal contribution to shrimp progeny. Altogether, our in silico and transcriptional data allowed to conclude that (1) each sub-type displays a specific amino acid signature at the C-terminal end holding both the cysteine-rich region and the whey acidic protein (WAP) domain, and that (2) shrimp Type II crustins evolved from a common ancestral gene that conserved a similar pattern of transcriptional regulation. PMID:29337853

Identification of a melanosomal membrane protein encoded by the pink-eyed dilution (type II oculocutaneous albinism) gene.

PubMed Central

Rosemblat, S; Durham-Pierre, D; Gardner, J M; Nakatsu, Y; Brilliant, M H; Orlow, S J

1994-01-01

The pink-eyed dilution (p) locus in the mouse is critical to melanogenesis; mutations in the homologous locus in humans, P, are a cause of type II oculocutaneous albinism. Although a cDNA encoded by the p gene has recently been identified, nothing is known about the protein product of this gene. To characterize the protein encoded by the p gene, we performed immunoblot analysis of extracts of melanocytes cultured from wild-type mice with an antiserum from rabbits immunized with a peptide corresponding to amino acids 285-298 of the predicted protein product of the murine p gene. This antiserum recognized a 110-kDa protein. The protein was absent from extracts of melanocytes cultured from mice with two mutations (pcp and p) in which transcripts of the p gene are absent or greatly reduced. Introduction of the cDNA for the p gene into pcp melanocytes by electroporation resulted in expression of the 3.3-kb mRNA and the 110-kDa protein. Upon subcellular fractionation of cultured melanocytes, the 110-kDa protein was found to be present in melanosomes but absent from the vesicular fraction; phase separation performed with the nonionic detergent Triton X-114 confirmed the predicted hydrophobic nature of the protein. These results demonstrate that the p gene encodes a 110-kDa integral melanosomal membrane protein and establish a framework by which mutations at this locus, which diminish pigmentation, can be analyzed at the cellular and biochemical levels. Images PMID:7991586

The zntA gene of Escherichia coli encodes a Zn(II)-translocating P-type ATPase

PubMed Central

Rensing, Christopher; Mitra, Bharati; Rosen, Barry P.

1997-01-01

The first Zn(II)-translocating P-type ATPase has been identified as the product of o732, a potential gene identified in the sequencing of the Escherichia coli genome. This gene, termed zntA, was disrupted by insertion of a kanamycin gene through homologous recombination. The mutant strain exhibited hypersensitivity to zinc and cadmium salts but not salts of other metals, suggesting a role in zinc homeostasis in E. coli. Everted membrane vesicles from a wild-type strain accumulated 65Zn(II) and 109Cd(II) by using ATP as an energy source. Transport was sensitive to vanadate, an inhibitor of P-type ATPases. Membrane vesicles from the zntA∷kan strain did not accumulate those metal ions. Both the sensitive phenotype and transport defect of the mutant were complemented by expression of zntA on a plasmid. PMID:9405611

Interferon-induced TRAIL-independent cell death in DNase II-/- embryos.

PubMed

Kitahara, Yusuke; Kawane, Kohki; Nagata, Shigekazu

2010-09-01

The chromosomal DNA of apoptotic cells and the nuclear DNA expelled from erythroid precursors is cleaved by DNase II in lysosomes after the cells or nuclei are engulfed by macrophages. DNase II(-/-) embryos suffer from lethal anemia due to IFN-beta produced in the macrophages carrying undigested DNA. Here, we show that Type I IFN induced a caspase-dependent cell death in human epithelial cells that were transformed to express a high level of IFN type I receptor. During this death process, a set of genes was strongly activated, one of which encoded TRAIL, a death ligand. A high level of TRAIL mRNA was also found in the fetal liver of the lethally anemic DNase II(-/-) embryos, and a lack of IFN type I receptor in the DNase II(-/-) IFN-IR(-/-) embryos blocked the expression of TRAIL mRNA. However, a null mutation in TRAIL did not rescue the lethal anemia of the DNase II(-/-) embryos, indicating that TRAIL is dispensable for inducing the apoptosis of erythroid cells in DNase II(-/-) embryos, and therefore, that there is a TRAIL-independent mechanism for the IFN-induced apoptosis.

Multi-functional acetyl-CoA carboxylase from Brassica napus is encoded by a multi-gene family: indication for plastidic localization of at least one isoform.

PubMed

Schulte, W; Töpfer, R; Stracke, R; Schell, J; Martini, N

1997-04-01

Three genes coding for different multifunctional acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) isoenzymes from Brassica napus were isolated and divided into two major classes according to structural features in their 5' regions: class I comprises two genes with an additional coding exon of approximately 300 bp at the 5' end, and class II is represented by one gene carrying an intron of 586 bp in its 5' untranslated region. Fusion of the peptide sequence encoded by the additional first exon of a class I ACCase gene to the jellyfish Aequorea victoria green fluorescent protein (GFP) and transient expression in tobacco protoplasts targeted GFP to the chloroplasts. In contrast to the deduced primary structure of the biotin carboxylase domain encoded by the class I gene, the corresponding amino acid sequence of the class II ACCase shows higher identity with that of the Arabidopsis ACCase, both lacking a transit peptide. The Arabidopsis ACCase has been proposed to be a cytosolic isoenzyme. These observations indicate that the two classes of ACCase genes encode plastidic and cytosolic isoforms of multi-functional, eukaryotic type, respectively, and that B. napus contains at least one multi-functional ACCase besides the multi-subunit, prokaryotic type located in plastids. Southern blot analysis of genomic DNA from B. napus, Brassica rapa, and Brassica oleracea, the ancestors of amphidiploid rapeseed, using a fragment of a multi-functional ACCase gene as a probe revealed that ACCase is encoded by a multi-gene family of at least five members.

Dysmorphic choroid plexuses and hydrocephalus associated with increased nuchal translucency: early ultrasound markers of de novo thanatophoric dysplasia type II with cloverleaf skull (Kleeblattschaedel).

PubMed

Tonni, Gabriele; Palmisano, Marcella; Ginocchi, Vladimiro; Ventura, Alessandro; Baldi, Maurizia; Baffico, Ave Maria

2014-11-01

Prenatal diagnosis of thanatophoric dysplasia (TD) type II presenting in the first trimester with increased nuchal translucency (NT) and cloverleaf skull (Kleeblattschaedel) have been scantly reported in the medical record. Abnormal choroid plexus has been seen in association with fetal anomalies. Here we described a case of increased NT associated with indented choroid plexuses, early onset hydrocephalus and cloverleaf skull in a fetus subsequently diagnosed at early second trimester to carry a de novo mutation encoding for TD type II. The findings of dysmorphic choroid plexus, early onset hydrocephalus and cloverleaf skull at first trimester scan may be early, useful ultrasound markers of TD type II. Molecular analysis to control for possible overlapping syndromes were performed and resulted negative. Postmortem X-ray and 3D-CT scan confirmed the cloverleaf skull, narrow thorax, straight femur with rhizomelic shortening of the limbs and the presence of a communicating hydrocephalus. © 2014 Japanese Teratology Society.

Draft genome sequence of Actinotignum schaalii DSM 15541T: Genetic insights into the lifestyle, cell fitness and virulence.

PubMed

Yassin, Atteyet F; Langenberg, Stefan; Huntemann, Marcel; Clum, Alicia; Pillay, Manoj; Palaniappan, Krishnaveni; Varghese, Neha; Mikhailova, Natalia; Mukherjee, Supratim; Reddy, T B K; Daum, Chris; Shapiro, Nicole; Ivanova, Natalia; Woyke, Tanja; Kyrpides, Nikos C

2017-01-01

The permanent draft genome sequence of Actinotignum schaalii DSM 15541T is presented. The annotated genome includes 2,130,987 bp, with 1777 protein-coding and 58 rRNA-coding genes. Genome sequence analysis revealed absence of genes encoding for: components of the PTS systems, enzymes of the TCA cycle, glyoxylate shunt and gluconeogensis. Genomic data revealed that A. schaalii is able to oxidize carbohydrates via glycolysis, the nonoxidative pentose phosphate and the Entner-Doudoroff pathways. Besides, the genome harbors genes encoding for enzymes involved in the conversion of pyruvate to lactate, acetate and ethanol, which are found to be the end products of carbohydrate fermentation. The genome contained the gene encoding Type I fatty acid synthase required for de novo FAS biosynthesis. The plsY and plsX genes encoding the acyltransferases necessary for phosphatidic acid biosynthesis were absent from the genome. The genome harbors genes encoding enzymes responsible for isoprene biosynthesis via the mevalonate (MVA) pathway. Genes encoding enzymes that confer resistance to reactive oxygen species (ROS) were identified. In addition, A. schaalii harbors genes that protect the genome against viral infections. These include restriction-modification (RM) systems, type II toxin-antitoxin (TA), CRISPR-Cas and abortive infection system. A. schaalii genome also encodes several virulence factors that contribute to adhesion and internalization of this pathogen such as the tad genes encoding proteins required for pili assembly, the nanI gene encoding exo-alpha-sialidase, genes encoding heat shock proteins and genes encoding type VII secretion system. These features are consistent with anaerobic and pathogenic lifestyles. Finally, resistance to ciprofloxacin occurs by mutation in chromosomal genes that encode the subunits of DNA-gyrase (GyrA) and topisomerase IV (ParC) enzymes, while resistant to metronidazole was due to the frxA gene, which encodes NADPH-flavin oxidoreductase.

A genetic marker of the ACKR1 gene is present in patients with Type II congenital smell loss who have type I hyposmia and hypogeusia

PubMed Central

Stateman, William A.; Knöppel, Alexandra B.; Flegel, Willy A.; Henkin, Robert I.

2015-01-01

PURPOSE Our previous study of Type II congenital smell loss patients revealed a statistically significant lower prevalence of an FY (ACKR1, formerly DARC) haplotype compared to controls. The present study correlates this genetic feature with subgroups of patients defined by specific smell and taste functions. METHODS Smell and taste function measurements were performed by use of olfactometry and gustometry to define degree of abnormality of smell and taste function. Smell loss was classified as anosmia or hyposmia (types I, II or III). Taste loss was similarly classified as ageusia or hypogeusia (types I, II or III). Based upon these results patient erythrocyte antigen expression frequencies were categorized by smell and taste loss with results compared between patients within the Type II group and published controls. RESULTS Comparison of antigen expression frequencies revealed a statistically significant decrease in incidence of an Fyb haplotype only among patients with type I hyposmia and any form of taste loss (hypogeusia). In all other patient groups erythrocyte antigens were expressed at normal frequencies. CONCLUSIONS Data suggest that Type II congenital smell loss patients who exhibit both type I hyposmia and hypogeusia are genetically distinct from all other patients with Type II congenital smell loss. This distinction is based on decreased Fyb expression which correlated with abnormalities in two sensory modalities (hyposmia type I and hypogeusia). Only patients with these two specific sensory abnormalities expressed the Fyb antigen (encoded by the ACKR1 gene on the long arm of chromosome 1) at frequencies different from controls. PMID:27968956

Spectroscopy of Cu(II)-PcoC and the multicopper oxidase function of PcoA, two essential components of Escherichia coli pco copper resistance operon.

PubMed

Huffman, David L; Huyett, Jennifer; Outten, F Wayne; Doan, Peter E; Finney, Lydia A; Hoffman, Brian M; O'Halloran, Thomas V

2002-08-06

The plasmid-encoded pco copper resistance operon in Escherichia coli consists of seven genes that are expressed from two pco promoters in response to elevated copper; however, little is known about how they mediate resistance to excess environmental copper. Two of the genes encode the soluble periplasmic proteins PcoA and PcoC. We show here that inactivation of PcoC, and PcoA to a lesser extent, causes cells to become more sensitive to copper than wild-type nonresistant strains, consistent with a tightly coupled detoxification pathway. Periplasmic extracts show copper-inducible oxidase activity, attributed to the multicopper oxidase function of PcoA. PcoC, a much smaller protein than PcoA, binds one Cu(II) and exhibits a weak electronic transition characteristic of a type II copper center. ENDOR and ESEEM spectroscopy of Cu(II)-PcoC and the (15)N- and Met-CD(3)-labeled samples are consistent with a tetragonal ligand environment of three nitrogens and one aqua ligand "in the plane". A weakly associated S-Met and aqua are likely axial ligands. At least one N is a histidine and is likely trans to the in-plane aqua ligand. The copper chemistry of PcoC and the oxidase function of PcoA are consistent with the emerging picture of the chromosomally encoded copper homeostasis apparatus in the E. coli cell envelope [Outten, F. W., Huffman, D. L., Hale, J. A., and O'Halloran, T. V. (2001) J. Biol. Chem. 276, 30670-30677]. We propose a model for the plasmid system in which Cu(I)-PcoC functions in this copper efflux pathway as a periplasmic copper binding protein that docks with the multiple repeats of Met-rich domains in PcoA to effect oxidation of Cu(I) to the less toxic Cu(II) form. The solvent accessibility of the Cu(II) in PcoC may allow for metal transfer to other plasmid and chromosomal factors and thus facilitate removal of Cu(II) from the cell envelope.

Keeping the Wolves at Bay: Antitoxins of Prokaryotic Type II Toxin-Antitoxin Systems.

PubMed

Chan, Wai Ting; Espinosa, Manuel; Yeo, Chew Chieng

2016-01-01

In their initial stages of discovery, prokaryotic toxin-antitoxin (TA) systems were confined to bacterial plasmids where they function to mediate the maintenance and stability of usually low- to medium-copy number plasmids through the post-segregational killing of any plasmid-free daughter cells that developed. Their eventual discovery as nearly ubiquitous and repetitive elements in bacterial chromosomes led to a wealth of knowledge and scientific debate as to their diversity and functionality in the prokaryotic lifestyle. Currently categorized into six different types designated types I-VI, type II TA systems are the best characterized. These generally comprised of two genes encoding a proteic toxin and its corresponding proteic antitoxin, respectively. Under normal growth conditions, the stable toxin is prevented from exerting its lethal effect through tight binding with the less stable antitoxin partner, forming a non-lethal TA protein complex. Besides binding with its cognate toxin, the antitoxin also plays a role in regulating the expression of the type II TA operon by binding to the operator site, thereby repressing transcription from the TA promoter. In most cases, full repression is observed in the presence of the TA complex as binding of the toxin enhances the DNA binding capability of the antitoxin. TA systems have been implicated in a gamut of prokaryotic cellular functions such as being mediators of programmed cell death as well as persistence or dormancy, biofilm formation, as defensive weapons against bacteriophage infections and as virulence factors in pathogenic bacteria. It is thus apparent that these antitoxins, as DNA-binding proteins, play an essential role in modulating the prokaryotic lifestyle whilst at the same time preventing the lethal action of the toxins under normal growth conditions, i.e., keeping the proverbial wolves at bay. In this review, we will cover the diversity and characteristics of various type II TA antitoxins. We shall also look into some interesting deviations from the canonical type II TA systems such as tripartite TA systems where the regulatory role is played by a third party protein and not the antitoxin, and a unique TA system encoding a single protein with both toxin as well as antitoxin domains.

Keeping the Wolves at Bay: Antitoxins of Prokaryotic Type II Toxin-Antitoxin Systems

PubMed Central

Chan, Wai Ting; Espinosa, Manuel; Yeo, Chew Chieng

2016-01-01

In their initial stages of discovery, prokaryotic toxin-antitoxin (TA) systems were confined to bacterial plasmids where they function to mediate the maintenance and stability of usually low- to medium-copy number plasmids through the post-segregational killing of any plasmid-free daughter cells that developed. Their eventual discovery as nearly ubiquitous and repetitive elements in bacterial chromosomes led to a wealth of knowledge and scientific debate as to their diversity and functionality in the prokaryotic lifestyle. Currently categorized into six different types designated types I–VI, type II TA systems are the best characterized. These generally comprised of two genes encoding a proteic toxin and its corresponding proteic antitoxin, respectively. Under normal growth conditions, the stable toxin is prevented from exerting its lethal effect through tight binding with the less stable antitoxin partner, forming a non-lethal TA protein complex. Besides binding with its cognate toxin, the antitoxin also plays a role in regulating the expression of the type II TA operon by binding to the operator site, thereby repressing transcription from the TA promoter. In most cases, full repression is observed in the presence of the TA complex as binding of the toxin enhances the DNA binding capability of the antitoxin. TA systems have been implicated in a gamut of prokaryotic cellular functions such as being mediators of programmed cell death as well as persistence or dormancy, biofilm formation, as defensive weapons against bacteriophage infections and as virulence factors in pathogenic bacteria. It is thus apparent that these antitoxins, as DNA-binding proteins, play an essential role in modulating the prokaryotic lifestyle whilst at the same time preventing the lethal action of the toxins under normal growth conditions, i.e., keeping the proverbial wolves at bay. In this review, we will cover the diversity and characteristics of various type II TA antitoxins. We shall also look into some interesting deviations from the canonical type II TA systems such as tripartite TA systems where the regulatory role is played by a third party protein and not the antitoxin, and a unique TA system encoding a single protein with both toxin as well as antitoxin domains. PMID:27047942

Myxobacterium-Produced Antibiotic TA (Myxovirescin) Inhibits Type II Signal Peptidase

PubMed Central

Xiao, Yao; Gerth, Klaus; Müller, Rolf

2012-01-01

Antibiotic TA is a macrocyclic secondary metabolite produced by myxobacteria that has broad-spectrum bactericidal activity. The structure of TA is unique, and its molecular target is unknown. Here, we sought to elucidate TA's mode of action (MOA) through two parallel genetic approaches. First, chromosomal Escherichia coli TA-resistant mutants were isolated. One mutant that showed specific resistance toward TA was mapped and resulted from an IS4 insertion in the lpp gene, which encodes an abundant outer membrane (Braun's) lipoprotein. In a second approach, the comprehensive E. coli ASKA plasmid library was screened for overexpressing clones that conferred TAr. This effort resulted in the isolation of the lspA gene, which encodes the type II signal peptidase that cleaves signal sequences from prolipoproteins. In whole cells, TA was shown to inhibit Lpp prolipoprotein processing, similar to the known LspA inhibitor globomycin. Based on genetic evidence and prior globomycin studies, a block in Lpp expression or prevention of Lpp covalent cell wall attachment confers TAr by alleviating a toxic buildup of mislocalized pro-Lpp. Taken together, these data argue that LspA is the molecular target of TA. Strikingly, the giant ta biosynthetic gene cluster encodes two lspA paralogs that we hypothesize play a role in producer strain resistance. PMID:22232277

Type II restriction modification system methylation subunit of Alicyclobacillus acidocaldarius

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

Lee, Brady D.; Newby, Deborah T.; Lacey, Jeffrey A.

2018-02-13

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods for modulating or altering recombination inside or outside of a cell using isolated and/or purified polypeptides and/or nucleic acid sequences from Alicyclobacillus acidocaldarius.

Type II restriction-modification system methylation subunit of Alicyclobacillus acidocaldarius

DOEpatents

Lee, Brady D; Newby, Deborah T; Lacey, Jeffrey A; Thompson, David N; Thompson, Vicki S; Apel, William A; Roberto, Francisco F; Reed, David W

2013-10-29

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods for modulating or altering recombination inside or outside of a cell using isolated and/or purified polypeptides and/or nucleic acid sequences from Alicyclobacillus acidocaldarius.

Type II restriction-modification system methylation subunit of Alicyclobacillus acidocaldarius

DOEpatents

Lee, Brady D; Newby, Deborah T; Lacey, Jeffrey A; Thompson, David N; Thompson, Vicki S; Apel, William A; Roberto, Francisco F; Reed, David W

2015-05-12

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods for modulating or altering recombination inside or outside of a cell using isolated and/or purified polypeptides and/or nucleic acid sequences from Alicyclobacillus acidocaldarius.

Type II restriction modification system methylation subunit of Alicyclobacillus acidocaldarius

DOEpatents

Lee, Brady D.; Newby, Deborah T.; Lacey, Jeffrey A.; Thompson, David N.; Thompson, Vicki S.; Apel, William A.; Roberto, Francisco F.; Reed, David W.

2017-02-14

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods for modulating or altering recombination inside or outside of a cell using isolated and/or purified polypeptides and/or nucleic acid sequences from Alicyclobacillus acidocaldarius.

Localization of a bacterial group II intron-encoded protein in eukaryotic nuclear splicing-related cell compartments.

PubMed

Nisa-Martínez, Rafael; Laporte, Philippe; Jiménez-Zurdo, José Ignacio; Frugier, Florian; Crespi, Martin; Toro, Nicolás

2013-01-01

Some bacterial group II introns are widely used for genetic engineering in bacteria, because they can be reprogrammed to insert into the desired DNA target sites. There is considerable interest in developing this group II intron gene targeting technology for use in eukaryotes, but nuclear genomes present several obstacles to the use of this approach. The nuclear genomes of eukaryotes do not contain group II introns, but these introns are thought to have been the progenitors of nuclear spliceosomal introns. We investigated the expression and subcellular localization of the bacterial RmInt1 group II intron-encoded protein (IEP) in Arabidopsis thaliana protoplasts. Following the expression of translational fusions of the wild-type protein and several mutant variants with EGFP, the full-length IEP was found exclusively in the nucleolus, whereas the maturase domain alone targeted EGFP to nuclear speckles. The distribution of the bacterial RmInt1 IEP in plant cell protoplasts suggests that the compartmentalization of eukaryotic cells into nucleus and cytoplasm does not prevent group II introns from invading the host genome. Furthermore, the trafficking of the IEP between the nucleolus and the speckles upon maturase inactivation is consistent with the hypothesis that the spliceosomal machinery evolved from group II introns.

Localization of a Bacterial Group II Intron-Encoded Protein in Eukaryotic Nuclear Splicing-Related Cell Compartments

PubMed Central

Nisa-Martínez, Rafael; Laporte, Philippe; Jiménez-Zurdo, José Ignacio; Frugier, Florian; Crespi, Martin; Toro, Nicolás

2013-01-01

Some bacterial group II introns are widely used for genetic engineering in bacteria, because they can be reprogrammed to insert into the desired DNA target sites. There is considerable interest in developing this group II intron gene targeting technology for use in eukaryotes, but nuclear genomes present several obstacles to the use of this approach. The nuclear genomes of eukaryotes do not contain group II introns, but these introns are thought to have been the progenitors of nuclear spliceosomal introns. We investigated the expression and subcellular localization of the bacterial RmInt1 group II intron-encoded protein (IEP) in Arabidopsis thaliana protoplasts. Following the expression of translational fusions of the wild-type protein and several mutant variants with EGFP, the full-length IEP was found exclusively in the nucleolus, whereas the maturase domain alone targeted EGFP to nuclear speckles. The distribution of the bacterial RmInt1 IEP in plant cell protoplasts suggests that the compartmentalization of eukaryotic cells into nucleus and cytoplasm does not prevent group II introns from invading the host genome. Furthermore, the trafficking of the IEP between the nucleolus and the speckles upon maturase inactivation is consistent with the hypothesis that the spliceosomal machinery evolved from group II introns. PMID:24391881

Three copies of a single protein II-encoding sequence in the genome of Neisseria gonorrhoeae JS3: evidence for gene conversion and gene duplication.

PubMed

van der Ley, P

1988-11-01

Gonococci express a family of related outer membrane proteins designated protein II (P.II). These surface proteins are subject to both phase variation and antigenic variation. The P.II gene repertoire of Neisseria gonorrhoeae strain JS3 was found to consist of at least ten genes, eight of which were cloned. Sequence analysis and DNA hybridization studies revealed that one particular P.II-encoding sequence is present in three distinct, but almost identical, copies in the JS3 genome. These genes encode the P.II protein that was previously identified as P.IIc. Comparison of their sequences shows that the multiple copies of this P.IIc-encoding gene might have been generated by both gene conversion and gene duplication.

Flexible Manufacturing System Handbook. Volume II. Description of the Technology

DTIC Science & Technology

1983-02-01

hubs, or wheels with considerable 4 FM5 Handbook, Volume II milling, drilling and/or tapping, are usually candidates for inclusion in a prismatic...0.06 inch) to transfer pallets to a machine or unload station. Wheel encoders can be used as less precise feedback for the drive system and its...must be used to control pallet transfer. The Cincinnati Milacron Variable Mission System uses this type of MHS, specifically the Eaton-Kenway Robo

Single cell analysis of voltage-gated potassium channels that determines neuronal types of rat hypothalamic paraventricular nucleus neurons.

PubMed

Lee, S K; Lee, S; Shin, S Y; Ryu, P D; Lee, S Y

2012-03-15

The hypothalamic paraventricular nucleus (PVN), a site for the integration of both the neuroendocrine and autonomic systems, has heterogeneous cell composition. These neurons are classified into type I and type II neurons based on their electrophysiological properties. In the present study, we investigated the molecular identification of voltage-gated K+ (Kv) channels, which determines a distinctive characteristic of type I PVN neurons, by means of single-cell reverse transcription-polymerase chain reaction (RT-PCR) along with slice patch clamp recordings. In order to determine the mRNA expression profiles, firstly, the PVN neurons of male rats were classified into type I and type II neurons, and then, single-cell RT-PCR and single-cell real-time RT-PCR analysis were performed using the identical cell. The single-cell RT-PCR analysis revealed that Kv1.2, Kv1.3, Kv1.4, Kv4.1, Kv4.2, and Kv4.3 were expressed both in type I and in type II neurons, and several Kv channels were co-expressed in a single PVN neuron. However, we found that the expression densities of Kv4.2 and Kv4.3 were significantly higher in type I neurons than in type II neurons. Taken together, several Kv channels encoding A-type K+ currents are present both in type I and in type II neurons, and among those, Kv4.2 and Kv4.3 are the major Kv subunits responsible for determining the distinct electrophysiological properties. Thus these 2 Kv subunits may play important roles in determining PVN cell types and regulating PVN neuronal excitability. This study further provides key molecular mechanisms for differentiating type I and type II PVN neurons. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

β-Lactamase Genes of the Penicillin-Susceptible Bacillus anthracis Sterne Strain

PubMed Central

Chen, Yahua; Succi, Janice; Tenover, Fred C.; Koehler, Theresa M.

2003-01-01

Susceptibility to penicillin and other β-lactam-containing compounds is a common trait of Bacillus anthracis. β-lactam agents, particularly penicillin, have been used worldwide to treat anthrax in humans. Nonetheless, surveys of clinical and soil-derived strains reveal penicillin G resistance in 2 to 16% of isolates tested. Bacterial resistance to β-lactam agents is often mediated by production of one or more types of β-lactamases that hydrolyze the β-lactam ring, inactivating the antimicrobial agent. Here, we report the presence of two β-lactamase (bla) genes in the penicillin-susceptible Sterne strain of B. anthracis. We identified bla1 by functional cloning with Escherichia coli. bla1 is a 927-nucleotide (nt) gene predicted to encode a protein with 93.8% identity to the type I β-lactamase gene of Bacillus cereus. A second gene, bla2, was identified by searching the unfinished B. anthracis chromosome sequence database of The Institute for Genome Research for open reading frames (ORFs) predicted to encode β-lactamases. We found a partial ORF predicted to encode a protein with significant similarity to the carboxy-terminal end of the type II β-lactamase of B. cereus. DNA adjacent to the 5′ end of the partial ORF was cloned using inverse PCR. bla2 is a 768-nt gene predicted to encode a protein with 92% identity to the B. cereus type II enzyme. The bla1 and bla2 genes confer ampicillin resistance to E. coli and Bacillus subtilis when cloned individually in these species. The MICs of various antimicrobial agents for the E. coli clones indicate that the two β-lactamase genes confer different susceptibility profiles to E. coli; bla1 is a penicillinase, while bla2 appears to be a cephalosporinase. The β-galactosidase activities of B. cereus group species harboring bla promoter-lacZ transcriptional fusions indicate that bla1 is poorly transcribed in B. anthracis, B. cereus, and B. thuringiensis. The bla2 gene is strongly expressed in B. cereus and B. thuringiensis and weakly expressed in B. anthracis. Taken together, these data indicate that the bla1 and bla2 genes of the B. anthracis Sterne strain encode functional β-lactamases of different types, but gene expression is usually not sufficient to confer resistance to β-lactam agents. PMID:12533457

Dual specificity of activin type II receptor ActRIIb in dorso-ventral patterning during zebrafish embryogenesis.

PubMed

Nagaso, H; Suzuki, A; Tada, M; Ueno, N

1999-04-01

Members of the transforming growth factor-beta (TGF-beta) superfamily are thought to regulate specification of a variety of tissue types in early embryogenesis. These effects are mediated through a cell surface receptor complex, consisting of two classes of ser/thr kinase receptor, type I and type II. In the present study, cDNA encoding zebrafish activin type II receptors, ActRIIa and ActRIIb was cloned and characterized. Overexpression of ActRIIb in zebrafish embryos caused dorsalization of embryos, as observed in activin-overexpressing embryos. However, in blastula stage embryos, ActRIIb induced formation of both dorsal and ventro-lateral mesoderm. It has been suggested that these inducing signals from ActRIIb are mediated through each specific type I receptor, TARAM-A and BMPRIA, depending on activin and bone morphogenetic protein (BMP), respectively. In addition, it was shown that a kinase-deleted form of ActRIIb (dnActRIIb) suppressed both activin- and BMP-like signaling pathways. These results suggest that ActRIIb at least has dual roles in both activin and BMP signaling pathways during zebrafish embryogenesis.

Ankle joint movements are encoded by both cutaneous and muscle afferents in humans.

PubMed

Aimonetti, Jean-Marc; Roll, Jean-Pierre; Hospod, Valérie; Ribot-Ciscar, Edith

2012-08-01

We analyzed the cutaneous encoding of two-dimensional movements by investigating the coding of movement velocity for differently oriented straight-line movements and the coding of complex trajectories describing cursive letters. The cutaneous feedback was then compared with that of the underlying muscle afferents previously recorded during the same "writing-like" movements. The unitary activity of 43 type II cutaneous afferents was recorded in the common peroneal nerve in healthy subjects during imposed ankle movements. These movements consisted first of ramp-and-hold movements imposed at two different and close velocities in seven directions and secondly of "writing-like" movements. In both cases, the responses were analyzed using the neuronal population vector model. The results show that movement velocity encoding depended on the direction of the ongoing movement. Discriminating between two velocities therefore involved processing the activity of afferent populations located in the various skin areas surrounding the moving joint, as shown by the statistically significant difference observed in the amplitude of the sum vectors. Secondly, "writing-like" movements induced cutaneous neuronal patterns of activity, which were reproducible and specific to each trajectory. Lastly, the "cutaneous neuronal trajectories," built by adding the sum vectors tip-to-tail, nearly matched both the movement trajectories and the "muscle neuronal trajectories," built from previously recorded muscle afferents. It was concluded that type II cutaneous and the underlying muscle afferents show similar encoding properties of two-dimensional movement parameters. This similarity is discussed in relation to a central gating process that would for instance increase the gain of cutaneous inputs when muscle information is altered by the fusimotor drive.

Current Understanding of Usher Syndrome Type II

PubMed Central

Yang, Jun; Wang, Le; Song, Hongman; Sokolov, Maxim

2012-01-01

Usher syndrome is the most common deafness-blindness caused by genetic mutations. To date, three genes have been identified underlying the most prevalent form of Usher syndrome, the type II form (USH2). The proteins encoded by these genes are demonstrated to form a complex in vivo. This complex is localized mainly at the periciliary membrane complex in photoreceptors and the ankle-link of the stereocilia in hair cells. Many proteins have been found to interact with USH2 proteins in vitro, suggesting that they are potential additional components of this USH2 complex and that the genes encoding these proteins may be the candidate USH2 genes. However, further investigations are critical to establish their existence in the USH2 complex in vivo. Based on the predicted functional domains in USH2 proteins, their cellular localizations in photoreceptors and hair cells, the observed phenotypes in USH2 mutant mice, and the known knowledge about diseases similar to USH2, putative biological functions of the USH2 complex have been proposed. Finally, therapeutic approaches for this group of diseases are now being actively explored. PMID:22201796

Determinants of affinity and mode of DNA binding at the carboxy terminus of the bacteriophage SPO1-encoded type II DNA-binding protein, TF1.

PubMed

Andera, L; Geiduschek, E P

1994-03-01

The role of the carboxy-terminal amino acids of the bacteriophage SPO1-encoded type II DNA-binding protein, TF1, in DNA binding was analyzed. Chain-terminating mutations truncating the normally 99-amino-acid TF1 at amino acids 96, 97, and 98 were constructed, as were missense mutations substituting cysteine, arginine, and serine for phenylalanine at amino acid 97 and tryptophan for lysine at amino acid 99. The binding of the resulting proteins to a synthetic 44-bp binding site in 5-(hydroxymethyl)uracil DNA, to binding sites in larger SPO1 [5-(hydroxymethyl)uracil-containing] DNA fragments, and to thymine-containing homologous DNA was analyzed by gel retardation and also by DNase I and hydroxy radical footprinting. We conclude that the C tail up to and including phenylalanine at amino acid 97 is essential for DNA binding and that the two C-terminal amino acids, 98 and 99, are involved in protein-protein interactions between TF1 dimers bound to DNA.

Genomes to natural products PRediction Informatics for Secondary Metabolomes (PRISM)

PubMed Central

Skinnider, Michael A.; Dejong, Chris A.; Rees, Philip N.; Johnston, Chad W.; Li, Haoxin; Webster, Andrew L. H.; Wyatt, Morgan A.; Magarvey, Nathan A.

2015-01-01

Microbial natural products are an invaluable source of evolved bioactive small molecules and pharmaceutical agents. Next-generation and metagenomic sequencing indicates untapped genomic potential, yet high rediscovery rates of known metabolites increasingly frustrate conventional natural product screening programs. New methods to connect biosynthetic gene clusters to novel chemical scaffolds are therefore critical to enable the targeted discovery of genetically encoded natural products. Here, we present PRISM, a computational resource for the identification of biosynthetic gene clusters, prediction of genetically encoded nonribosomal peptides and type I and II polyketides, and bio- and cheminformatic dereplication of known natural products. PRISM implements novel algorithms which render it uniquely capable of predicting type II polyketides, deoxygenated sugars, and starter units, making it a comprehensive genome-guided chemical structure prediction engine. A library of 57 tailoring reactions is leveraged for combinatorial scaffold library generation when multiple potential substrates are consistent with biosynthetic logic. We compare the accuracy of PRISM to existing genomic analysis platforms. PRISM is an open-source, user-friendly web application available at http://magarveylab.ca/prism/. PMID:26442528

Invasion of Epithelial Cells and Proteolysis of Cellular Focal Adhesion Components by Distinct Types of Porphyromonas gingivalis Fimbriae

PubMed Central

Nakagawa, Ichiro; Inaba, Hiroaki; Yamamura, Taihei; Kato, Takahiro; Kawai, Shinji; Ooshima, Takashi; Amano, Atsuo

2006-01-01

Porphyromonas gingivalis fimbriae are classified into six types (types I to V and Ib) based on the fimA genes encoding FimA (a subunit of fimbriae), and they play a critical role in bacterial interactions with host tissues. In this study, we compared the efficiencies of P. gingivalis strains with distinct types of fimbriae for invasion of epithelial cells and for degradation of cellular focal adhesion components, paxillin, and focal adhesion kinase (FAK). Six representative strains with the different types of fimbriae were tested, and P. gingivalis with type II fimbriae (type II P. gingivalis) adhered to and invaded epithelial cells at significantly greater levels than the other strains. There were negligible differences in gingipain activities among the six strains; however, type II P. gingivalis apparently degraded intracellular paxillin in association with a loss of phosphorylation 30 min after infection. Degradation was blocked with cytochalasin D or in mutants with fimA disrupted. Paxillin was degraded by the mutant with Lys-gingipain disrupted, and this degradation was prevented by inhibition of Arg-gingipain activity by Nα-p-tosyl-l-lysine chloromethyl ketone. FAK was also degraded by type II P. gingivalis. Cellular focal adhesions with green fluorescent protein-paxillin macroaggregates were clearly destroyed, and this was associated with cellular morphological changes and microtubule disassembly. In an in vitro wound closure assay, type II P. gingivalis significantly inhibited cellular migration and proliferation compared to the cellular migration and proliferation observed with the other types. These results suggest that type II P. gingivalis efficiently invades epithelial cells and degrades focal adhesion components with Arg-gingipain, which results in cellular impairment during wound healing and periodontal tissue regeneration. PMID:16790749

Qubit and fermionic Fock spaces from type II superstring black holes

NASA Astrophysics Data System (ADS)

Belhaj, A.; Bensed, M.; Benslimane, Z.; Sedra, M. B.; Segui, A.

Using Hodge diagram combinatorial data, we study qubit and fermionic Fock spaces from the point of view of type II superstring black holes based on complex compactifications. Concretely, we establish a one-to-one correspondence between qubits, fermionic spaces and extremal black holes in maximally supersymmetric supergravity obtained from type II superstring on complex toroidal and Calabi-Yau compactifications. We interpret the differential forms of the n-dimensional complex toroidal compactification as states of n-qubits encoding information on extremal black hole charges. We show that there are 2n copies of n qubit systems which can be split as 2n = 2n-1 + 2n-1. More precisely, 2n-1 copies are associated with even D-brane charges in type IIA superstring and the other 2n-1 ones correspond to odd D-brane charges in IIB superstring. This correspondence is generalized to a class of Calabi-Yau manifolds. In connection with black hole charges in type IIA superstring, an n-qubit system has been obtained from a canonical line bundle of n factors of one-dimensional projective space ℂℙ1.

Fructose Degradation in the Haloarchaeon Haloferax volcanii Involves a Bacterial Type Phosphoenolpyruvate-Dependent Phosphotransferase System, Fructose-1-Phosphate Kinase, and Class II Fructose-1,6-Bisphosphate Aldolase

PubMed Central

Pickl, Andreas; Johnsen, Ulrike

2012-01-01

The halophilic archaeon Haloferax volcanii utilizes fructose as a sole carbon and energy source. Genes and enzymes involved in fructose uptake and degradation were identified by transcriptional analyses, deletion mutant experiments, and enzyme characterization. During growth on fructose, the gene cluster HVO_1495 to HVO_1499, encoding homologs of the five bacterial phosphotransferase system (PTS) components enzyme IIB (EIIB), enzyme I (EI), histidine protein (HPr), EIIA, and EIIC, was highly upregulated as a cotranscript. The in-frame deletion of HVO_1499, designated ptfC (ptf stands for phosphotransferase system for fructose) and encoding the putative fructose-specific membrane component EIIC, resulted in a loss of growth on fructose, which could be recovered by complementation in trans. Transcripts of HVO_1500 (pfkB) and HVO_1494 (fba), encoding putative fructose-1-phosphate kinase (1-PFK) and fructose-1,6-bisphosphate aldolase (FBA), respectively, as well as 1-PFK and FBA activities were specifically upregulated in fructose-grown cells. pfkB and fba knockout mutants did not grow on fructose, whereas growth on glucose was not inhibited, indicating the functional involvement of both enzymes in fructose catabolism. Recombinant 1-PFK and FBA obtained after homologous overexpression were characterized as having kinetic properties indicative of functional 1-PFK and a class II type FBA. From these data, we conclude that fructose uptake in H. volcanii involves a fructose-specific PTS generating fructose-1-phosphate, which is further converted via fructose-1,6-bisphosphate to triose phosphates by 1-PFK and FBA. This is the first report of the functional involvement of a bacterial-like PTS and of class II FBA in the sugar metabolism of archaea. PMID:22493022

Antigen I/II encoded by integrative and conjugative elements of Streptococcus agalactiae and role in biofilm formation.

PubMed

Chuzeville, Sarah; Dramsi, Shaynoor; Madec, Jean-Yves; Haenni, Marisa; Payot, Sophie

2015-11-01

Streptococcus agalactiae (i.e. Group B streptococcus, GBS) is a major human and animal pathogen. Genes encoding putative surface proteins and in particular an antigen I/II have been identified on Integrative and Conjugative Elements (ICEs) found in GBS. Antigens I/II are multimodal adhesins promoting colonization of the oral cavity by streptococci such as Streptococcus gordonii and Streptococcus mutans. The prevalence and diversity of antigens I/II in GBS were studied by a bioinformatic analysis. It revealed that antigens I/II, which are acquired by horizontal transfer via ICEs, exhibit diversity and are widespread in GBS, in particular in the serotype Ia/ST23 invasive strains. This study aimed at characterizing the impact on GBS biology of proteins encoded by a previously characterized ICE of S. agalactiae (ICE_515_tRNA(Lys)). The production and surface exposition of the antigen I/II encoded by this ICE was examined using RT-PCR and immunoblotting experiments. Surface proteins of ICE_515_tRNA(Lys) were found to contribute to GBS biofilm formation and to fibrinogen binding. Contribution of antigen I/II encoded by SAL_2056 to biofilm formation was also demonstrated. These results highlight the potential for ICEs to spread microbial adhesins between species. Copyright © 2015 Elsevier Ltd. All rights reserved.

cDNAs encoding [D-Ala2]deltorphin precursors from skin of Phyllomedusa bicolor also contain genetic information for three dermorphin-related opioid peptides.

PubMed

Richter, K; Egger, R; Negri, L; Corsi, R; Severini, C; Kreil, G

1990-06-01

We present the structure of four precursors for [D-Ala2]deltorphins I and II as deduced from cDNAs cloned from skin of the frog Phyllomedusa bicolor. These contain the genetic information for one copy of [D-Ala2]deltorphin II and zero, one, or three copies of [D-Ala2]deltorphin I. In each case, the D-alanine of the end product is encoded by a normal GCG codon for L-alanine. In addition, the existence of three peptides related to dermorphin was predicted from the amino acid sequence of the precursors. These peptides were synthesized with a D-alanine in position 2 and their pharmacological properties were tested. Two of them, [Lys7]dermorphin-OH and [Trp4,Asn7]dermorphin-OH, were found to have roughly the same affinity and selectivity for mu-type opioid receptors as dermorphin.

cDNAs encoding [D-Ala2]deltorphin precursors from skin of Phyllomedusa bicolor also contain genetic information for three dermorphin-related opioid peptides.

PubMed Central

Richter, K; Egger, R; Negri, L; Corsi, R; Severini, C; Kreil, G

1990-01-01

We present the structure of four precursors for [D-Ala2]deltorphins I and II as deduced from cDNAs cloned from skin of the frog Phyllomedusa bicolor. These contain the genetic information for one copy of [D-Ala2]deltorphin II and zero, one, or three copies of [D-Ala2]deltorphin I. In each case, the D-alanine of the end product is encoded by a normal GCG codon for L-alanine. In addition, the existence of three peptides related to dermorphin was predicted from the amino acid sequence of the precursors. These peptides were synthesized with a D-alanine in position 2 and their pharmacological properties were tested. Two of them, [Lys7]dermorphin-OH and [Trp4,Asn7]dermorphin-OH, were found to have roughly the same affinity and selectivity for mu-type opioid receptors as dermorphin. PMID:2352951

Reduction of nuclear encoded enzymes of mitochondrial energy metabolism in cells devoid of mitochondrial DNA

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

Mueller, Edith E., E-mail: ed.mueller@salk.at; Mayr, Johannes A., E-mail: h.mayr@salk.at; Zimmermann, Franz A., E-mail: f.zimmermann@salk.at

2012-01-20

Highlights: Black-Right-Pointing-Pointer We examined OXPHOS and citrate synthase enzyme activities in HEK293 cells devoid of mtDNA. Black-Right-Pointing-Pointer Enzymes partially encoded by mtDNA show reduced activities. Black-Right-Pointing-Pointer Also the entirely nuclear encoded complex II and citrate synthase exhibit reduced activities. Black-Right-Pointing-Pointer Loss of mtDNA induces a feedback mechanism that downregulates complex II and citrate synthase. -- Abstract: Mitochondrial DNA (mtDNA) depletion syndromes are generally associated with reduced activities of oxidative phosphorylation (OXPHOS) enzymes that contain subunits encoded by mtDNA. Conversely, entirely nuclear encoded mitochondrial enzymes in these syndromes, such as the tricarboxylic acid cycle enzyme citrate synthase (CS) and OXPHOS complexmore » II, usually exhibit normal or compensatory enhanced activities. Here we report that a human cell line devoid of mtDNA (HEK293 {rho}{sup 0} cells) has diminished activities of both complex II and CS. This finding indicates the existence of a feedback mechanism in {rho}{sup 0} cells that downregulates the expression of entirely nuclear encoded components of mitochondrial energy metabolism.« less

Molecular Cloning and Expression Analysis of Eight PgWRKY Genes in Panax ginseng Responsive to Salt and Hormones.

PubMed

Xiu, Hao; Nuruzzaman, Mohammed; Guo, Xiangqian; Cao, Hongzhe; Huang, Jingjia; Chen, Xianghui; Wu, Kunlu; Zhang, Ru; Huang, Yuzhao; Luo, Junli; Luo, Zhiyong

2016-03-04

Despite the importance of WRKY genes in plant physiological processes, little is known about their roles in Panax ginseng C.A. Meyer. Forty-eight unigenes on this species were previously reported as WRKY transcripts using the next-generation sequencing (NGS) technology. Subsequently, one gene that encodes PgWRKY1 protein belonging to subgroup II-d was cloned and functionally characterized. In this study, eight WRKY genes from the NGS-based transcriptome sequencing dataset designated as PgWRKY2-9 have been cloned and characterized. The genes encoding WRKY proteins were assigned to WRKY Group II (one subgroup II-c, four subgroup II-d, and three subgroup II-e) based on phylogenetic analysis. The cDNAs of the cloned PgWRKYs encode putative proteins ranging from 194 to 358 amino acid residues, each of which includes one WRKYGQK sequence motif and one C₂H₂-type zinc-finger motif. Quantitative real-time PCR (qRT-PCR) analysis demonstrated that the eight analyzed PgWRKY genes were expressed at different levels in various organs including leaves, roots, adventitious roots, stems, and seeds. Importantly, the transcription responses of these PgWRKYs to methyl jasmonate (MeJA) showed that PgWRKY2, PgWRKY3, PgWRKY4, PgWRKY5, PgWRKY6, and PgWRKY7 were downregulated by MeJA treatment, while PgWRKY8 and PgWRKY9 were upregulated to varying degrees. Moreover, the PgWRKY genes increased or decreased by salicylic acid (SA), abscisic acid (ABA), and NaCl treatments. The results suggest that the PgWRKYs may be multiple stress-inducible genes responding to both salt and hormones.

Tubulin homolog TubZ in a phage-encoded partition system

PubMed Central

Oliva, María A.; Martin-Galiano, Antonio J.; Sakaguchi, Yoshihiko; Andreu, José M.

2012-01-01

Partition systems are responsible for the process whereby large and essential plasmids are accurately positioned to daughter cells during bacterial division. They are typically made of three components: a centromere-like DNA zone, an adaptor protein, and an assembling protein that is either a Walker-box ATPase (type I) or an actin-like ATPase (type II). A recently described type III segregation system has a tubulin/FtsZ-like protein, called TubZ, for plasmid movement. Here, we present the 2.3 Å structure and dynamic assembly of a TubZ tubulin homolog from a bacteriophage and unravel the Clostridium botulinum phage c-st type III partition system. Using biochemical and biophysical approaches, we prove that a gene upstream from tubZ encodes the partner TubR and localize the centromeric region (tubS), both of which are essential for anchoring phage DNA to the motile TubZ filaments. Finally, we describe a conserved fourth component, TubY, which modulates the TubZ-R-S complex interaction. PMID:22538818

The genome sequence of the facultative intracellular pathogen Brucella melitensis.

PubMed

DelVecchio, Vito G; Kapatral, Vinayak; Redkar, Rajendra J; Patra, Guy; Mujer, Cesar; Los, Tamara; Ivanova, Natalia; Anderson, Iain; Bhattacharyya, Anamitra; Lykidis, Athanasios; Reznik, Gary; Jablonski, Lynn; Larsen, Niels; D'Souza, Mark; Bernal, Axel; Mazur, Mikhail; Goltsman, Eugene; Selkov, Eugene; Elzer, Philip H; Hagius, Sue; O'Callaghan, David; Letesson, Jean-Jacques; Haselkorn, Robert; Kyrpides, Nikos; Overbeek, Ross

2002-01-08

Brucella melitensis is a facultative intracellular bacterial pathogen that causes abortion in goats and sheep and Malta fever in humans. The genome of B. melitensis strain 16M was sequenced and found to contain 3,294,935 bp distributed over two circular chromosomes of 2,117,144 bp and 1,177,787 bp encoding 3,197 ORFs. By using the bioinformatics suite ERGO, 2,487 (78%) ORFs were assigned functions. The origins of replication of the two chromosomes are similar to those of other alpha-proteobacteria. Housekeeping genes, including those involved in DNA replication, transcription, translation, core metabolism, and cell wall biosynthesis, are distributed on both chromosomes. Type I, II, and III secretion systems are absent, but genes encoding sec-dependent, sec-independent, and flagella-specific type III, type IV, and type V secretion systems as well as adhesins, invasins, and hemolysins were identified. Several features of the B. melitensis genome are similar to those of the symbiotic Sinorhizobium meliloti.

The genome sequence of the facultative intracellular pathogen Brucella melitensis

PubMed Central

DelVecchio, Vito G.; Kapatral, Vinayak; Redkar, Rajendra J.; Patra, Guy; Mujer, Cesar; Los, Tamara; Ivanova, Natalia; Anderson, Iain; Bhattacharyya, Anamitra; Lykidis, Athanasios; Reznik, Gary; Jablonski, Lynn; Larsen, Niels; D'Souza, Mark; Bernal, Axel; Mazur, Mikhail; Goltsman, Eugene; Selkov, Eugene; Elzer, Philip H.; Hagius, Sue; O'Callaghan, David; Letesson, Jean-Jacques; Haselkorn, Robert; Kyrpides, Nikos; Overbeek, Ross

2002-01-01

Brucella melitensis is a facultative intracellular bacterial pathogen that causes abortion in goats and sheep and Malta fever in humans. The genome of B. melitensis strain 16M was sequenced and found to contain 3,294,935 bp distributed over two circular chromosomes of 2,117,144 bp and 1,177,787 bp encoding 3,197 ORFs. By using the bioinformatics suite ERGO, 2,487 (78%) ORFs were assigned functions. The origins of replication of the two chromosomes are similar to those of other α-proteobacteria. Housekeeping genes, including those involved in DNA replication, transcription, translation, core metabolism, and cell wall biosynthesis, are distributed on both chromosomes. Type I, II, and III secretion systems are absent, but genes encoding sec-dependent, sec-independent, and flagella-specific type III, type IV, and type V secretion systems as well as adhesins, invasins, and hemolysins were identified. Several features of the B. melitensis genome are similar to those of the symbiotic Sinorhizobium meliloti. PMID:11756688

Type II flavohemoglobin of Mycobacterium smegmatis oxidizes d-lactate and mediate electron transfer.

PubMed

Thakur, Naveen; Kumar, Ashwani; Dikshit, Kanak L

2018-06-01

Two distantly related flavohemoglobins (FHbs), MsFHbI and MsFHbII, having crucial differences in their heme and reductase domains, co-exist in Mycobacterium smegmatis. Function of MsFHbI is associated with nitric-oxide detoxification but physiological relevance of MsFHbII remains unknown. This study unravels some unique spectral and functional characteristics of MsFHbII. Unlike conventional type I FHbs, MsFHbII lacks nitric-oxide dioxygenase and NADH oxidase activities but utilizes d-lactate as an electron donor to mediate electron transfer. MsFHbII carries a d-lactate dehydrogenase type FAD binding motif in its reductase domain and oxidizes d-lactate in a FAD dependent manner to reduce the heme iron, suggesting that the globin is acting as an electron acceptor. Importantly, expression of MsFHbII in Escherichia coli imparted protection under oxidative stress, suggesting its important role in stress management of its host. Since M. smegmatis lacks the gene encoding for d-lactate dehydrogenase and d-lactate is produced during aerobic metabolism and also as a by-product of lipid peroxidation, the ability of MsFHbII to metabolize d-lactate may provide it a unique ability to balance the oxidative stress generated due to accumulation of d-lactate in the cell and at the same time sequester electrons and pass it to the respiratory apparatus. Copyright © 2018 Elsevier B.V. All rights reserved.

Inventory of Extended-Spectrum-β-Lactamase-Producing Enterobacteriaceae in France as Assessed by a Multicenter Study.

PubMed

Robin, F; Beyrouthy, R; Bonacorsi, S; Aissa, N; Bret, L; Brieu, N; Cattoir, V; Chapuis, A; Chardon, H; Degand, N; Doucet-Populaire, F; Dubois, V; Fortineau, N; Grillon, A; Lanotte, P; Leyssene, D; Patry, I; Podglajen, I; Recule, C; Ros, A; Colomb-Cotinat, M; Ponties, V; Ploy, M C; Bonnet, R

2017-03-01

The objective of this study was to perform an inventory of the extended-spectrum-β-lactamase (ESBL)-producing Enterobacteriaceae isolates responsible for infections in French hospitals and to assess the mechanisms associated with ESBL diffusion. A total of 200 nonredundant ESBL-producing Enterobacteriaceae strains isolated from clinical samples were collected during a multicenter study performed in 18 representative French hospitals. Antibiotic resistance genes were identified by PCR and sequencing experiments. The clonal relatedness between isolates was investigated by the use of the DiversiLab system. ESBL-encoding plasmids were compared by PCR-based replicon typing and plasmid multilocus sequence typing. CTX-M-15, CTX-M-1, CTX-M-14, and SHV-12 were the most prevalent ESBLs (8% to 46.5%). The three CTX-M-type EBSLs were significantly observed in Escherichia coli (37.1%, 24.2%, and 21.8%, respectively), and CTX-M-15 was the predominant ESBL in Klebsiella pneumoniae (81.1%). SHV-12 was associated with ESBL-encoding Enterobacter cloacae strains (37.9%). qnrB , aac(6 ' )-Ib-cr , and aac(3)-II genes were the main plasmid-mediated resistance genes, with prevalences ranging between 19.5% and 45% according to the ESBL results. Molecular typing did not identify wide clonal diffusion. Plasmid analysis suggested the diffusion of low numbers of ESBL-encoding plasmids, especially in K. pneumoniae and E. cloacae However, the ESBL-encoding genes were observed in different plasmid replicons according to the bacterial species. The prevalences of ESBL subtypes differ according to the Enterobacteriaceae species. Plasmid spread is a key determinant of this epidemiology, and the link observed between the ESBL-encoding plasmids and the bacterial host explains the differences observed in the Enterobacteriaceae species. Copyright © 2017 American Society for Microbiology.

Definition of RNA Polymerase II CoTC Terminator Elements in the Human Genome

PubMed Central

Nojima, Takayuki; Dienstbier, Martin; Murphy, Shona; Proudfoot, Nicholas J.; Dye, Michael J.

2013-01-01

Summary Mammalian RNA polymerase II (Pol II) transcription termination is an essential step in protein-coding gene expression that is mediated by pre-mRNA processing activities and DNA-encoded terminator elements. Although much is known about the role of pre-mRNA processing in termination, our understanding of the characteristics and generality of terminator elements is limited. Whereas promoter databases list up to 40,000 known and potential Pol II promoter sequences, fewer than ten Pol II terminator sequences have been described. Using our knowledge of the human β-globin terminator mechanism, we have developed a selection strategy for mapping mammalian Pol II terminator elements. We report the identification of 78 cotranscriptional cleavage (CoTC)-type terminator elements at endogenous gene loci. The results of this analysis pave the way for the full understanding of Pol II termination pathways and their roles in gene expression. PMID:23562152

Endoribonuclease type II toxin-antitoxin systems: functional or selfish?

PubMed

Ramisetty, Bhaskar Chandra Mohan; Santhosh, Ramachandran Sarojini

2017-07-01

Most bacterial genomes have multiple type II toxin-antitoxin systems (TAs) that encode two proteins which are referred to as a toxin and an antitoxin. Toxins inhibit a cellular process, while the interaction of the antitoxin with the toxin attenuates the toxin's activity. Endoribonuclease-encoding TAs cleave RNA in a sequence-dependent fashion, resulting in translational inhibition. To account for their prevalence and retention by bacterial genomes, TAs are credited with clinically significant phenomena, such as bacterial programmed cell death, persistence, biofilms and anti-addiction to plasmids. However, the programmed cell death and persistence hypotheses have been challenged because of conceptual, methodological and/or strain issues. In an alternative view, chromosomal TAs seem to be retained by virtue of addiction at two levels: via a poison-antidote combination (TA proteins) and via transcriptional reprogramming of the downstream core gene (due to integration). Any perturbation in the chromosomal TA operons could cause fitness loss due to polar effects on the downstream genes and hence be detrimental under natural conditions. The endoribonucleases encoding chromosomal TAs are most likely selfish DNA as they are retained by bacterial genomes, even though TAs do not confer a direct advantage via the TA proteins. TAs are likely used by various replicons as 'genetic arms' that allow the maintenance of themselves and associated genetic elements. TAs seem to be the 'selfish arms' that make the best use of the 'arms race' between bacterial genomes and plasmids.

External Loops at the C Terminus of Erwinia chrysanthemi Pectate Lyase C Are Required for Species-Specific Secretion through the Out Type II Pathway

PubMed Central

Lindeberg, Magdalen; Boyd, Carol M.; Keen, Noel T.; Collmer, Alan

1998-01-01

The type II secretion system (main terminal branch of the general secretion pathway) is used by diverse gram-negative bacteria to secrete extracellular proteins. Proteins secreted by this pathway are synthesized with an N-terminal signal peptide which is removed upon translocation across the inner membrane, but the signals which target the mature proteins for secretion across the outer membrane are unknown. The plant pathogens Erwinia chrysanthemi and Erwinia carotovora secrete several isozymes of pectate lyase (Pel) by the out-encoded type II pathway. However, these two bacteria cannot secrete Pels encoded by heterologously expressed pel genes from the other species, suggesting the existence of species-specific secretion signals within these proteins. The functional cluster of E. chrysanthemi out genes carried on cosmid pCPP2006 enables Escherichia coli to secrete E. chrysanthemi, but not E. carotovora, Pels. We exploited the high sequence similarity between E. chrysanthemi PelC and E. carotovora Pel1 to construct 15 hybrid proteins in which different regions of PelC were replaced with homologous sequences from Pel1. The differential secretion of these hybrid proteins by E. coli(pCPP2006) revealed M118 to D175 and V215 to C329 as regions required for species-specific secretion of PelC. We propose that the primary targeting signal is contained within the external loops formed by G274 to C329 but is dependent on residues in M118 to D170 and V215 to G274 for proper positioning. PMID:9515910

Transcriptional Profiling of Type II Toxin-Antitoxin Genes of Helicobacter pylori under Different Environmental Conditions: Identification of HP0967-HP0968 System.

PubMed

Cárdenas-Mondragón, María G; Ares, Miguel A; Panunzi, Leonardo G; Pacheco, Sabino; Camorlinga-Ponce, Margarita; Girón, Jorge A; Torres, Javier; De la Cruz, Miguel A

2016-01-01

Helicobacter pylori is a Gram-negative bacterium that colonizes the human gastric mucosa and is responsible for causing peptic ulcers and gastric carcinoma. The expression of virulence factors allows the persistence of H. pylori in the stomach, which results in a chronic, sometimes uncontrolled inflammatory response. Type II toxin-antitoxin (TA) systems have emerged as important virulence factors in many pathogenic bacteria. Three type II TA systems have previously been identified in the genome of H. pylori 26695: HP0315-HP0316, HP0892-HP0893, and HP0894-HP0895. Here we characterized a heretofore undescribed type II TA system in H. pylori , HP0967-HP0968, which is encoded by the bicistronic operon hp0968-hp0967 and belongs to the Vap family. The predicted HP0967 protein is a toxin with ribonuclease activity whereas HP0968 is an antitoxin that binds to its own regulatory region. We found that all type II TA systems were expressed in H. pylori during early stationary growth phase, and differentially expressed in the presence of urea, nickel, and iron, although, the hp0968-hp0967 pair was the most affected under these environmental conditions. Transcription of hp0968-hp0967 was strongly induced in a mature H. pylori biofilm and when the bacteria interacted with AGS epithelial cells. Kanamycin and chloramphenicol considerably boosted transcription levels of all the four type II TA systems. The hp0968-hp0967 TA system was the most frequent among 317 H. pylori strains isolated from all over the world. This study is the first report on the transcription of type II TA genes in H. pylori under different environmental conditions. Our data show that the HP0967 and HP0968 proteins constitute a bona fide type II TA system in H. pylori , whose expression is regulated by environmental cues, which are relevant in the context of infection of the human gastric mucosa.

A newly discovered Bordetella species carries a transcriptionally active CRISPR-Cas with a small Cas9 endonuclease

USDA-ARS?s Scientific Manuscript database

The Cas9 endonuclease of the Type II-a clustered regularly interspersed short palindromic repeats (CRISPR), of Streptococcus pyogenes (SpCas9) has been adapted as a widely used tool for genome editing and genome engineering. Herein, we describe a gene encoding a novel Cas9 ortholog (BpsuCas9) and th...

Genomes to natural products PRediction Informatics for Secondary Metabolomes (PRISM).

PubMed

Skinnider, Michael A; Dejong, Chris A; Rees, Philip N; Johnston, Chad W; Li, Haoxin; Webster, Andrew L H; Wyatt, Morgan A; Magarvey, Nathan A

2015-11-16

Microbial natural products are an invaluable source of evolved bioactive small molecules and pharmaceutical agents. Next-generation and metagenomic sequencing indicates untapped genomic potential, yet high rediscovery rates of known metabolites increasingly frustrate conventional natural product screening programs. New methods to connect biosynthetic gene clusters to novel chemical scaffolds are therefore critical to enable the targeted discovery of genetically encoded natural products. Here, we present PRISM, a computational resource for the identification of biosynthetic gene clusters, prediction of genetically encoded nonribosomal peptides and type I and II polyketides, and bio- and cheminformatic dereplication of known natural products. PRISM implements novel algorithms which render it uniquely capable of predicting type II polyketides, deoxygenated sugars, and starter units, making it a comprehensive genome-guided chemical structure prediction engine. A library of 57 tailoring reactions is leveraged for combinatorial scaffold library generation when multiple potential substrates are consistent with biosynthetic logic. We compare the accuracy of PRISM to existing genomic analysis platforms. PRISM is an open-source, user-friendly web application available at http://magarveylab.ca/prism/. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Whole transcriptome profiling of taste bud cells.

PubMed

Sukumaran, Sunil K; Lewandowski, Brian C; Qin, Yumei; Kotha, Ramana; Bachmanov, Alexander A; Margolskee, Robert F

2017-08-08

Analysis of single-cell RNA-Seq data can provide insights into the specific functions of individual cell types that compose complex tissues. Here, we examined gene expression in two distinct subpopulations of mouse taste cells: Tas1r3-expressing type II cells and physiologically identified type III cells. Our RNA-Seq libraries met high quality control standards and accurately captured differential expression of marker genes for type II (e.g. the Tas1r genes, Plcb2, Trpm5) and type III (e.g. Pkd2l1, Ncam, Snap25) taste cells. Bioinformatics analysis showed that genes regulating responses to stimuli were up-regulated in type II cells, while pathways related to neuronal function were up-regulated in type III cells. We also identified highly expressed genes and pathways associated with chemotaxis and axon guidance, providing new insights into the mechanisms underlying integration of new taste cells into the taste bud. We validated our results by immunohistochemically confirming expression of selected genes encoding synaptic (Cplx2 and Pclo) and semaphorin signalling pathway (Crmp2, PlexinB1, Fes and Sema4a) components. The approach described here could provide a comprehensive map of gene expression for all taste cell subpopulations and will be particularly relevant for cell types in taste buds and other tissues that can be identified only by physiological methods.

Flagellin diversity in Clostridium botulinum groups I and II: a new strategy for strain identification.

PubMed

Paul, Catherine J; Twine, Susan M; Tam, Kevin J; Mullen, James A; Kelly, John F; Austin, John W; Logan, Susan M

2007-05-01

Strains of Clostridium botulinum are traditionally identified by botulinum neurotoxin type; however, identification of an additional target for typing would improve differentiation. Isolation of flagellar filaments and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that C. botulinum produced multiple flagellin proteins. Nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) analysis of in-gel tryptic digests identified peptides in all flagellin bands that matched two homologous tandem flagellin genes identified in the C. botulinum Hall A genome. Designated flaA1 and flaA2, these open reading frames encode the major structural flagellins of C. botulinum. Colony PCR and sequencing of flaA1/A2 variable regions classified 80 environmental and clinical strains into group I or group II and clustered isolates into 12 flagellar types. Flagellar type was distinct from neurotoxin type, and epidemiologically related isolates clustered together. Sequencing a larger PCR product, obtained during amplification of flaA1/A2 from type E strain Bennett identified a second flagellin gene, flaB. LC-MS analysis confirmed that flaB encoded a large type E-specific flagellin protein, and the predicted molecular mass for FlaB matched that observed by SDS-PAGE. In contrast, the molecular mass of FlaA was 2 to 12 kDa larger than the mass predicted by the flaA1/A2 sequence of a given strain, suggesting that FlaA is posttranslationally modified. While identification of FlaB, and the observation by SDS-PAGE of different masses of the FlaA proteins, showed the flagellin proteins of C. botulinum to be diverse, the presence of the flaA1/A2 gene in all strains examined facilitates single locus sequence typing of C. botulinum using the flagellin variable region.

FLAVODIIRON2 and FLAVODIIRON4 Proteins Mediate an Oxygen-Dependent Alternative Electron Flow in Synechocystis sp. PCC 6803 under CO2-Limited Conditions1[OPEN

PubMed Central

Shimakawa, Ginga; Shaku, Keiichiro; Nishi, Akiko; Hayashi, Ryosuke; Yamamoto, Hiroshi; Sakamoto, Katsuhiko; Makino, Amane; Miyake, Chikahiro

2015-01-01

This study aims to elucidate the molecular mechanism of an alternative electron flow (AEF) functioning under suppressed (CO2-limited) photosynthesis in the cyanobacterium Synechocystis sp. PCC 6803. Photosynthetic linear electron flow, evaluated as the quantum yield of photosystem II [Y(II)], reaches a maximum shortly after the onset of actinic illumination. Thereafter, Y(II) transiently decreases concomitantly with a decrease in the photosynthetic oxygen evolution rate and then recovers to a rate that is close to the initial maximum. These results show that CO2 limitation suppresses photosynthesis and induces AEF. In contrast to the wild type, Synechocystis sp. PCC 6803 mutants deficient in the genes encoding FLAVODIIRON2 (FLV2) and FLV4 proteins show no recovery of Y(II) after prolonged illumination. However, Synechocystis sp. PCC 6803 mutants deficient in genes encoding proteins functioning in photorespiration show AEF activity similar to the wild type. In contrast to Synechocystis sp. PCC 6803, the cyanobacterium Synechococcus elongatus PCC 7942 has no FLV proteins with high homology to FLV2 and FLV4 in Synechocystis sp. PCC 6803. This lack of FLV2/4 may explain why AEF is not induced under CO2-limited photosynthesis in S. elongatus PCC 7942. As the glutathione S-transferase fusion protein overexpressed in Escherichia coli exhibits NADH-dependent oxygen reduction to water, we suggest that FLV2 and FLV4 mediate oxygen-dependent AEF in Synechocystis sp. PCC 6803 when electron acceptors such as CO2 are not available. PMID:25540330

CRISPR/cas Loci of Type II Propionibacterium acnes Confer Immunity against Acquisition of Mobile Elements Present in Type I P. acnes

PubMed Central

Brüggemann, Holger; Lomholt, Hans B.; Tettelin, Hervé; Kilian, Mogens

2012-01-01

Propionibacterium acnes is a skin commensal that occasionally acts as an opportunistic pathogen. The population structure of this species shows three main lineages (I–III). While type I strains are mainly associated with sebaceous follicles of human skin and inflammatory acne, types II and III strains are more often associated with deep tissue infections. We investigated the occurrence and distribution of the clustered regularly interspaced short palindromic repeats (CRISPR) in P. acnes, assessed their immunological memory, and addressed the question if such a system could account for type-specific properties of the species. A collection of 108 clinical isolates covering all known phylotypes of P. acnes was screened for the existence of CRISPR/cas loci. We found that CRISPR loci are restricted to type II P. acnes strains. Sequence analyses of the CRISPR spacers revealed that the system confers immunity to P. acnes-specific phages and to two mobile genetic elements. These elements are found almost exclusively in type I P. acnes strains. Genome sequencing of a type I P. acnes isolate revealed that one element, 54 kb in size, encodes a putative secretion/tight adherence (TAD) system. Thus, CRISPR/cas loci in P. acnes recorded the exposure of type II strains to mobile genetic elements of type I strains. The CRISPR/cas locus is deleted in type I strains, which conceivably accounts for their ability to horizontally acquire fitness or virulence traits and might indicate that type I strains constitute a younger subpopulation of P. acnes. PMID:22479553

The Bacillus subtilis ywjI (glpX) gene encodes a class II fructose-1,6-bisphosphatase, functionally equivalent to the class III Fbp enzyme.

PubMed

Jules, Matthieu; Le Chat, Ludovic; Aymerich, Stéphane; Le Coq, Dominique

2009-05-01

We present here experimental evidence that the Bacillus subtilis ywjI gene encodes a class II fructose-1,6-bisphosphatase, functionally equivalent to the fbp-encoded class III enzyme, and constitutes with the upstream gene, murAB, an operon transcribed at the same level under glycolytic or gluconeogenic conditions.

The Bacillus subtilis ywjI (glpX) Gene Encodes a Class II Fructose-1,6-Bisphosphatase, Functionally Equivalent to the Class III Fbp Enzyme▿

PubMed Central

Jules, Matthieu; Le Chat, Ludovic; Aymerich, Stéphane; Le Coq, Dominique

2009-01-01

We present here experimental evidence that the Bacillus subtilis ywjI gene encodes a class II fructose-1,6-bisphosphatase, functionally equivalent to the fbp-encoded class III enzyme, and constitutes with the upstream gene, murAB, an operon transcribed at the same level under glycolytic or gluconeogenic conditions. PMID:19270101

STL-based Analysis of TRAIL-induced Apoptosis Challenges the Notion of Type I/Type II Cell Line Classification

PubMed Central

Bertaux, François; Maler, Oded; Batt, Gregory

2013-01-01

Extrinsic apoptosis is a programmed cell death triggered by external ligands, such as the TNF-related apoptosis inducing ligand (TRAIL). Depending on the cell line, the specific molecular mechanisms leading to cell death may significantly differ. Precise characterization of these differences is crucial for understanding and exploiting extrinsic apoptosis. Cells show distinct behaviors on several aspects of apoptosis, including (i) the relative order of caspases activation, (ii) the necessity of mitochondria outer membrane permeabilization (MOMP) for effector caspase activation, and (iii) the survival of cell lines overexpressing Bcl2. These differences are attributed to the activation of one of two pathways, leading to classification of cell lines into two groups: type I and type II. In this work we challenge this type I/type II cell line classification. We encode the three aforementioned distinguishing behaviors in a formal language, called signal temporal logic (STL), and use it to extensively test the validity of a previously-proposed model of TRAIL-induced apoptosis with respect to experimental observations made on different cell lines. After having solved a few inconsistencies using STL-guided parameter search, we show that these three criteria do not define consistent cell line classifications in type I or type II, and suggest mutants that are predicted to exhibit ambivalent behaviors. In particular, this finding sheds light on the role of a feedback loop between caspases, and reconciliates two apparently-conflicting views regarding the importance of either upstream or downstream processes for cell-type determination. More generally, our work suggests that these three distinguishing behaviors should be merely considered as type I/II features rather than cell-type defining criteria. On the methodological side, this work illustrates the biological relevance of STL-diagrams, STL population data, and STL-guided parameter search implemented in the tool Breach. Such tools are well-adapted to the ever-increasing availability of heterogeneous knowledge on complex signal transduction pathways. PMID:23675292

Molecular and phenotypic characteristics of methicillin-resistant Staphylococcus aureus isolated from hospitalized patients.

PubMed

de Oliveira, Caio Ferreira; Morey, Alexandre Tadachi; Santos, Jussevania Pereira; Gomes, Ludmila Vilela Pereira; Cardoso, Juscélio Donizete; Pinge-Filho, Phileno; Perugini, Márcia Regina Eches; Yamauchi, Lucy Megumi; Yamada-Ogatta, Sueli Fumie

2015-07-30

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of infections acquired in both community and hospital settings. In this study, MRSA isolated from different sources of hospitalized patients was characterized by molecular and phenotypic methods. A total of 123 S. aureus isolates were characterized according to their genetic relatedness by repetitive element sequence based-PCR (REP-PCR), in vitro antimicrobial susceptibility profile, SCCmec typing and presence of seven virulence factor-encoding genes. REP-PCR fingerprinting showed low relatedness between the isolates, and the predominance of one specific lineage or clonal group was not observed. All isolates were susceptible to teicoplanin and linezolide. All isolates were resistant to cefoxitin and penicillin, and the majority were also resistant to one or more other antimicrobials. Fifty isolates (41.7%) were intermediately resistant to vancomycin. Most isolates harbored SCCmec type II (53.7%), followed by type I (22.8%), type IV (8.1%) and type III (1.6%). All isolates harbored at least two virulence factor-encoding genes, and the prevalence was as follows: coa, 100%; icaA, 100%; hla, 13.0%; hlb, 91.1%, hld, 91.1%; lukS-PV and lukF-PV, 2.4%; and tst, 34.1%. A positive association with the presence of hla and SCCmec type II, and tst and SCCmec type I was observed. This study showed the high virulence potential of multidrug-resistant MRSA circulating in a teaching hospital. A high prevalence of MRSA showing intermediate vancomycin resistance was also observed, indicating the urgent need to improve strategies for controlling the use of antimicrobials for appropriate management of S. aureus infections.

Type II Toxin–Antitoxin Systems in the Unicellular Cyanobacterium Synechocystis sp. PCC 6803

PubMed Central

Kopfmann, Stefan; Roesch, Stefanie K.; Hess, Wolfgang R.

2016-01-01

Bacterial toxin–antitoxin (TA) systems are genetic elements, which are encoded by plasmid as well as chromosomal loci. They mediate plasmid and genomic island maintenance through post-segregational killing mechanisms but may also have milder effects, acting as mobile stress response systems that help certain cells of a population in persisting adverse growth conditions. Very few cyanobacterial TA system have been characterized thus far. In this work, we focus on the cyanobacterium Synechocystis 6803, a widely used model organism. We expand the number of putative Type II TA systems from 36 to 69 plus seven stand-alone components. Forty-seven TA pairs are located on the chromosome and 22 are plasmid-located. Different types of toxins are associated with various antitoxins in a mix and match principle. According to protein domains and experimental data, 81% of all toxins in Synechocystis 6803 likely exhibit RNase activity, suggesting extensive potential for toxicity-related RNA degradation and toxin-mediated transcriptome remodeling. Of particular interest is the Ssr8013–Slr8014 system encoded on plasmid pSYSG, which is part of a larger defense island or the pSYSX system Slr6056–Slr6057, which is linked to a bacterial ubiquitin-like system. Consequently, Synechocystis 6803 is one of the most prolific sources of new information about these genetic elements. PMID:27455323

EcpA, an extracellular protease, is a specific virulence factor required by Xanthomonas oryzae pv. oryzicola but not by X. oryzae pv. oryzae in rice

USDA-ARS?s Scientific Manuscript database

Previously, twelve protease-deficient mutants of Xanthomonas oryzae pv. oryzicola (Xoc) RS105 strain were recovered from a Tn5-tagged mutant library. In the current study, the Tn5 insertion site in each mutant was mapped. Mutations in genes encoding components of the type II secretion apparatus, cAM...

Cloning, purification, crystallization and preliminary X-ray crystallographic analysis of MCAT from Staphylococcus aureus.

PubMed

Hong, Seung Kon; Kim, Kook Han; Kim, Eunice EunKyeong

2010-01-01

Malonyl-CoA:acyl-carrier protein transacylase (MCAT), encoded by the fabd gene, is a key enzyme in type II fatty-acid biosynthesis. It is responsible for transferring the malonyl group from malonyl-CoA to the holo acyl-carrier protein (ACP). Since the type II system differs from the type I system that mammals use, it has received enormous attention as a possible antibiotic target. In particular, only a single isoform of MCAT has been reported and a continuous coupled enzyme assay has been developed. MCAT from Staphylococcus aureus was overexpressed in Escherichia coli and the protein was purified and crystallized. Diffraction data were collected to 1.2 A resolution. The crystals belonged to space group P2(1), with unit-cell parameters a = 41.608, b = 86.717, c = 43.163 A, alpha = gamma = 90, beta = 106.330 degrees . The asymmetric unit contains one SaMCAT molecule.

Distribution and regulation of the mobile genetic element-encoded phenol-soluble modulin PSM-mec in methicillin-resistant Staphylococcus aureus.

PubMed

Chatterjee, Som S; Chen, Liang; Joo, Hwang-Soo; Cheung, Gordon Y C; Kreiswirth, Barry N; Otto, Michael

2011-01-01

The phenol-soluble modulin PSM-mec is the only known staphylococcal toxin that is encoded on a mobile antibiotic resistance determinant, namely the staphylococcal cassette chromosome (SCC) element mec encoding resistance to methicillin. Here we show that the psm-mec gene is found frequently among methicillin-resistant Staphylococcus aureus (MRSA) strains of SCCmec types II, III, and VIII, and is a conserved part of the class A mec gene complex. Controlled expression of AgrA versus RNAIII in agr mutants of all 3 psm-mec-positive SCCmec types demonstrated that expression of psm-mec, which is highly variable, is controlled by AgrA in an RNAIII-independent manner. Furthermore, psm-mec isogenic deletion mutants showed only minor changes in PSMα peptide production and unchanged (or, as previously described, diminished) virulence compared to the corresponding wild-type strains in a mouse model of skin infection. This indicates that the recently reported regulatory impact of the psm-mec locus on MRSA virulence, which is opposite to that of the PSM-mec peptide and likely mediated by a regulatory RNA, is minor when analyzed in the original strain background. Our study gives new insight in the distribution, regulation, and role in virulence of the PSM-mec peptide and the psm-mec gene locus.

Definition of RNA polymerase II CoTC terminator elements in the human genome.

PubMed

Nojima, Takayuki; Dienstbier, Martin; Murphy, Shona; Proudfoot, Nicholas J; Dye, Michael J

2013-04-25

Mammalian RNA polymerase II (Pol II) transcription termination is an essential step in protein-coding gene expression that is mediated by pre-mRNA processing activities and DNA-encoded terminator elements. Although much is known about the role of pre-mRNA processing in termination, our understanding of the characteristics and generality of terminator elements is limited. Whereas promoter databases list up to 40,000 known and potential Pol II promoter sequences, fewer than ten Pol II terminator sequences have been described. Using our knowledge of the human β-globin terminator mechanism, we have developed a selection strategy for mapping mammalian Pol II terminator elements. We report the identification of 78 cotranscriptional cleavage (CoTC)-type terminator elements at endogenous gene loci. The results of this analysis pave the way for the full understanding of Pol II termination pathways and their roles in gene expression. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Organization of Genes Required for the Oxidation of Methanol to Formaldehyde in Three Type II Methylotrophs

PubMed Central

Bastien, C.; Machlin, S.; Zhang, Y.; Donaldson, K.; Hanson, R. S.

1989-01-01

Restriction maps of genes required for the synthesis of active methanol dehydrogenase in Methylobacterium organophilum XX and Methylobacterium sp. strain AM1 have been completed and compared. In these two species of pink-pigmented, type II methylotrophs, 15 genes were identified that were required for the expression of methanol dehydrogenase activity. None of these genes were required for the synthesis of the prosthetic group of methanol dehydrogenase, pyrroloquinoline quinone. The structural gene required for the synthesis of cytochrome cL, an electron acceptor uniquely required for methanol dehydrogenase, and the genes encoding small basic peptides that copurified with methanol dehydrogenases were closely linked to the methanol dehydrogenase structural genes. A cloned 22-kilobase DNA insert from Methylsporovibrio methanica 81Z, an obligate type II methanotroph, complemented mutants that contained lesions in four genes closely linked to the methanol dehydrogenase structural genes. The methanol dehydrogenase and cytochrome cL structural genes were found to be transcribed independently in M. organophilum XX. Only two of the genes required for methanol dehydrogenase synthesis in this bacterium were found to be cotranscribed. PMID:16348074

Functional analysis of environmental DNA-derived type II polyketide synthases reveals structurally diverse secondary metabolites.

PubMed

Feng, Zhiyang; Kallifidas, Dimitris; Brady, Sean F

2011-08-02

A single gram of soil is predicted to contain thousands of unique bacterial species. The majority of these species remain recalcitrant to standard culture methods, prohibiting their use as sources of unique bioactive small molecules. The cloning and analysis of DNA extracted directly from environmental samples (environmental DNA, eDNA) provides a means of exploring the biosynthetic capacity of natural bacterial populations. Environmental DNA libraries contain large reservoirs of bacterial genetic diversity from which new secondary metabolite gene clusters can be systematically recovered and studied. The identification and heterologous expression of type II polyketide synthase-containing eDNA clones is reported here. Functional analysis of three soil DNA-derived polyketide synthase systems in Streptomyces albus revealed diverse metabolites belonging to well-known, rare, and previously uncharacterized structural families. The first of these systems is predicted to encode the production of the known antibiotic landomycin E. The second was found to encode the production of a metabolite with a previously uncharacterized pentacyclic ring system. The third was found to encode the production of unique KB-3346-5 derivatives, which show activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis. These results, together with those of other small-molecule-directed metagenomic studies, suggest that culture-independent approaches are capable of accessing biosynthetic diversity that has not yet been extensively explored using culture-based methods. The large-scale functional screening of eDNA clones should be a productive strategy for generating structurally previously uncharacterized chemical entities for use in future drug development efforts.

Characterization, adaptive traffic shaping, and multiplexing of real-time MPEG II video

NASA Astrophysics Data System (ADS)

Agrawal, Sanjay; Barry, Charles F.; Binnai, Vinay; Kazovsky, Leonid G.

1997-01-01

We obtain network traffic model for real-time MPEG-II encoded digital video by analyzing video stream samples from real-time encoders from NUKO Information Systems. MPEG-II sample streams include a resolution intensive movie, City of Joy, an action intensive movie, Aliens, a luminance intensive (black and white) movie, Road To Utopia, and a chrominance intensive (color) movie, Dick Tracy. From our analysis we obtain a heuristic model for the encoded video traffic which uses a 15-stage Markov process to model the I,B,P frame sequences within a group of pictures (GOP). A jointly-correlated Gaussian process is used to model the individual frame sizes. Scene change arrivals are modeled according to a gamma process. Simulations show that our MPEG-II traffic model generates, I,B,P frame sequences and frame sizes that closely match the sample MPEG-II stream traffic characteristics as they relate to latency and buffer occupancy in network queues. To achieve high multiplexing efficiency we propose a traffic shaping scheme which sets preferred 1-frame generation times among a group of encoders so as to minimize the overall variation in total offered traffic while still allowing the individual encoders to react to scene changes. Simulations show that our scheme results in multiplexing gains of up to 10% enabling us to multiplex twenty 6 Mbps MPEG-II video streams instead of 18 streams over an ATM/SONET OC3 link without latency or cell loss penalty. This scheme is due for a patent.

Transcriptional Profiling of Type II Toxin–Antitoxin Genes of Helicobacter pylori under Different Environmental Conditions: Identification of HP0967–HP0968 System

PubMed Central

Cárdenas-Mondragón, María G.; Ares, Miguel A.; Panunzi, Leonardo G.; Pacheco, Sabino; Camorlinga-Ponce, Margarita; Girón, Jorge A.; Torres, Javier; De la Cruz, Miguel A.

2016-01-01

Helicobacter pylori is a Gram-negative bacterium that colonizes the human gastric mucosa and is responsible for causing peptic ulcers and gastric carcinoma. The expression of virulence factors allows the persistence of H. pylori in the stomach, which results in a chronic, sometimes uncontrolled inflammatory response. Type II toxin–antitoxin (TA) systems have emerged as important virulence factors in many pathogenic bacteria. Three type II TA systems have previously been identified in the genome of H. pylori 26695: HP0315–HP0316, HP0892–HP0893, and HP0894–HP0895. Here we characterized a heretofore undescribed type II TA system in H. pylori, HP0967–HP0968, which is encoded by the bicistronic operon hp0968–hp0967 and belongs to the Vap family. The predicted HP0967 protein is a toxin with ribonuclease activity whereas HP0968 is an antitoxin that binds to its own regulatory region. We found that all type II TA systems were expressed in H. pylori during early stationary growth phase, and differentially expressed in the presence of urea, nickel, and iron, although, the hp0968–hp0967 pair was the most affected under these environmental conditions. Transcription of hp0968–hp0967 was strongly induced in a mature H. pylori biofilm and when the bacteria interacted with AGS epithelial cells. Kanamycin and chloramphenicol considerably boosted transcription levels of all the four type II TA systems. The hp0968–hp0967 TA system was the most frequent among 317 H. pylori strains isolated from all over the world. This study is the first report on the transcription of type II TA genes in H. pylori under different environmental conditions. Our data show that the HP0967 and HP0968 proteins constitute a bona fide type II TA system in H. pylori, whose expression is regulated by environmental cues, which are relevant in the context of infection of the human gastric mucosa. PMID:27920769

Variola virus immune evasion proteins.

PubMed

Dunlop, Lance R; Oehlberg, Katherine A; Reid, Jeremy J; Avci, Dilek; Rosengard, Ariella M

2003-09-01

Variola virus, the causative agent of smallpox, encodes approximately 200 proteins. Over 80 of these proteins are located in the terminal regions of the genome, where proteins associated with host immune evasion are encoded. To date, only two variola proteins have been characterized. Both are located in the terminal regions and demonstrate immunoregulatory functions. One protein, the smallpox inhibitor of complement enzymes (SPICE), is homologous to a vaccinia virus virulence factor, the vaccinia virus complement-control protein (VCP), which has been found experimentally to be expressed early in the course of vaccinia infection. Both SPICE and VCP are similar in structure and function to the family of mammalian complement regulatory proteins, which function to prevent inadvertent injury to adjacent cells and tissues during complement activation. The second variola protein is the variola virus high-affinity secreted chemokine-binding protein type II (CKBP-II, CBP-II, vCCI), which binds CC-chemokine receptors. The vaccinia homologue of CKBP-II is secreted both early and late in infection. CKBP-II proteins are highly conserved among orthopoxviruses, sharing approximately 85% homology, but are absent in eukaryotes. This characteristic sets it apart from other known virulence factors in orthopoxviruses, which share sequence homology with known mammalian immune regulatory gene products. Future studies of additional variola proteins may help illuminate factors associated with its virulence, pathogenesis and strict human tropism. In addition, these studies may also assist in the development of targeted therapies for the treatment of both smallpox and human immune-related diseases.

Evidence for an apical Na-Cl cotransporter involved in ion uptake in a teleost fish

USGS Publications Warehouse

Hiroi, J.; Yasumasu, S.; McCormick, S.D.; Hwang, P.-P.; Kaneko, T.

2008-01-01

Cation-chloride cotransporters, such as the Na+/K +/2Cl- cotransporter (NKCC) and Na+/Cl - cotransporter (NCC), are localized to the apical or basolateral plasma membranes of epithelial cells and are involved in active ion absorption or secretion. The objectives of this study were to clone and identify 'freshwater-type' and 'seawater-type' cation-chloride cotransporters of euryhaline Mozambique tilapia (Oreochromis mossambicus) and to determine their intracellular localization patterns within mitochondria-rich cells (MRCs). From tilapia gills, we cloned four full-length cDNAs homologous to human cation-chloride cotransporters and designated them as tilapia NKCC1a, NKCC1b, NKCC2 and NCC. Out of the four candidates, the mRNA encoding NKCC1a was highly expressed in the yolk-sac membrane and gills (sites of the MRC localization) of seawater-acclimatized fish, whereas the mRNA encoding NCC was exclusively expressed in the yolk-sac membrane and gills of freshwater-acclimatized fish. We then generated antibodies specific for tilapia NKCC1a and NCC and conducted whole-mount immunofluorescence staining for NKCC1a and NCC, together with Na+/K+-ATPase, cystic fibrosis transmembrane conductance regulator (CFTR) and Na+/H+ exchanger 3 (NHE3), on the yolk-sac membrane of tilapia embryos acclimatized to freshwater or seawater. The simultaneous quintuple-color immunofluorescence staining allowed us to classify MRCs clearly into four types: types I, II, III and IV. The NKCC1a immunoreactivity was localized to the basolateral membrane of seawater-specific type-IV MRCs, whereas the NCC immunoreactivity was restricted to the apical membrane of freshwater-specific type-II MRCs. Taking account of these data at the level of both mRNA and protein, we deduce that NKCC1a is the seawater-type cotransporter involved in ion secretion by type-IV MRCs and that NCC is the freshwater-type cotransporter involved in ion absorption by type-II MRCs. We propose a novel ion-uptake model by MRCs in freshwater that incorporates apically located NCC. We also reevaluate a traditional ion-uptake model incorporating NHE3; the mRNA was highly expressed in freshwater, and the immunoreactivity was found at the apical membrane of other freshwater-specific MRCs.

Multiple Scenarios of Transition to Chaos in the Alternative Splicing Model

NASA Astrophysics Data System (ADS)

Kogai, Vladislav V.; Likhoshvai, Vitaly A.; Fadeev, Stanislav I.; Khlebodarova, Tamara M.

We have investigated the scenarios of transition to chaos in the mathematical model of a genetic system constituted by a single transcription factor-encoding gene, the expression of which is self-regulated by a feedback loop that involves protein isoforms. Alternative splicing results in the synthesis of protein isoforms providing opposite regulatory outcomes — activation or repression. The model is represented by a differential equation with two delayed arguments. The possibility of transition to chaos dynamics via all classical scenarios: a cascade of period-doubling bifurcations, quasiperiodicity and type-I, type-II and type-III intermittencies, has been numerically demonstrated. The parametric features of each type of transition to chaos have been described.

Involvement of the pagR gene of pXO2 in anthrax pathogenesis

PubMed Central

Liang, Xudong; Zhang, Enmin; Zhang, Huijuan; Wei, Jianchun; Li, Wei; Zhu, Jin; Wang, Bingxiang; Dong, Shulin

2016-01-01

Anthrax is a disease caused by Bacillus anthracis. Specifically, the anthrax toxins and capsules encoded by the pXO1 and pXO2 plasmids, respectively, are the major virulence factors. We previously reported that the pXO1 plasmid was retained in the attenuated strain of B. anthracis vaccine strains even after subculturing at high temperatures. In the present study, we reinvestigate the attenuation mechanism of Pasteur II. Sequencing of pXO1 and pXO2 from Pasteur II strain revealed mutations in these plasmids as compared to the reference sequences. Two deletions on these plasmids, one each on pXO1 and pXO2, were confirmed to be unique to the Pasteur II strain as compared to the wild-type strains. Gene replacement with homologous recombination revealed that the mutation in the promoter region of the pagR gene on pXO2, but not the mutation on pXO1, contributes to lethal levels of toxin production. This result was further confirmed by RT-PCR, western blot, and animal toxicity assays. Taken together, our results signify that the attenuation of the Pasteur II vaccine strain is caused by a mutation in the pagR gene on its pXO2 plasmid. Moreover, these data suggest that pXO2 plasmid encoded proteins are involved in the virulence of B. anthracis. PMID:27363681

Transformation to continuous growth of primary human T lymphocytes by human T-cell leukemia virus type I X-region genes transduced by a herpesvirus saimiri vector

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

Grassmann, R.; Dengler, C.; Mueller-Fleckenstein, I.

1989-05-01

The role of the X region of the genome of the human T-cell leukemia virus type I (HTLV-I) in the immortalization of lymphocytes has been difficult to distinguish from its role in viral replication as this region encodes at least two genes, tax and rex, required for replication and the expression of viral proteins. To determine whether the X region does encode immortalizing functions, a fragment of the HTLV-I provirus capable of expressing known X-region proteins was inserted into the genome of a transformation-defective, replication-competent Herpesvirus saimiri. Infection of fresh mitogen-activated human cord blood and thymocytes yielded immortal T-cell linesmore » that had the same phenotype (CD4{sup +}, Cd5{sup +}, HLA class II{sup +}, interleukin 2 receptor {alpha}-chain +) as lymphocytes transformed by cocultivation with HTLV-I. These experiments demonstrate that the X region encodes the functions of HTLV-I that immortalize a distinct subpopulation of human T cells. The experiments also demonstrate the utility of the H. saimiri vector for the transduction of heterologous genes into human T cells.« less

The RNA-binding protein Spo5 promotes meiosis II by regulating cyclin Cdc13 in fission yeast.

PubMed

Arata, Mayumi; Sato, Masamitsu; Yamashita, Akira; Yamamoto, Masayuki

2014-03-01

Meiosis comprises two consecutive nuclear divisions, meiosis I and II. Despite this unique progression through the cell cycle, little is known about the mechanisms controlling the sequential divisions. In this study, we carried out a genetic screen to identify factors that regulate the initiation of meiosis II in the fission yeast Schizosaccharomyces pombe. We identified mutants deficient in meiosis II progression and repeatedly isolated mutants defective in spo5, which encodes an RNA-binding protein. Using fluorescence microscopy to visualize YFP-tagged protein, we found that spo5 mutant cells precociously lost Cdc13, the major B-type cyclin in fission yeast, before meiosis II. Importantly, the defect in meiosis II was rescued by increasing CDK activity. In wild-type cells, cdc13 transcripts increased during meiosis II, but this increase in cdc13 expression was weaker in spo5 mutants. Thus, Spo5 is a novel regulator of meiosis II that controls the level of cdc13 expression and promotes de novo synthesis of Cdc13. We previously reported that inhibition of Cdc13 degradation is necessary to initiate meiosis II; together with the previous information, the current findings indicate that the dual control of Cdc13 by de novo synthesis and suppression of proteolysis ensures the progression of meiosis II. © 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

Regulation of Sulfur Assimilation Pathways in Burkholderia cenocepacia through Control of Genes by the SsuR Transcription Factor▿

PubMed Central

Łochowska, Anna; Iwanicka-Nowicka, Roksana; Zielak, Agata; Modelewska, Anna; Thomas, Mark S.; Hryniewicz, Monika M.

2011-01-01

The genome of Burkholderia cenocepacia contains two genes encoding closely related LysR-type transcriptional regulators, CysB and SsuR, involved in control of sulfur assimilation processes. In this study we show that the function of SsuR is essential for the utilization of a number of organic sulfur sources of either environmental or human origin. Among the genes upregulated by SsuR identified here are the tauABC operon encoding a predicted taurine transporter, three tauD-type genes encoding putative taurine dioxygenases, and atsA encoding a putative arylsulfatase. The role of SsuR in expression of these genes/operons was characterized through (i) construction of transcriptional reporter fusions to candidate promoter regions and analysis of their expression in the presence/absence of SsuR and (ii) testing the ability of SsuR to bind SsuR-responsive promoter regions. We also demonstrate that expression of SsuR-activated genes is not repressed in the presence of inorganic sulfate. A more detailed analysis of four SsuR-responsive promoter regions indicated that ∼44 bp of the DNA sequence preceding and/or overlapping the predicted −35 element of such promoters is sufficient for SsuR binding. The DNA sequence homology among SsuR “recognition motifs” at different responsive promoters appears to be limited. PMID:21317335

Molecular and biochemical characterization of two tungsten- and selenium-containing formate dehydrogenases from Eubacterium acidaminophilum that are associated with components of an iron-only hydrogenase.

PubMed

Graentzdoerffer, Andrea; Rauh, David; Pich, Andreas; Andreesen, Jan R

2003-01-01

Two gene clusters encoding similar formate dehydrogenases (FDH) were identified in Eubacterium acidaminophilum. Each cluster is composed of one gene coding for a catalytic subunit ( fdhA-I, fdhA-II) and one for an electron-transferring subunit ( fdhB-I, fdhB-II). Both fdhA genes contain a TGA codon for selenocysteine incorporation and the encoded proteins harbor five putative iron-sulfur clusters in their N-terminal region. Both FdhB subunits resemble the N-terminal region of FdhA on the amino acid level and contain five putative iron-sulfur clusters. Four genes thought to encode the subunits of an iron-only hydrogenase are located upstream of the FDH gene cluster I. By sequence comparison, HymA and HymB are predicted to contain one and four iron-sulfur clusters, respectively, the latter protein also binding sites for FMN and NAD(P). Thus, HymA and HymB seem to represent electron-transferring subunits, and HymC the putative catalytic subunit containing motifs for four iron-sulfur clusters and one H-cluster specific for Fe-only hydrogenases. HymD has six predicted transmembrane helices and might be an integral membrane protein. Viologen-dependent FDH activity was purified from serine-grown cells of E. acidaminophilum and the purified protein complex contained four subunits, FdhA and FdhB, encoded by FDH gene cluster II, and HymA and HymB, identified after determination of their N-terminal sequences. Thus, this complex might represent the most simple type of a formate hydrogen lyase. The purified formate dehydrogenase fraction contained iron, tungsten, a pterin cofactor, and zinc, but no molybdenum. FDH-II had a two-fold higher K(m) for formate (0.37 mM) than FDH-I and also catalyzed CO(2) reduction to formate. Reverse transcription (RT)-PCR pointed to increased expression of FDH-II in serine-grown cells, supporting the isolation of this FDH isoform. The fdhA-I gene was expressed as inactive protein in Escherichia coli. The in-frame UGA codon for selenocysteine incorporation was read in the heterologous system only as stop codon, although its potential SECIS element exhibited a quite high similarity to that of E. coli FDH.

Structural and functional dissection reveals distinct roles of Ca2+-binding sites in the giant adhesin SiiE of Salmonella enterica

PubMed Central

Klingl, Stefan; Sandmann, Achim; Taccardi, Nicola; Sticht, Heinrich; Muller, Yves A.; Hensel, Michael

2017-01-01

The giant non-fimbrial adhesin SiiE of Salmonella enterica mediates the first contact to the apical site of epithelial cells and enables subsequent invasion. SiiE is a 595 kDa protein composed of 53 repetitive bacterial immunoglobulin (BIg) domains and the only known substrate of the SPI4-encoded type 1 secretion system (T1SS). The crystal structure of BIg50-52 of SiiE revealed two distinct Ca2+-binding sites per BIg domain formed by conserved aspartate or glutamate residues. In a mutational analysis Ca2+-binding sites were disrupted by aspartate to serine exchange at various positions in the BIg domains of SiiE. Amounts of secreted SiiE diminish with a decreasing number of intact Ca2+-binding sites. BIg domains of SiiE contain distinct Ca2+-binding sites, with type I sites being similar to other T1SS-secreted proteins and type II sites newly identified in SiiE. We functionally and structurally dissected the roles of type I and type II Ca2+-binding sites in SiiE, as well as the importance of Ca2+-binding sites in various positions of SiiE. Type I Ca2+-binding sites were critical for efficient secretion of SiiE and a decreasing number of type I sites correlated with reduced secretion. Type II sites were less important for secretion, stability and surface expression of SiiE, however integrity of type II sites in the C-terminal portion was required for the function of SiiE in mediating adhesion and invasion. PMID:28558023

Phylogenomics of MADS-Box Genes in Plants - Two Opposing Life Styles in One Gene Family.

PubMed

Gramzow, Lydia; Theißen, Günter

2013-09-12

The development of multicellular eukaryotes, according to their body plan, is often directed by members of multigene families that encode transcription factors. MADS (for MINICHROMOSOME MAINTENANCE1, AGAMOUS, DEFICIENS and SERUM RESPONSE FACTOR)-box genes form one of those families controlling nearly all major aspects of plant development. Knowing the complete complement of MADS-box genes in sequenced plant genomes will allow a better understanding of the evolutionary patterns of these genes and the association of their evolution with the evolution of plant morphologies. Here, we have applied a combination of automatic and manual annotations to identify the complete set of MADS-box genes in 17 plant genomes. Furthermore, three plant genomes were reanalyzed and published datasets were used for four genomes such that more than 2,600 genes from 24 species were classified into the two types of MADS-box genes, Type I and Type II. Our results extend previous studies, highlighting the remarkably different evolutionary patterns of Type I and Type II genes and provide a basis for further studies on the evolution and function of MADS-box genes.

Role of Accessory Proteins of HTLV-1 in Viral Replication, T Cell Activation, and Cellular Gene Expression

PubMed Central

Michael, Bindhu; Nair, Amithraj; Lairmore, Michael D.

2010-01-01

Human T-cell lymphotropic virus type 1 (HTLV-1), causes adult T cell leukemia/lymphoma (ATLL), and initiates a variety of immune mediated disorders. The viral genome encodes common structural and enzymatic proteins characteristic of all retroviruses and utilizes alternative splicing and alternate codon usage to make several regulatory and accessory proteins encoded in the pX region (pX ORF I to IV). Recent studies indicate that the accessory proteins p12I, p27I, p13II, and p30II, encoded by pX ORF I and II, contribute to viral replication and the ability of the virus to maintain typical in vivo expression levels. Proviral clones that are mutated in either pX ORF I or II, while fully competent in cell culture, are severely limited in their replicative capacity in a rabbit model. These HTLV-1 accessory proteins are critical for establishment of viral infectivity, enhance T- lymphocyte activation and potentially alter gene transcription and mitochondrial function. HTLV-1 pX ORF I expression is critical to the viral infectivity in resting primary lymphocytes suggesting a role for the calcineurin-binding protein p12I in lymphocyte activation. The endoplasmic reticulum and cis-Golgi localizing p12I activates NFAT, a key T cell transcription factor, through calcium-mediated signaling pathways and may lower the threshold of lymphocyte activation via the JAK/STAT pathway. In contrast p30II localizes to the nucleus and represses viral promoter activity, but may regulate cellular gene expression through p300/CBP or related co-activators of transcription. The mitochondrial localizing p13II induces morphologic changes in the organelle and may influence energy metabolism infected cells. Future studies of the molecular details HTLV-1 “accessory” proteins interactions will provide important new directions for investigations of HTLV-1 and related viruses associated with lymphoproliferative diseases. Thus, the accessory proteins of HTLV-1, once thought to be dispensable for viral replication, have proven to be directly involved in viral spread in vivo and represent potential targets for therapeutic intervention against HTLV-1 infection and disease. PMID:15358581

Flagellin Diversity in Clostridium botulinum Groups I and II: a New Strategy for Strain Identification▿

PubMed Central

Paul, Catherine J.; Twine, Susan M.; Tam, Kevin J.; Mullen, James A.; Kelly, John F.; Austin, John W.; Logan, Susan M.

2007-01-01

Strains of Clostridium botulinum are traditionally identified by botulinum neurotoxin type; however, identification of an additional target for typing would improve differentiation. Isolation of flagellar filaments and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that C. botulinum produced multiple flagellin proteins. Nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) analysis of in-gel tryptic digests identified peptides in all flagellin bands that matched two homologous tandem flagellin genes identified in the C. botulinum Hall A genome. Designated flaA1 and flaA2, these open reading frames encode the major structural flagellins of C. botulinum. Colony PCR and sequencing of flaA1/A2 variable regions classified 80 environmental and clinical strains into group I or group II and clustered isolates into 12 flagellar types. Flagellar type was distinct from neurotoxin type, and epidemiologically related isolates clustered together. Sequencing a larger PCR product, obtained during amplification of flaA1/A2 from type E strain Bennett identified a second flagellin gene, flaB. LC-MS analysis confirmed that flaB encoded a large type E-specific flagellin protein, and the predicted molecular mass for FlaB matched that observed by SDS-PAGE. In contrast, the molecular mass of FlaA was 2 to 12 kDa larger than the mass predicted by the flaA1/A2 sequence of a given strain, suggesting that FlaA is posttranslationally modified. While identification of FlaB, and the observation by SDS-PAGE of different masses of the FlaA proteins, showed the flagellin proteins of C. botulinum to be diverse, the presence of the flaA1/A2 gene in all strains examined facilitates single locus sequence typing of C. botulinum using the flagellin variable region. PMID:17351097

Assignment of electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) to human chromosome 4q33 by fluorescence in situ hybridization and somatic cell hybridization.

PubMed

Spector, E B; Seltzer, W K; Goodman, S I

1999-08-01

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is a nuclear-encoded protein located in the inner mitochondrial membrane. Inherited defects of ETF-QO cause glutaric acidemia type II. We here describe the localization of the ETF-QO gene to human chromosome 4q33 by somatic cell hybridization and fluorescence in situ hybridization. Copyright 1999 Academic Press.

Molecular characterization of a nuclear topoisomerase II from Nicotiana tabacum that functionally complements a temperature-sensitive topoisomerase II yeast mutant.

PubMed

Singh, B N; Mudgil, Yashwanti; Sopory, S K; Reddy, M K

2003-07-01

We have successfully expressed enzymatically active plant topoisomerase II in Escherichia coli for the first time, which has enabled its biochemical characterization. Using a PCR-based strategy, we obtained a full-length cDNA and the corresponding genomic clone of tobacco topoisomerase II. The genomic clone has 18 exons interrupted by 17 introns. Most of the 5' and 3' splice junctions follow the typical canonical consensus dinucleotide sequence GU-AG present in other plant introns. The position of introns and phasing with respect to primary amino acid sequence in tobacco TopII and Arabidopsis TopII are highly conserved, suggesting that the two genes are evolved from the common ancestral type II topoisomerase gene. The cDNA encodes a polypeptide of 1482 amino acids. The primary amino acid sequence shows a striking sequence similarity, preserving all the structural domains that are conserved among eukaryotic type II topoisomerases in an identical spatial order. We have expressed the full-length polypeptide in E. coli and purified the recombinant protein to homogeneity. The full-length polypeptide relaxed supercoiled DNA and decatenated the catenated DNA in a Mg(2+)- and ATP-dependent manner, and this activity was inhibited by 4'-(9-acridinylamino)-3'-methoxymethanesulfonanilide (m-AMSA). The immunofluorescence and confocal microscopic studies, with antibodies developed against the N-terminal region of tobacco recombinant topoisomerase II, established the nuclear localization of topoisomerase II in tobacco BY2 cells. The regulated expression of tobacco topoisomerase II gene under the GAL1 promoter functionally complemented a temperature-sensitive TopII(ts) yeast mutant.

Toxin-Antitoxin Systems in the Mobile Genome of Acidithiobacillus ferrooxidans

PubMed Central

Bustamante, Paula; Tello, Mario; Orellana, Omar

2014-01-01

Toxin-antitoxin (TA) systems are genetic modules composed of a pair of genes encoding a stable toxin and an unstable antitoxin that inhibits toxin activity. They are widespread among plasmids and chromosomes of bacteria and archaea. TA systems are known to be involved in the stabilization of plasmids but there is no consensus about the function of chromosomal TA systems. To shed light on the role of chromosomally encoded TA systems we analyzed the distribution and functionality of type II TA systems in the chromosome of two strains from Acidithiobacillus ferrooxidans (ATCC 23270 and 53993), a Gram-negative, acidophilic, environmental bacterium that participates in the bioleaching of minerals. As in other environmental microorganisms, A. ferrooxidans has a high content of TA systems (28-29) and in twenty of them the toxin is a putative ribonuclease. According to the genetic context, some of these systems are encoded near or within mobile genetic elements. Although most TA systems are shared by both strains, four of them, which are encoded in the active mobile element ICEAfe1, are exclusive to the type strain ATCC 23270. We demostrated that two TA systems from ICEAfe1 are functional in E. coli cells, since the toxins inhibit growth and the antitoxins counteract the effect of their cognate toxins. All the toxins from ICEAfe1, including a novel toxin, are RNases with different ion requirements. The data indicate that some of the chromosomally encoded TA systems are actually part of the A. ferrooxidans mobile genome and we propose that could be involved in the maintenance of these integrated mobile genetic elements. PMID:25384039

Characterization of a recombinant type II 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Helicobacter pylori.

PubMed

Webby, Celia J; Patchett, Mark L; Parker, Emily J

2005-08-15

DAH7P (3-Deoxy-D-arabino-heptulosonate 7-phosphate) synthase catalyses the condensation reaction between phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P) as the first committed step in the biosynthesis of aromatic compounds in plants and micro-organisms. Previous work has identified two families of DAH7P synthases based on sequence similarity and molecular mass, with the majority of the mechanistic and structural studies being carried out on the type I paralogues from Escherichia coli. Whereas a number of organisms possess genes encoding both type I and type II DAH7P synthases, the pathogen Helicobacter pylori has only a single, type II, enzyme. Recombinant DAH7P synthase from H. pylori was partially solubilized by co-expression with chaperonins GroEL/GroES in E. coli, and purified to homogeneity. The enzyme reaction follows an ordered sequential mechanism with the following kinetic parameters: K(m) (PEP), 3 microM; K(m) (E4P), 6 microM; and kcat, 3.3 s(-1). The enzyme reaction involves interaction of the si face of PEP with the re face of E4P. H. pylori DAH7P synthase is not inhibited by phenylalanine, tyrosine, tryptophan or chorismate. EDTA inactivates the enzyme, and activity is restored by a range of bivalent metal ions, including (in order of decreasing effectiveness) Co2+, Mn2+, Ca2+, Mg2+, Cu2+ and Zn2+. Analysis of type II DAH7P synthase sequences reveals several highly conserved motifs, and comparison with the type I enzymes suggests that catalysis by these two enzyme types occurs on a similar active-site scaffold and that the two DAH7P synthase families may indeed be distantly related.

Restriction Fragment Length Polymorphisms of Virulence Plasmids in Rhodococcus equi

PubMed Central

Takai, Shinji; Shoda, Masato; Sasaki, Yukako; Tsubaki, Shiro; Fortier, Guillaume; Pronost, Stephane; Rahal, Karim; Becu, Teotimo; Begg, Angela; Browning, Glenn; Nicholson, Vivian M.; Prescott, John F.

1999-01-01

Virulent Rhodococcus equi, which is a well-known cause of pyogranulomatous pneumonia in foals, possesses a large plasmid encoding virulence-associated 15- to 17-kDa antigens. Foal and soil isolates from five countries—Argentina, Australia, Canada, France, and Japan—were investigated for the presence of 15- to 17-kDa antigens by colony blotting, using the monoclonal antibody 10G5, and the gene coding for 15- to 17-kDa antigens by PCR. Plasmid DNAs extracted from positive isolates were digested with restriction endonucleases BamHI, EcoRI, EcoT22I, and HindIII, and the digestion patterns that resulted divided the plasmids of virulent isolates into five closely related types. Three of the five types had already been reported in Canadian and Japanese isolates, and the two new types had been found in French and Japanese isolates. Therefore, we tentatively designated these five types 85-kb type I (pREAT701), 85-kb type II (a new type), 87-kb type I (EcoRI and BamHI type 2 [V. M. Nicholson and J. F. Prescott, J. Clin. Microbiol. 35:738–740, 1997]), 87-kb type II (a new type), and 90-kb (pREL1) plasmids. The 85-kb type I plasmid was found in isolates from Argentina, Australia, Canada, and France. Plasmid 87-kb type I was isolated in specimens from Argentina, Canada, and France. The 85-kb type II plasmid appeared in isolates from France. On the other hand, plasmids 87-kb type II and 90-kb were found only in isolates from Japan. These results revealed geographic differences in the distribution of the virulence plasmids found in the five countries and suggested that the restriction fragment length polymorphism of virulence plasmids might be useful to elucidate the molecular epidemiology of virulent R. equi in the world. PMID:10488224

Organization of genes required for the oxidation of methanol to formaldehyde in three type II methylotrophs

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

Bastien, C.; Machlin, S.; Zhang, Y.

Restriction maps of genes required for the synthesis of active methanol dehydrogenase in Methylobacterium organophilum XX and Methylobacterium sp. strain AM1 have been completed and compared. In these two species of pink-pigmented, type II methylotrophs, 15 genes were identified that were required for the expression of methanol dehydrogenase activity. None of these genes were required for the synthesis of the prosthetic group of methanol dehydrogenase, pyrroloquinoline quinone. The structural gene required for the synthesis of cytochrome c{sub L}, an electron acceptor uniquely required for methanol dehydrogenase, and the genes encoding small basic peptides that copurified with methanol dehydrogenases were closelymore » linked to the methanol dehydrogenase structural genes. A cloned 22-kilobase DNA insert from Methylsporovibrio methanica 81Z, an obligate type II methanotroph, complemented mutants that contained lesions in four genes closely linked to the methanol dehydrogenase structural genes. The methanol dehydrogenase and cytochrome c{sub L} structural genes were found to be transcribed independently in M. organophilum XX. Only two of the genes required for methanol dehydrogenase synthesis in this bacterium were found to be cotranscribed.« less

The oxygenating constituent of 3,6-diketocamphane monooxygenase from the CAM plasmid of Pseudomonas putida: the first crystal structure of a type II Baeyer–Villiger monooxygenase

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

Isupov, Michail N.; Schröder, Ewald; Gibson, Robert P.

The first crystal structure of a type II Baeyer–Villiger monooxygenase reveals a different ring orientation of its FMN cofactor compared with other related bacterial luciferase-family enzymes. The three-dimensional structures of the native enzyme and the FMN complex of the overexpressed form of the oxygenating component of the type II Baeyer–Villiger 3,6-diketocamphane monooxygenase have been determined to 1.9 Å resolution. The structure of this dimeric FMN-dependent enzyme, which is encoded on the large CAM plasmid of Pseudomonas putida, has been solved by a combination of multiple anomalous dispersion from a bromine crystal soak and molecular replacement using a bacterial luciferase model.more » The orientation of the isoalloxazine ring of the FMN cofactor in the active site of this TIM-barrel fold enzyme differs significantly from that previously observed in enzymes of the bacterial luciferase-like superfamily. The Ala77 residue is in a cis conformation and forms a β-bulge at the C-terminus of β-strand 3, which is a feature observed in many proteins of this superfamily.« less

Complementary DNA sequences encoding the multimammate rat MHC class II DQ alpha and beta chains and cross-species sequence comparison in rodents.

PubMed

de Bellocq, J Goüy; Leirs, H

2009-09-01

Sequences of the complete open reading frame (ORF) for rodents major histocompatibility complex (MHC) class II genes are rare. Multimammate rat (Mastomys natalensis) complementary DNA (cDNA) encoding the alpha and beta chains of MHC class II DQ gene was cloned from a rapid amplifications of cDNA Emds (RACE) cDNA library. The ORFs consist of 801 and 771 bp encoding 266 and 256 amino acid residues for DQB and DQA, respectively. The genomic structure of Mana-DQ genes is globally analogous to that described for other rodents except for the insertion of a serine residue in the signal peptide of Mana-DQB, which is unique among known rodents.

Novel Pelagic Iron-Oxidizing Zetaproteobacteria from the Chesapeake Bay Oxic-Anoxic Transition Zone.

PubMed

Chiu, Beverly K; Kato, Shingo; McAllister, Sean M; Field, Erin K; Chan, Clara S

2017-01-01

Chemolithotrophic iron-oxidizing bacteria (FeOB) could theoretically inhabit any environment where Fe(II) and O 2 (or nitrate) coexist. Until recently, marine Fe-oxidizing Zetaproteobacteria had primarily been observed in benthic and subsurface settings, but not redox-stratified water columns. This may be due to the challenges that a pelagic lifestyle would pose for Zetaproteobacteria, given low Fe(II) concentrations in modern marine waters and the possibility that Fe oxyhydroxide biominerals could cause cells to sink. However, we recently cultivated Zetaproteobacteria from the Chesapeake Bay oxic-anoxic transition zone, suggesting that they can survive and contribute to biogeochemical cycling in a stratified estuary. Here we describe the isolation, characterization, and genomes of two new species, Mariprofundus aestuarium CP-5 and Mariprofundus ferrinatatus CP-8, which are the first Zetaproteobacteria isolates from a pelagic environment. We looked for adaptations enabling strains CP-5 and CP-8 to overcome the challenges of living in a low Fe redoxcline with frequent O 2 fluctuations due to tidal mixing. We found that the CP strains produce distinctive dreadlock-like Fe oxyhydroxide structures that are easily shed, which would help cells maintain suspension in the water column. These oxides are by-products of Fe(II) oxidation, likely catalyzed by the putative Fe(II) oxidase encoded by the cyc2 gene, present in both CP-5 and CP-8 genomes; the consistent presence of cyc2 in all microaerophilic FeOB and other FeOB genomes supports its putative role in Fe(II) oxidation. The CP strains also have two gene clusters associated with biofilm formation (Wsp system and the Widespread Colonization Island) that are absent or rare in other Zetaproteobacteria. We propose that biofilm formation enables the CP strains to attach to FeS particles and form flocs, an advantageous strategy for scavenging Fe(II) and developing low [O 2 ] microenvironments within more oxygenated waters. However, the CP strains appear to be adapted to somewhat higher concentrations of O 2 , as indicated by the presence of genes encoding aa 3 -type cytochrome c oxidases, but not the cbb 3 -type found in all other Zetaproteobacteria isolate genomes. Overall, our results reveal adaptations for life in a physically dynamic, low Fe(II) water column, suggesting that niche-specific strategies can enable Zetaproteobacteria to live in any environment with Fe(II).

Novel Pelagic Iron-Oxidizing Zetaproteobacteria from the Chesapeake Bay Oxic–Anoxic Transition Zone

PubMed Central

Chiu, Beverly K.; Kato, Shingo; McAllister, Sean M.; Field, Erin K.; Chan, Clara S.

2017-01-01

Chemolithotrophic iron-oxidizing bacteria (FeOB) could theoretically inhabit any environment where Fe(II) and O2 (or nitrate) coexist. Until recently, marine Fe-oxidizing Zetaproteobacteria had primarily been observed in benthic and subsurface settings, but not redox-stratified water columns. This may be due to the challenges that a pelagic lifestyle would pose for Zetaproteobacteria, given low Fe(II) concentrations in modern marine waters and the possibility that Fe oxyhydroxide biominerals could cause cells to sink. However, we recently cultivated Zetaproteobacteria from the Chesapeake Bay oxic–anoxic transition zone, suggesting that they can survive and contribute to biogeochemical cycling in a stratified estuary. Here we describe the isolation, characterization, and genomes of two new species, Mariprofundus aestuarium CP-5 and Mariprofundus ferrinatatus CP-8, which are the first Zetaproteobacteria isolates from a pelagic environment. We looked for adaptations enabling strains CP-5 and CP-8 to overcome the challenges of living in a low Fe redoxcline with frequent O2 fluctuations due to tidal mixing. We found that the CP strains produce distinctive dreadlock-like Fe oxyhydroxide structures that are easily shed, which would help cells maintain suspension in the water column. These oxides are by-products of Fe(II) oxidation, likely catalyzed by the putative Fe(II) oxidase encoded by the cyc2 gene, present in both CP-5 and CP-8 genomes; the consistent presence of cyc2 in all microaerophilic FeOB and other FeOB genomes supports its putative role in Fe(II) oxidation. The CP strains also have two gene clusters associated with biofilm formation (Wsp system and the Widespread Colonization Island) that are absent or rare in other Zetaproteobacteria. We propose that biofilm formation enables the CP strains to attach to FeS particles and form flocs, an advantageous strategy for scavenging Fe(II) and developing low [O2] microenvironments within more oxygenated waters. However, the CP strains appear to be adapted to somewhat higher concentrations of O2, as indicated by the presence of genes encoding aa3-type cytochrome c oxidases, but not the cbb3-type found in all other Zetaproteobacteria isolate genomes. Overall, our results reveal adaptations for life in a physically dynamic, low Fe(II) water column, suggesting that niche-specific strategies can enable Zetaproteobacteria to live in any environment with Fe(II). PMID:28769885

Tolerance to MHC class II disparate allografts through genetic modification of bone marrow

PubMed Central

Jindra, Peter T.; Tripathi, Sudipta; Tian, Chaorui; Iacomini, John; Bagley, Jessamyn

2012-01-01

Induction of molecular chimerism through genetic modification of bone marrow is a powerful tool for the induction of tolerance. Here we demonstrate for the first time that expression of an allogeneic MHC class II gene in autologous bone marrow cells, resulting in a state of molecular chimerism, induces tolerance to MHC class II mismatched skin grafts, a stringent test of transplant tolerance. Reconstitution of recipients with syngeneic bone marrow transduced with retrovirus encoding H-2I-Ab (I-Ab) resulted the long-term expression of the retroviral gene product on the surface of MHC class II-expressing bone marrow derived cell types. Mechanistically, tolerance was maintained by the presence of regulatory T cells, which prevented proliferation and cytokine production by alloreactive host T cells. Thus, the introduction of MHC class II genes into bone marrow derived cells through genetic engineering results in tolerance. These results have the potential to extend the clinical applicability of molecular chimerism for tolerance induction. PMID:22833118

Substitution of aspartate for glycine 103 of the type II collagen triple helical domain: Identification of the minimal mutation which can produce Kniest dysplasia

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

Wilkin, D.J.; Rimoin, D.L.; Cohn, D.H.

1994-09-01

Kniest dysplasia is an autosomal dominant chondrodysplasia which results from mutations in the gene for type II collagen, COL2A1. Characteristics of the disorder include a short trunk and extremities, mid-face hypoplasia, cleft palate, myopia, retinal detachment, and hearing loss. Recently, deletions of all or part of exon 12 have been identified in individuals with Kniest dysplasia, suggesting that mutations within this region of the protein may primarily result in the Kniest dysplasia phenotype. We used SSCP to analyze an amplified genomic DNA fragment containing exon 12 from 7 individuals with Kniest dysplasia. An abnormality was identified in one patient. DNAmore » sequence analysis demonstrated that the patient was heterozygous for a G to A transition that implied substitution of glycine{sup 103} of the triple helix by aspartate. The mutation was not observed in DNA from either of the proband`s parents. Protein microsequencing demonstrated expression of the abnormal allele in the proband`s cartilage, indicating that the Kniest phenotype results from the presence of abnormal type II collagen molecules in the extracellular matrix. These data demonstrate the minimal mutation which can produce Kniest dysplasia and further support the hypothesis that alteration of a domain which includes the region encoded by exon 12 in the type II collagen protein leads to this disorder. Experiments designed to identify specific effects that mutations in this region have on intermolecular interactions among abnormal type II collagen molecules and other components of the cartilage extracellular matrix may clarify the underlying pathophysiology of Kniest dysplasia.« less

Omega-3 fatty acid supplementation decreases DNA damage in brain of rats subjected to a chemically induced chronic model of Tyrosinemia type II.

PubMed

Carvalho-Silva, Milena; Gomes, Lara M; Scaini, Giselli; Rebelo, Joyce; Damiani, Adriani P; Pereira, Maiara; Andrade, Vanessa M; Gava, Fernanda F; Valvassori, Samira S; Schuck, Patricia F; Ferreira, Gustavo C; Streck, Emilio L

2017-08-01

Tyrosinemia type II is an inborn error of metabolism caused by a mutation in a gene encoding the enzyme tyrosine aminotransferase leading to an accumulation of tyrosine in the body, and is associated with neurologic and development difficulties in numerous patients. Because the accumulation of tyrosine promotes oxidative stress and DNA damage, the main aim of this study was to investigate the possible antioxidant and neuroprotective effects of omega-3 treatment in a chemically-induced model of Tyrosinemia type II in hippocampus, striatum and cerebral cortex of rats. Our results showed chronic administration of L-tyrosine increased the frequency and the index of DNA damage, as well as the 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in the hippocampus, striatum and cerebral cortex. Moreover, omega-3 fatty acid treatment totally prevented increased DNA damage in the striatum and hippocampus, and partially prevented in the cerebral cortex, whereas the increase in 8-OHdG levels was totally prevented by omega-3 fatty acid treatment in hippocampus, striatum and cerebral cortex. In conclusion, the present study demonstrated that the main accumulating metabolite in Tyrosinemia type II induce DNA damage in hippocampus, striatum and cerebral cortex, possibly mediated by free radical production, and the supplementation with omega-3 fatty acids was able to prevent this damage, suggesting that could be involved in the prevention of oxidative damage to DNA in this disease. Thus, omega-3 fatty acids supplementation to Tyrosinemia type II patients may represent a new therapeutic approach and a possible adjuvant to the curren t treatment of this disease.

Identification of InuR, a new Zn(II)2Cys6 transcriptional activator involved in the regulation of inulinolytic genes in Aspergillus niger.

PubMed

Yuan, Xiao-Lian; Roubos, Johannes A; van den Hondel, Cees A M J J; Ram, Arthur F J

2008-01-01

The expression of inulinolytic genes in Aspergillus niger is co-regulated and induced by inulin and sucrose. We have identified a positive acting transcription factor InuR, which is required for the induced expression of inulinolytic genes. InuR is a member of the fungal specific class of transcription factors of the Zn(II)2Cys6 type. Involvement of InuR in inulin and sucrose metabolism was suspected because of the clustering of inuR gene with sucB, which encodes an intracellular invertase with transfructosylation activity and a putative sugar transporter encoding gene (An15g00310). Deletion of the inuR gene resulted in a strain displaying a severe reduction in growth on inulin and sucrose medium. Northern analysis revealed that expression of inulinolytic and sucrolytic genes, e.g., inuE, inuA, sucA, as well as the putative sugar transporter gene (An15g00310) is dependent on InuR. Genome-wide expression analysis revealed, three additional putative sugar transporters encoding genes (An15g04060, An15g03940 and An17g01710), which were strongly induced by sucrose in an InuR dependent way. In silico analysis of the promoter sequences of strongly InuR regulated genes suggests that InuR might bind as dimer to two CGG triplets, which are separated by eight nucleotides.

Distinct prophase arrest mechanisms in human male meiosis.

PubMed

Jan, Sabrina Z; Jongejan, Aldo; Korver, Cindy M; van Daalen, Saskia K M; van Pelt, Ans M M; Repping, Sjoerd; Hamer, Geert

2018-04-16

To prevent chromosomal aberrations being transmitted to the offspring, strict meiotic checkpoints are in place to remove aberrant spermatocytes. However, in about 1% of males these checkpoints cause complete meiotic arrest leading to azoospermia and subsequent infertility. Here, we unravel two clearly distinct meiotic arrest mechanisms that occur during prophase of human male meiosis. Type I arrested spermatocytes display severe asynapsis of the homologous chromosomes, disturbed XY-body formation and increased expression of the Y chromosome-encoded gene ZFY and seem to activate a DNA damage pathway leading to induction of p63, possibly causing spermatocyte apoptosis. Type II arrested spermatocytes display normal chromosome synapsis, normal XY-body morphology and meiotic crossover formation but have a lowered expression of several cell cycle regulating genes and fail to silence the X chromosome-encoded gene ZFX Discovery and understanding of these meiotic arrest mechanisms increases our knowledge of how genomic stability is guarded during human germ cell development. © 2018. Published by The Company of Biologists Ltd.

Organellar maturases: A window into the evolution of the spliceosome.

PubMed

Schmitz-Linneweber, Christian; Lampe, Marie-Kristin; Sultan, Laure D; Ostersetzer-Biran, Oren

2015-09-01

During the evolution of eukaryotic genomes, many genes have been interrupted by intervening sequences (introns) that must be removed post-transcriptionally from RNA precursors to form mRNAs ready for translation. The origin of nuclear introns is still under debate, but one hypothesis is that the spliceosome and the intron-exon structure of genes have evolved from bacterial-type group II introns that invaded the eukaryotic genomes. The group II introns were most likely introduced into the eukaryotic genome from an α-proteobacterial predecessor of mitochondria early during the endosymbiosis event. These self-splicing and mobile introns spread through the eukaryotic genome and later degenerated. Pieces of introns became part of the general splicing machinery we know today as the spliceosome. In addition, group II introns likely brought intron maturases with them to the nucleus. Maturases are found in most bacterial introns, where they act as highly specific splicing factors for group II introns. In the spliceosome, the core protein Prp8 shows homology to group II intron-encoded maturases. While maturases are entirely intron specific, their descendant of the spliceosomal machinery, the Prp8 protein, is an extremely versatile splicing factor with multiple interacting proteins and RNAs. How could such a general player in spliceosomal splicing evolve from the monospecific bacterial maturases? Analysis of the organellar splicing machinery in plants may give clues on the evolution of nuclear splicing. Plants encode various proteins which are closely related to bacterial maturases. The organellar genomes contain one maturase each, named MatK in chloroplasts and MatR in mitochondria. In addition, several maturase genes have been found in the nucleus as well, which are acting on mitochondrial pre-RNAs. All plant maturases show sequence deviation from their progenitor bacterial maturases, and interestingly are all acting on multiple organellar group II intron targets. Moreover, they seem to function in the splicing of group II introns together with a number of additional nuclear-encoded splicing factors, possibly acting as an organellar proto-spliceosome. Together, this makes them interesting models for the early evolution of nuclear spliceosomal splicing. In this review, we summarize recent advances in our understanding of the role of plant maturases and their accessory factors in plants. This article is part of a Special Issue entitled: Chloroplast Biogenesis. Copyright © 2015 Elsevier B.V. All rights reserved.

Generation of the first Autosomal Dominant Osteopetrosis Type II (ADO2) disease models

PubMed Central

Alam, Imranul; Gray, Amie K.; Chu, Kang; Ichikawa, Shoji; Mohammad, Khalid S.; Capannolo, Marta; Capulli, Mattia; Maurizi, Antonio; Muraca, Maurizio; Teti, Anna; Econs, Michael J.; Fattore, Andrea Del

2013-01-01

Autosomal Dominant Osteopetrosis Type II (ADO2) is a heritable osteosclerotic disorder dependent on osteoclast impairment. In most patients it results from heterozygous missense mutations in the chloride channel 7 (CLCN7) gene, encoding for a 2Cl−/1H+ antiporter. By a knock-in strategy inserting a missense mutation in the Clcn7 gene, our two research groups independently generated mouse models of ADO2 on different genetic backgrounds carrying the homolog of the most frequent heterozygous mutation (p.G213R) in the Clcn7 gene found in humans. Our results demonstrate that the heterozygous model holds true presenting with higher bone mass, increased numbers of poorly resorbing osteoclasts and a lethal phenotype in the homozygous state. Considerable variability is observed in the heterozygous mice according with the mouse background, suggesting that modifier genes could influence the penetrance of the disease gene. PMID:24185277

Molecular characterization of the major membrane skeletal protein in the ciliate Tetrahymena pyriformis suggests n-plication of an early evolutionary intermediate filament protein subdomain.

PubMed

Bouchard, P; Chomilier, J; Ravet, V; Mornon, J P; Viguès, B

2001-01-01

Epiplasmin C is the major protein component of the membrane skeleton in the ciliate Tetrahymena pyriformis. Cloning and analysis of the gene encoding epiplasmin C showed this protein to be a previously unrecognized protein. In particular, epiplasmin C was shown to lack the canonical features of already known epiplasmic proteins in ciliates and flagellates. By means of hydrophobic cluster analysis (HCA), it has been shown that epiplasmin C is constituted of a repeat of 25 domains of 40 residues each. These domains are related and can be grouped in two families called types I and types II. Connections between types I and types II present rules that can be evidenced in the sequence itself, thus enforcing the validity of the splitting of the domains. Using these repeated domains as queries, significant structural similarities were demonstrated with an extra six heptads shared by nuclear lamins and invertebrate cytoplasmic intermediate filament proteins and deleted in the cytoplasmic intermediate filament protein lineage at the protostome-deuterostome branching in the eukaryotic phylogenetic tree.

Utilization of Natural Fucosylated Oligosaccharides by Three Novel α-l-Fucosidases from a Probiotic Lactobacillus casei Strain ▿

PubMed Central

Rodríguez-Díaz, Jesús; Monedero, Vicente; Yebra, María J.

2011-01-01

Three putative α-l-fucosidases encoded in the Lactobacillus casei BL23 genome were cloned and purified. The proteins displayed different abilities to hydrolyze natural fucosyloligosaccharides like 2′-fucosyllactose, H antigen disaccharide, H antigen type II trisaccharide, and 3′-, 4′-, and 6′-fucosyl-GlcNAc. This indicated a possible role in the utilization of oligosaccharides present in human milk and intestinal mucosa. PMID:21097595

Mutations in TMPRSS6 cause iron-refractory iron deficiency anemia (IRIDA)

PubMed Central

Finberg, Karin E; Heeney, Matthew M; Campagna, Dean R; Aydınok, Yeşim; Pearson, Howard A; Hartman, Kip R; Mayo, Mary M; Samuel, Stewart M; Strouse, John J; Markianos, Kyriacos; Andrews, Nancy C; Fleming, Mark D

2011-01-01

Iron deficiency is usually attributed to chronic blood loss or inadequate dietary intake. Here, we show that iron deficiency anemia refractory to oral iron therapy can be caused by germline mutations in TMPRSS6, which encodes a type II transmembrane serine protease produced by the liver that regulates the expression of the systemic iron regulatory hormone hepcidin. These findings demonstrate that TMPRSS6 is essential for normal systemic iron homeostasis in humans. PMID:18408718

Systemic correction of the muscle disorder glycogen storage disease type II after hepatic targeting of a modified adenovirus vector encoding human acid-α-glucosidase

PubMed Central

Amalfitano, A.; McVie-Wylie, A. J.; Hu, H.; Dawson, T. L.; Raben, N.; Plotz, P.; Chen, Y. T.

1999-01-01

This report demonstrates that a single intravenous administration of a gene therapy vector can potentially result in the correction of all affected muscles in a mouse model of a human genetic muscle disease. These results were achieved by capitalizing both on the positive attributes of modified adenovirus-based vectoring systems and receptor-mediated lysosomal targeting of enzymes. The muscle disease treated, glycogen storage disease type II, is a lysosomal storage disorder that manifests as a progressive myopathy, secondary to massive glycogen accumulations in the skeletal and/or cardiac muscles of affected individuals. We demonstrated that a single intravenous administration of a modified Ad vector encoding human acid α-glucosidase (GAA) resulted in efficient hepatic transduction and secretion of high levels of the precursor GAA proenzyme into the plasma of treated animals. Subsequently, systemic distribution and uptake of the proenzyme into the skeletal and cardiac muscles of the GAA-knockout mouse was confirmed. As a result, systemic decreases (and correction) of the glycogen accumulations in a variety of muscle tissues was demonstrated. This model can potentially be expanded to include the treatment of other lysosomal enzyme disorders. Lessons learned from systemic genetic therapy of muscle disorders also should have implications for other muscle diseases, such as the muscular dystrophies. PMID:10430861

Primary structure and mapping of the hupA gene of Salmonella typhimurium.

PubMed Central

Higgins, N P; Hillyard, D

1988-01-01

In bacteria, the complex nucleoid structure is folded and maintained by negative superhelical tension and a set of type II DNA-binding proteins, also called histonelike proteins. The most abundant type II DNA-binding protein is HU. Southern blot analysis showed that Salmonella typhimurium contained two HU genes that corresponded to Escherichia coli genes hupA (encoding HU-2 protein) and hupB (encoding HU-1). Salmonella hupA was cloned, and the nucleotide sequence of the gene was determined. Comparison of hupA of E. coli and S. typhimurium revealed that the HU-2 proteins were identical and that there was high conservation of nucleotide sequences outside the coding frames of the genes. A 300-member genomic library of S. typhimurium was constructed by using random transposition of MudP, a specialized chimeric P22-Mu phage that packages chromosomal DNA unidirectionally from its insertion point. Oligonucleotide hybridization against the library identified one MudP insertion that lies within 28 kilobases of hupA; the MudP was 12% linked to purH at 90.5 min on the standard map. Plasmids expressing HU-2 had a surprising phenotype; they caused growth arrest when they were introduced into E. coli strains bearing a himA or hip mutation. These results suggest that IHF and HU have interactive roles in bacteria. Images PMID:3056912

Localization of human T-cell lymphotropic virus type II Tax protein is dependent upon a nuclear localization determinant in the N-terminal region.

PubMed

Turci, Marco; Romanelli, Maria Grazia; Lorenzi, Pamela; Righi, Paola; Bertazzoni, Umberto

2006-01-03

Human T-cell lymphotropic viruses (HTLV) types I and II are closely related oncogenic retroviruses that have been associated with lymphoproliferative and neurological disorders. The proviral genome encodes a trans-regulatory Tax protein that activates viral genes and upregulates various cellular genes involved in both cell growth and transformation. Tax proteins of HTLV-I (Tax-I) and HTLV-II (Tax-II) exhibit more than 77% aa homology and expression of either Tax-I or Tax-II is sufficient for immortalization of cultured T lymphocytes. Tax-I shuttles from the nucleus to the cytoplasm and accumulates within the nucleus, whereas Tax-II is found mainly in the cytoplasm. In the present study we have used recombinant vectors to analyze the size and structure of the nuclear localization domain within the Tax-II protein sequence. The Tax-II protein was expressed in HeLa cells either as the complete protein, or regions thereof, that were individually fused to the green fluorescent protein (GFP). Immunoblot analysis of the fused Tax-II products confirmed their expression and size. Fluorescence microscopy studies indicated that the complete Tax-II as well as N-truncated forms presented a punctuate cytoplasmic distribution and that a nuclear localization determinant is confined to within the first 60 aa of Tax-II. Accordingly, site directed mutagenesis and deletion of specific sequences within the first 60 aa showed that the nuclear determinant lies within the first 41 residues of Tax-II. These results point to a direct involvement of the amino-terminal residues of Tax-II protein in determining its nuclear functionality.

[Molecular epidemiology of methicillin-resistant Staphylococcus aureus isolates with toxic shock syndrome toxin and staphylococcal enterotoxin C genes].

PubMed

Kim, Jae Seok; Kim, Han Sung; Song, Wonkeun; Cho, Hyoun Chan; Lee, Kyu Man; Kim, Eui Chong

2007-04-01

Many methicillin-resistant Staphylococcus aureus (MRSA) isolates in Korea possess a specific profile of staphylococcal enterotoxins in that the toxic shock syndrome toxin gene (tst) coexists with the staphylococcal enterotoxin C gene (sec). Because the analysis of staphylococcal cassette chromosome mec (SCCmec), a mobile genetic element mecA gene encoding methicillin resistance, showed that majority of these are SCCmec type II, these MRSA isolates with tst and sec may be genetically related with each other. This study was performed to investigate the genetic relatedness of tstand sec-harboring MRSA strains isolated in Korea by using pulsed-field gel electrophoresis (PFGE). A total of 59 strains of MRSA isolates of SCCmec type II possessing tst and sec were selected for PFGE and phylogenetic analyses. These isolates were collected from 13 health care facilities during nationwide surveillance of antimicrobial resistance in 2002. The 59 MRSA isolates were clustered into 11 PFGE types, including one major group of 26 strains (44.1%) isolated from 7 healthcare facilities. Seven PFGE types contained 2 or more isolates each, comprising 55 isolates in total. Most of SCCmec type II MRSA isolates containing tst and sec showed closely related PFGE patterns. Moreover, MRSA isolates collected from different healthcare facilities showed identical PFGE patterns. These findings suggested a clonal spread of MRSA strains possessing tst and sec in Korean hospitals.

The polymorphism of YWHAE, a gene encoding 14-3-3epsilon, and orbitofrontal sulcogyral pattern in patients with schizophrenia and healthy subjects.

PubMed

Takahashi, Tsutomu; Nakamura, Yumiko; Nakamura, Yukako; Aleksic, Branko; Takayanagi, Yoichiro; Furuichi, Atsushi; Kido, Mikio; Nakamura, Mihoko; Sasabayashi, Daiki; Ikeda, Masashi; Noguchi, Kyo; Kaibuchi, Kozo; Iwata, Nakao; Ozaki, Norio; Suzuki, Michio

2014-06-03

An altered sulcogyral pattern in the orbitofrontal cortex (OFC) has been implicated in schizophrenia as a possible marker of abnormal neurodevelopment, while its genetic mechanism remains unknown. This magnetic resonance imaging study investigated the relationship between the polymorphism of YWHAE (rs28365859), a gene encoding 14-3-3epsilon that is a Disrupted-in-Schizophrenia 1 (DISC1)-interacting molecule associated with neuronal development, and the OFC subtypes of the 'H-shaped' sulcus (Types I, II, and III) in a Japanese sample of 72 schizophrenia patients and 86 healthy controls. The schizophrenia patients had significantly increased Type III (p = 0.004) and decreased Type I (p = 0.013) expression on the right hemisphere compared to the controls. The subjects carrying the protective C allele showed a decrease in Type III (p = 0.005) and an increase in Type I (p = 0.017) compared to the G allele homozygotes, especially for the healthy subjects in the left hemisphere. These results suggest a possible role for the YWHAE genotype in the early development of the OFC sulcogyral pattern, but its effect alone is not likely to explain the altered sulcogyral pattern in schizophrenia. Copyright © 2014 Elsevier Inc. All rights reserved.

New steroid 5alpha-reductase type I (SRD5A1) homologous sequences on human chromosomes 6 and 8.

PubMed

Eminović, I; Liović, M; Prezelj, J; Kocijancic, A; Rozman, D; Komel, R

2001-01-01

To date, two genes encoding 5alpha-reductase isoenzymes are known (type I, type II), and one type I pseudogene. The divergent localization of these genes and the still not fully understood function of the encoded enzymes as well as the perplexing results we obtained after sequencing PCR-amplified SRD5A1 gene fragments (out of genomic DNA), made us assume that, in addition to the known SRD5A1 gene, one or more different human 5alpha-reductase type I coding genes may exist. Our research provide the first evidence for the existence of two new SRD5A1 related, previously unidentified sequences in the human genome. These sequences which were localized to chromosomes 6 and 8 are highly homologous (> 99%) to SRD5A1, and also do not contain any deletions or insertions that are otherwise a characteristic of the SRD5API pseudogene. Our results imply that these sequences may be either coding parts of yet unknown, active SRD5A1 genes, and/or of previously unidentified pseudogenes. These findings additionally support data of Chen et al. who confirmed the existence of various SRD5A1 proteins in cultured human skin cells.

Two functionally distinct NADP+-dependent ferredoxin oxidoreductases maintain the primary redox balance of Pyrococcus furiosus.

PubMed

Nguyen, Diep M N; Schut, Gerrit J; Zadvornyy, Oleg A; Tokmina-Lukaszewska, Monika; Poudel, Saroj; Lipscomb, Gina L; Adams, Leslie A; Dinsmore, Jessica T; Nixon, William J; Boyd, Eric S; Bothner, Brian; Peters, John W; Adams, Michael W W

2017-09-01

Electron bifurcation has recently gained acceptance as the third mechanism of energy conservation in which energy is conserved through the coupling of exergonic and endergonic reactions. A structure-based mechanism of bifurcation has been elucidated recently for the flavin-based enzyme NADH-dependent ferredoxin NADP + oxidoreductase I (NfnI) from the hyperthermophillic archaeon Pyrococcus furiosus. NfnI is thought to be involved in maintaining the cellular redox balance, producing NADPH for biosynthesis by recycling the two other primary redox carriers, NADH and ferredoxin. The P. furiosus genome encodes an NfnI paralog termed NfnII, and the two are differentially expressed, depending on the growth conditions. In this study, we show that deletion of the genes encoding either NfnI or NfnII affects the cellular concentrations of NAD(P)H and particularly NADPH. This results in a moderate to severe growth phenotype in deletion mutants, demonstrating a key role for each enzyme in maintaining redox homeostasis. Despite their similarity in primary sequence and cofactor content, crystallographic, kinetic, and mass spectrometry analyses reveal that there are fundamental structural differences between the two enzymes, and NfnII does not catalyze the NfnI bifurcating reaction. Instead, it exhibits non-bifurcating ferredoxin NADP oxidoreductase-type activity. NfnII is therefore proposed to be a bifunctional enzyme and also to catalyze a bifurcating reaction, although its third substrate, in addition to ferredoxin and NADP(H), is as yet unknown. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Virulence factors encoded by Legionella longbeachae identified on the basis of the genome sequence analysis of clinical isolate D-4968.

PubMed

Kozak, Natalia A; Buss, Meghan; Lucas, Claressa E; Frace, Michael; Govil, Dhwani; Travis, Tatiana; Olsen-Rasmussen, Melissa; Benson, Robert F; Fields, Barry S

2010-02-01

Legionella longbeachae causes most cases of legionellosis in Australia and may be underreported worldwide due to the lack of L. longbeachae-specific diagnostic tests. L. longbeachae displays distinctive differences in intracellular trafficking, caspase 1 activation, and infection in mouse models compared to Legionella pneumophila, yet these two species have indistinguishable clinical presentations in humans. Unlike other legionellae, which inhabit freshwater systems, L. longbeachae is found predominantly in moist soil. In this study, we sequenced and annotated the genome of an L. longbeachae clinical isolate from Oregon, isolate D-4968, and compared it to the previously published genomes of L. pneumophila. The results revealed that the D-4968 genome is larger than the L. pneumophila genome and has a gene order that is different from that of the L. pneumophila genome. Genes encoding structural components of type II, type IV Lvh, and type IV Icm/Dot secretion systems are conserved. In contrast, only 42/140 homologs of genes encoding L. pneumophila Icm/Dot substrates have been found in the D-4968 genome. L. longbeachae encodes numerous proteins with eukaryotic motifs and eukaryote-like proteins unique to this species, including 16 ankyrin repeat-containing proteins and a novel U-box protein. We predict that these proteins are secreted by the L. longbeachae Icm/Dot secretion system. In contrast to the L. pneumophila genome, the L. longbeachae D-4968 genome does not contain flagellar biosynthesis genes, yet it contains a chemotaxis operon. The lack of a flagellum explains the failure of L. longbeachae to activate caspase 1 and trigger pyroptosis in murine macrophages. These unique features of L. longbeachae may reflect adaptation of this species to life in soil.

HTLV-I and HTLV-II Tax: differences in induction of micronuclei in cells and transcriptional activation of viral LTRs.

PubMed

Semmes, O J; Majone, F; Cantemir, C; Turchetto, L; Hjelle, B; Jeang, K T

1996-03-01

Human T-cell leukemia virus (HTLV) types I and II are highly related viruses that differ in disease manifestations. HTLV-I has been linked unmistakably to adult T-cell leukemia-lymphoma. On the other hand, there is little evidence that prior infection with HTLV-II increases risk for lymphoproliferative disorders. Both viruses encode homologous transcriptional-activating proteins (respectively designated as Tax1 and Tax2) which have been suggested to be important mediators of viral pathogenesis. Previously, we reported that Tax1 is a potent inducer of micronuclei formation in cells. Here, we present evidence that Tax2 lacks micronuclei inductive ability. We contrast this phenotypic difference between Tax1 and Tax2 at the cellular level with their similarities at the molecular level in transcriptional activation.

Histopathological evaluation of the diversity of cells susceptible to H5N1 virulent avian influenza virus.

PubMed

Ogiwara, Haru; Yasui, Fumihiko; Munekata, Keisuke; Takagi-Kamiya, Asako; Munakata, Tsubasa; Nomura, Namiko; Shibasaki, Futoshi; Kuwahara, Kazuhiko; Sakaguchi, Nobuo; Sakoda, Yoshihiro; Kida, Hiroshi; Kohara, Michinori

2014-01-01

Patients infected with highly pathogenic avian influenza A H5N1 viruses (H5N1 HPAIV) show diffuse alveolar damage. However, the temporal progression of tissue damage and repair after viral infection remains poorly defined. Therefore, we assessed the sequential histopathological characteristics of mouse lung after intranasal infection with H5N1 HPAIV or H1N1 2009 pandemic influenza virus (H1N1 pdm). We determined the amount and localization of virus in the lung through IHC staining and in situ hybridization. IHC used antibodies raised against the virus protein and antibodies specific for macrophages, type II pneumocytes, or proliferating cell nuclear antigen. In situ hybridization used RNA probes against both viral RNA and mRNA encoding the nucleoprotein and the hemagglutinin protein. H5N1 HPAIV infection and replication were observed in multiple lung cell types and might result in rapid progression of lung injury. Both type II pneumocytes and macrophages proliferated after H5N1 HPAIV infection. However, the abundant macrophages failed to block the viral attack, and proliferation of type II pneumocytes failed to restore the damaged alveoli. In contrast, mice infected with H1N1 pdm exhibited modest proliferation of type II pneumocytes and macrophages and slight alveolar damage. These results suggest that the virulence of H5N1 HPAIV results from the wide range of cell tropism of the virus, excessive virus replication, and rapid development of diffuse alveolar damage. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Histopathological Evaluation of the Diversity of Cells Susceptible to H5N1 Virulent Avian Influenza Virus

PubMed Central

Ogiwara, Haru; Yasui, Fumihiko; Munekata, Keisuke; Takagi-Kamiya, Asako; Munakata, Tsubasa; Nomura, Namiko; Shibasaki, Futoshi; Kuwahara, Kazuhiko; Sakaguchi, Nobuo; Sakoda, Yoshihiro; Kida, Hiroshi; Kohara, Michinori

2015-01-01

Patients infected with highly pathogenic avian influenza A H5N1 viruses (H5N1 HPAIV) show diffuse alveolar damage. However, the temporal progression of tissue damage and repair after viral infection remains poorly defined. Therefore, we assessed the sequential histopathological characteristics of mouse lung after intranasal infection with H5N1 HPAIV or H1N1 2009 pandemic influenza virus (H1N1 pdm). We determined the amount and localization of virus in the lung through IHC staining and in situ hybridization. IHC used antibodies raised against the virus protein and antibodies specific for macrophages, type II pneumocytes, or proliferating cell nuclear antigen. In situ hybridization used RNA probes against both viral RNA and mRNA encoding the nucleoprotein and the hemagglutinin protein. H5N1 HPAIV infection and replication were observed in multiple lung cell types and might result in rapid progression of lung injury. Both type II pneumocytes and macrophages proliferated after H5N1 HPAIV infection. However, the abundant macrophages failed to block the viral attack, and proliferation of type II pneumocytes failed to restore the damaged alveoli. In contrast, mice infected with H1N1 pdm exhibited modest proliferation of type II pneumocytes and macrophages and slight alveolar damage. These results suggest that the virulence of H5N1 HPAIV results from the wide range of cell tropism of the virus, excessive virus replication, and rapid development of diffuse alveolar damage. PMID:24200852

A novel angiotensin II type 1 receptor-associated protein induces cellular hypertrophy in rat vascular smooth muscle and renal proximal tubular cells.

PubMed

Guo, Deng-Fu; Tardif, Valerie; Ghelima, Karin; Chan, John S D; Ingelfinger, Julie R; Chen, XiangMei; Chenier, Isabelle

2004-05-14

Angiotensin II stimulates cellular hypertrophy in cultured vascular smooth muscle and renal proximal tubular cells. This effect is believed to be one of earliest morphological changes of heart and renal failure. However, the precise molecular mechanism involved in angiotensin II-induced hypertrophy is poorly understood. In the present study we report the isolation of a novel angiotensin II type 1 receptor-associated protein. It encodes a 531-amino acid protein. Its mRNA is detected in all human tissues examined but highly expressed in the human kidney, pancreas, heart, and human embryonic kidney cells as well as rat vascular smooth muscle and renal proximal tubular cells. Protein synthesis and relative cell size analyzed by flow cytometry studies indicate that overexpression of the novel angiotensin II type 1 receptor-associated protein induces cellular hypertrophy in cultured rat vascular smooth muscle and renal proximal tubular cells. In contrast, the hypertrophic effects was reversed in renal proximal tubular cell lines expressing the novel gene in the antisense orientation and its dominant negative mutant, which lacks the last 101 amino acids in its carboxyl-terminal tail. The hypertrophic effects are at least in part mediated via protein kinase B activation or cyclin-dependent kinase inhibitor, p27(kip1) protein expression level in vascular smooth muscle, and renal proximal tubular cells. Moreover, angiotensin II could not stimulate cellular hypertrophy in renal proximal tubular cells expressing the novel gene in the antisense orientation and its mutant. These findings may provide new molecular mechanisms to understand hypertrophic agents such as angiotensin II-induced cellular hypertrophy.

Molecular Gene Profiling of Clostridium botulinum Group III and Its Detection in Naturally Contaminated Samples Originating from Various European Countries

PubMed Central

Woudstra, Cedric; Le Maréchal, Caroline; Souillard, Rozenn; Bayon-Auboyer, Marie-Hélène; Anniballi, Fabrizio; Auricchio, Bruna; De Medici, Dario; Bano, Luca; Koene, Miriam; Sansonetti, Marie-Hélène; Desoutter, Denise; Hansbauer, Eva-Maria; Dorner, Martin B.; Dorner, Brigitte G.

2015-01-01

We report the development of real-time PCR assays for genotyping Clostridium botulinum group III targeting the newly defined C. novyi sensu lato group; the nontoxic nonhemagglutinin (NTNH)-encoding gene ntnh; the botulinum neurotoxin (BoNT)-encoding genes bont/C, bont/C/D, bont/D, and bont/D/C; and the flagellin (fliC) gene. The genetic diversity of fliC among C. botulinum group III strains resulted in the definition of five major subgroups named fliC-I to fliC-V. Investigation of fliC subtypes in 560 samples, with various European origins, showed that fliC-I was predominant and found exclusively in samples contaminated by C. botulinum type C/D, fliC-II was rarely detected, no sample was recorded as fliC-III or fliC-V, and only C. botulinum type D/C samples tested positive for fliC-IV. The lack of genetic diversity of the flagellin gene of C. botulinum type C/D would support a clonal spread of type C/D strains in different geographical areas. fliC-I to fliC-III are genetically related (87% to 92% sequence identity), whereas fliC-IV from C. botulinum type D/C is more genetically distant from the other fliC types (with only 50% sequence identity). These findings suggest fliC-I to fliC-III have evolved in a common environment and support a different genetic evolution for fliC-IV. A combination of the C. novyi sensu lato, ntnh, bont, and fliC PCR assays developed in this study allowed better characterization of C. botulinum group III and showed the group to be less genetically diverse than C. botulinum groups I and II, supporting a slow genetic evolution of the strains belonging to C. botulinum group III. PMID:25636839

Discovery of the type VII ESX-1 secretion needle?

PubMed

Ates, Louis S; Brosch, Roland

2017-01-01

Mycobacterium tuberculosis, the etiological agent of human tuberculosis, harbours five ESAT-6/type VII secretion (ESX/T7S) systems. The first esx gene clusters were identified during the genome-sequencing project of M. tuberculosis H37Rv. Follow-up studies revealed additional genes playing important roles in ESX/T7S systems. Among the latter genes, one can find those that encode Pro-Glu (PE) and Pro-Pro-Glu (PPE) proteins as well as a gene cluster that is encoded >260 kb upstream of the esx-1 locus and encodes ESX-1 secretion-associated proteins EspA (Rv3616c), EspC (Rv3615c) and EspD (Rv3614c). The espACD cluster has been suggested to have an important function in ESX-1 secretion since EspA-EspC and EsxA-EsxB are mutually co-dependent on each other for secretion. However, the molecular mechanism of this co-dependence and interaction between the substrates remained unknown. In this issue of Molecular Microbiology, Lou and colleagues show that EspC forms high-molecular weight polymerization complexes that resemble selected components of type II, III and/or IV secretion systems of Gram-negative bacteria. Indeed, EspC-multimeric complexes form filamentous structures that could well represent a secretion needle of ESX-1 type VII secretion systems. This exciting observation opens new avenues for research to discover and characterize ESX/T7S components and elucidates the co-dependence of EsxA/B secretion with EspA/C. © 2016 John Wiley & Sons Ltd.

Isolation and characterization of a cDNA from Cuphea lanceolata encoding a beta-ketoacyl-ACP reductase.

PubMed

Klein, B; Pawlowski, K; Höricke-Grandpierre, C; Schell, J; Töpfer, R

1992-05-01

A cDNA encoding beta-ketoacyl-ACP reductase (EC 1.1.1.100), an integral part of the fatty acid synthase type II, was cloned from Cuphea lanceolata. This cDNA of 1276 bp codes for a polypeptide of 320 amino acids with 63 N-terminal residues presumably representing a transit peptide and 257 residues corresponding to the mature protein of 27 kDa. The encoded protein shows strong homology with the amino-terminal sequence and two tryptic peptides from avocado mesocarp beta-ketoacyl-ACP reductase, and its total amino acid composition is highly similar to those of the beta-ketoacyl-ACP reductases of avocado and spinach. Amino acid sequence homologies to polyketide synthase, beta-ketoreductases and short-chain alcohol dehydrogenases are discussed. An engineered fusion protein lacking most of the transit peptide, which was produced in Escherichia coli, was isolated and proved to possess beta-ketoacyl-ACP reductase activity. Hybridization studies revealed that in C. lanceolata beta-ketoacyl-ACP reductase is encoded by a small family of at least two genes and that members of this family are expressed in roots, leaves, flowers and seeds.

Novel Humanized mice to test Therapeutics for Human Type 1 Diabetes

DTIC Science & Technology

2014-01-06

performed in non- obese diabetic (NOD) mice, the closest animal model for human T1D, have identified different immune cells involved in pancreatic β-cell...periphery. Each MHC class II chain contributes to the formation of a groove where the peptide is embedded (78). 17 The polygenetic factor underlying...diabetes mellitus in non- obese diabetic mice by transgenes encoding modified I-A beta-chain or normal I-E alpha-chain. Nature 345:727-9 63. Marek

Proteomic analysis reveals novel extracellular virulence-associated proteins and functions regulated by the diffusible signal factor (DSF) in Xanthomonas oryzae pv. oryzicola.

PubMed

Qian, Guoliang; Zhou, Yijing; Zhao, Yancun; Song, Zhiwei; Wang, Suyan; Fan, Jiaqin; Hu, Baishi; Venturi, Vittorio; Liu, Fengquan

2013-07-05

Quorum sensing (QS) in Xanthomonas oryzae pv. oryzicola (Xoc), the causal agent of bacterial leaf streak, is mediated by the diffusible signal factor (DSF). DSF-mediating QS has been shown to control virulence and a set of virulence-related functions; however, the expression profiles and functions of extracellular proteins controlled by DSF signal remain largely unclear. In the present study, 33 DSF-regulated extracellular proteins, whose functions include small-protein mediating QS, oxidative adaptation, macromolecule metabolism, cell structure, biosynthesis of small molecules, intermediary metabolism, cellular process, protein catabolism, and hypothetical function, were identified by proteomics in Xoc. Of these, 15 protein encoding genes were in-frame deleted, and 4 of them, including three genes encoding type II secretion system (T2SS)-dependent proteins and one gene encoding an Ax21 (activator of XA21-mediated immunity)-like protein (a novel small-protein type QS signal) were determined to be required for full virulence in Xoc. The contributions of these four genes to important virulence-associated functions, including bacterial colonization, extracellular polysaccharide, cell motility, biofilm formation, and antioxidative ability, are presented. To our knowledge, our analysis is the first complete list of DSF-regulated extracellular proteins and functions in a Xanthomonas species. Our results show that DSF-type QS played critical roles in regulation of T2SS and Ax21-mediating QS, which sheds light on the role of DSF signaling in Xanthomonas.

Revisiting the NaCl cotransporter regulation by with-no-lysine kinases

PubMed Central

Bazúa-Valenti, Silvana

2015-01-01

The renal thiazide-sensitive Na+-Cl− cotransporter (NCC) is the salt transporter in the distal convoluted tubule. Its activity is fundamental for defining blood pressure levels. Decreased NCC activity is associated with salt-remediable arterial hypotension with hypokalemia (Gitelman disease), while increased activity results in salt-sensitive arterial hypertension with hyperkalemia (pseudohypoaldosteronism type II; PHAII). The discovery of four different genes causing PHAII revealed a complex multiprotein system that regulates the activity of NCC. Two genes encode for with-no-lysine (K) kinases WNK1 and WNK4, while two encode for kelch-like 3 (KLHL3) and cullin 3 (CUL3) proteins that form a RING type E3 ubiquitin ligase complex. Extensive research has shown that WNK1 and WNK4 are the targets for the KLHL3-CUL3 complex and that WNKs modulate the activity of NCC by means of intermediary Ste20-type kinases known as SPAK or OSR1. The understanding of the effect of WNKs on NCC is a complex issue, but recent evidence discussed in this review suggests that we could be reaching the end of the dark ages regarding this matter. PMID:25788573

Arabidopsis Class I and Class II TCP Transcription Factors Regulate Jasmonic Acid Metabolism and Leaf Development Antagonistically1[C][W

PubMed Central

Danisman, Selahattin; van der Wal, Froukje; Dhondt, Stijn; Waites, Richard; de Folter, Stefan; Bimbo, Andrea; van Dijk, Aalt DJ; Muino, Jose M.; Cutri, Lucas; Dornelas, Marcelo C.; Angenent, Gerco C.; Immink, Richard G.H.

2012-01-01

TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1 (TCP) transcription factors control developmental processes in plants. The 24 TCP transcription factors encoded in the Arabidopsis (Arabidopsis thaliana) genome are divided into two classes, class I and class II TCPs, which are proposed to act antagonistically. We performed a detailed phenotypic analysis of the class I tcp20 mutant, showing an increase in leaf pavement cell sizes in 10-d-old seedlings. Subsequently, a glucocorticoid receptor induction assay was performed, aiming to identify potential target genes of the TCP20 protein during leaf development. The LIPOXYGENASE2 (LOX2) and class I TCP9 genes were identified as TCP20 targets, and binding of TCP20 to their regulatory sequences could be confirmed by chromatin immunoprecipitation analyses. LOX2 encodes for a jasmonate biosynthesis gene, which is also targeted by class II TCP proteins that are under the control of the microRNA JAGGED AND WAVY (JAW), although in an antagonistic manner. Mutation of TCP9, the second identified TCP20 target, resulted in increased pavement cell sizes during early leaf developmental stages. Analysis of senescence in the single tcp9 and tcp20 mutants and the tcp9tcp20 double mutants showed an earlier onset of this process in comparison with wild-type control plants in the double mutant only. Both the cell size and senescence phenotypes are opposite to the known class II TCP mutant phenotype in JAW plants. Altogether, these results point to an antagonistic function of class I and class II TCP proteins in the control of leaf development via the jasmonate signaling pathway. PMID:22718775

Diversity of Survival Patterns among Escherichia coli O157:H7 Genotypes Subjected to Food-Related Stress Conditions.

PubMed

Elhadidy, Mohamed; Álvarez-Ordóñez, Avelino

2016-01-01

The purpose of this study was to evaluate the resistance patterns to food-related stresses of Shiga toxin producing Escherichia coli O157:H7 strains belonging to specific genotypes. A total of 33 E. coli O157:H7 strains were exposed to seven different stress conditions acting as potential selective pressures affecting the transmission of E. coli O157:H7 to humans through the food chain. These stress conditions included cold, oxidative, osmotic, acid, heat, freeze-thaw, and starvation stresses. The genotypes used for comparison included lineage-specific polymorphism, Shiga-toxin-encoding bacteriophage insertion sites, clade type, tir (A255T) polymorphism, Shiga toxin 2 subtype, and antiterminator Q gene allele. Bacterial resistance to different stressors was calculated by determining D-values (times required for inactivation of 90% of the bacterial population), which were then subjected to univariate and multivariate analyses. In addition, a relative stress resistance value, integrating resistance values to all tested stressors, was calculated for each bacterial strain and allowed for a ranking-type classification of E. coli O157:H7 strains according to their environmental robustness. Lineage I/II strains were found to be significantly more resistant to acid, cold, and starvation stress than lineage II strains. Similarly, tir (255T) and clade 8 encoding strains were significantly more resistant to acid, heat, cold, and starvation stress than tir (255A) and non-clade 8 strains. Principal component analysis, which allows grouping of strains with similar stress survival characteristics, separated strains of lineage I and I/II from strains of lineage II, which in general showed reduced survival abilities. Results obtained suggest that lineage I/II, tir (255T), and clade 8 strains, which have been previously reported to be more frequently associated with human disease cases, have greater multiple stress resistance than strains of other genotypes. The results from this study provide a better insight into how selective pressures encountered through the food chain may play a role in the epidemiology of STEC O157:H7 through controlling the transmission of highly adapted strains to humans.

Diversity of Survival Patterns among Escherichia coli O157:H7 Genotypes Subjected to Food-Related Stress Conditions

PubMed Central

Elhadidy, Mohamed; Álvarez-Ordóñez, Avelino

2016-01-01

The purpose of this study was to evaluate the resistance patterns to food-related stresses of Shiga toxin producing Escherichia coli O157:H7 strains belonging to specific genotypes. A total of 33 E. coli O157:H7 strains were exposed to seven different stress conditions acting as potential selective pressures affecting the transmission of E. coli O157:H7 to humans through the food chain. These stress conditions included cold, oxidative, osmotic, acid, heat, freeze-thaw, and starvation stresses. The genotypes used for comparison included lineage-specific polymorphism, Shiga-toxin-encoding bacteriophage insertion sites, clade type, tir (A255T) polymorphism, Shiga toxin 2 subtype, and antiterminator Q gene allele. Bacterial resistance to different stressors was calculated by determining D-values (times required for inactivation of 90% of the bacterial population), which were then subjected to univariate and multivariate analyses. In addition, a relative stress resistance value, integrating resistance values to all tested stressors, was calculated for each bacterial strain and allowed for a ranking-type classification of E. coli O157:H7 strains according to their environmental robustness. Lineage I/II strains were found to be significantly more resistant to acid, cold, and starvation stress than lineage II strains. Similarly, tir (255T) and clade 8 encoding strains were significantly more resistant to acid, heat, cold, and starvation stress than tir (255A) and non-clade 8 strains. Principal component analysis, which allows grouping of strains with similar stress survival characteristics, separated strains of lineage I and I/II from strains of lineage II, which in general showed reduced survival abilities. Results obtained suggest that lineage I/II, tir (255T), and clade 8 strains, which have been previously reported to be more frequently associated with human disease cases, have greater multiple stress resistance than strains of other genotypes. The results from this study provide a better insight into how selective pressures encountered through the food chain may play a role in the epidemiology of STEC O157:H7 through controlling the transmission of highly adapted strains to humans. PMID:27014242

LCR-initiated rearrangements at the IDS locus, completed with Alu-mediated recombination or non-homologous end joining.

PubMed

Oshima, Junko; Lee, Jennifer A; Breman, Amy M; Fernandes, Priscilla H; Babovic-Vuksanovic, Dusica; Ward, Patricia A; Wolfe, Lynne A; Eng, Christine M; Del Gaudio, Daniela

2011-07-01

Mucopolysaccharidosis type II (MPS II) is caused by mutations in the IDS gene, which encodes the lysosomal enzyme iduronate-2-sulfatase. In ∼20% of MPS II patients the disorder is caused by gross IDS structural rearrangements. We identified two male cases harboring complex rearrangements involving the IDS gene and the nearby pseudogene, IDSP1, which has been annotated as a low-copy repeat (LCR). In both cases the rearrangement included a partial deletion of IDS and an inverted insertion of the neighboring region. In silico analyses revealed the presence of repetitive elements as well as LCRs at the junctions of rearrangements. Our models illustrate two alternative consequences of rearrangements initiated by non-allelic homologous recombination of LCRs: resolution by a second recombination event (that is, Alu-mediated recombination), or resolution by non-homologous end joining repair. These complex rearrangements have the potential to be recurrent and may be present among those MSP II cases with previously uncharacterized aberrations involving IDS.

Genetic Manipulation of Lactococcus lactis by Using Targeted Group II Introns: Generation of Stable Insertions without Selection

PubMed Central

Frazier, Courtney L.; San Filippo, Joseph; Lambowitz, Alan M.; Mills, David A.

2003-01-01

Despite their commercial importance, there are relatively few facile methods for genomic manipulation of the lactic acid bacteria. Here, the lactococcal group II intron, Ll.ltrB, was targeted to insert efficiently into genes encoding malate decarboxylase (mleS) and tetracycline resistance (tetM) within the Lactococcus lactis genome. Integrants were readily identified and maintained in the absence of a selectable marker. Since splicing of the Ll.ltrB intron depends on the intron-encoded protein, targeted invasion with an intron lacking the intron open reading frame disrupted TetM and MleS function, and MleS activity could be partially restored by expressing the intron-encoded protein in trans. Restoration of splicing from intron variants lacking the intron-encoded protein illustrates how targeted group II introns could be used for conditional expression of any gene. Furthermore, the modified Ll.ltrB intron was used to separately deliver a phage resistance gene (abiD) and a tetracycline resistance marker (tetM) into mleS, without the need for selection to drive the integration or to maintain the integrant. Our findings demonstrate the utility of targeted group II introns as a potential food-grade mechanism for delivery of industrially important traits into the genomes of lactococci. PMID:12571038

Supplementing healthy rats with a high-niacin dose has no effect on muscle fiber distribution and muscle metabolic phenotype.

PubMed

Scholz, Kristen; Kynast, Anna Marie; Couturier, Aline; Mooren, Frank-Christoph; Krüger, Karsten; Most, Erika; Eder, Klaus; Ringseis, Robert

2014-08-01

It was recently shown that niacin prevents the obesity-induced type I to type II fiber switching in skeletal muscle of obese rats and favors the development of a more oxidative metabolic phenotype and thereby increases whole body utilization of fatty acids. Whether niacin also causes type II to type I fiber switching in skeletal muscle of healthy rats has not been investigated yet. Thus, the present study aimed to investigate whether niacin supplementation influences fiber distribution and metabolic phenotype of different skeletal muscles with a distinct type I-to-type II fiber ratio in healthy rats. Twenty-four male, 10-week-old Sprague-Dawley rats were randomly assigned into two groups of 12 rats each and fed either a control diet with 30 mg supplemented niacin/kg diet (control group) or a high-niacin diet with 780 mg supplemented niacin/kg diet (high-niacin group). After 27 days of treatment, the percentage number of type I fibers in rectus femoris, gastrocnemius, and tibialis anterior muscles was 5-10% greater in the niacin group than in the control group, but did not differ between groups in soleus and vastus intermedius muscles. Transcript levels of genes encoding transcription factors regulating fiber switching, fiber-specific myosin heavy chain isoforms, and proteins involved in fatty acid utilization, oxidative phosphorylation, and angiogenesis did not differ between groups. The results show that niacin has only negligible effects on fiber distribution and its regulation as well as the metabolic phenotype of skeletal muscle in healthy rats.

Identification of four type II toxin-antitoxin systems in Actinobacillus pleuropneumoniae.

PubMed

Zheng, Chengkun; Zhao, Xigong; Zeng, Ting; Cao, Manman; Xu, Jiali; Shi, Guolin; Li, Jinquan; Chen, Huanchun; Bei, Weicheng

2017-07-03

Toxin-antitoxin (TA) systems are small genetic elements that are widely prevalent in the genomes of bacteria and archaea. These modules have been identified in various bacteria and proposed to play an important role in bacterial physiology and virulence. However, their presence in the genomes of Actinobacillus species has received no attention. In this study, we describe the identification of four type II TA systems in Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia. Reverse transcription PCR analysis revealed that the genes encoding the toxin and antitoxin are co-transcribed. Overexpression of each toxin inhibited the growth of Escherichia coli, and the toxic effect could be counteracted by its cognate antitoxin. The pull-down experiments demonstrated that each toxin interacts with its cognate antitoxin in vivo. The promoter activity assays showed that each antitoxin could autoregulate either positively or negatively the TA operon transcription. In addition, the APJL_0660/0659 TA system is present in half of the detected serovars of A. pleuropneumoniae, while the others are present in all. Collectively, we identified four type II TA systems in A. pleuropneumoniae, and this study has laid the foundation for further functional study of these TA systems. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Occurrence and activity of a type II CRISPR-Cas system in Lactobacillus gasseri.

PubMed

Sanozky-Dawes, Rosemary; Selle, Kurt; O'Flaherty, Sarah; Klaenhammer, Todd; Barrangou, Rodolphe

2015-09-01

Bacteria encode clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated genes (cas), which collectively form an RNA-guided adaptive immune system against invasive genetic elements. In silico surveys have revealed that lactic acid bacteria harbour a prolific and diverse set of CRISPR-Cas systems. Thus, the natural evolutionary role of CRISPR-Cas systems may be investigated in these ecologically, industrially, scientifically and medically important microbes. In this study, 17 Lactobacillus gasseri strains were investigated and 6 harboured a type II-A CRISPR-Cas system, with considerable diversity in array size and spacer content. Several of the spacers showed similarity to phage and plasmid sequences, which are typical targets of CRISPR-Cas immune systems. Aligning the protospacers facilitated inference of the protospacer adjacent motif sequence, determined to be 5'-NTAA-3' flanking the 3' end of the protospacer. The system in L. gasseri JV-V03 and NCK 1342 interfered with transforming plasmids containing sequences matching the most recently acquired CRISPR spacers in each strain. We report the distribution and function of a native type II-A CRISPR-Cas system in the commensal species L. gasseri. Collectively, these results open avenues for applications for bacteriophage protection and genome modification in L. gasseri, and contribute to the fundamental understanding of CRISPR-Cas systems in bacteria.

Cloning, overexpression, purification and preliminary crystallographic studies of a mitochondrial type II peroxiredoxin from Pisum sativum

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

Barranco-Medina, Sergio; López-Jaramillo, Francisco Javier, E-mail: fjljara@ugr.es; Bernier-Villamor, Laura

2006-07-01

The isolation, purification, crystallization and molecular-replacement solution of mitochondrial type II peroxiredoxin from P. sativum is reported. A cDNA encoding an open reading frame of 199 amino acids corresponding to a type II peroxiredoxin from Pisum sativum with its transit peptide was isolated by RT-PCR. The 171-amino-acid mature protein (estimated molecular weight 18.6 kDa) was cloned into the pET3d vector and overexpressed in Escherichia coli. The recombinant protein was purified and crystallized by the hanging-drop vapour-diffusion technique. A full data set (98.2% completeness) was collected using a rotating-anode generator to a resolution of 2.8 Å from a single crystal flash-cooledmore » at 100 K. X-ray data revealed that the protein crystallizes in space group P1, with unit-cell parameters a = 61.88, b = 66.40, c = 77.23 Å, α = 102.90, β = 104.40, γ = 99.07°, and molecular replacement using a theoretical model predicted from the primary structure as a search model confirmed the presence of six molecules in the unit cell as expected from the Matthews coefficient. Refinement of the structure is in progress.« less

Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells

PubMed Central

Min, Irene M.; Waterfall, Joshua J.; Core, Leighton J.; Munroe, Robert J.; Schimenti, John; Lis, John T.

2011-01-01

Transitions between pluripotent stem cells and differentiated cells are executed by key transcription regulators. Comparative measurements of RNA polymerase distribution over the genome's primary transcription units in different cell states can identify the genes and steps in the transcription cycle that are regulated during such transitions. To identify the complete transcriptional profiles of RNA polymerases with high sensitivity and resolution, as well as the critical regulated steps upon which regulatory factors act, we used genome-wide nuclear run-on (GRO-seq) to map the density and orientation of transcriptionally engaged RNA polymerases in mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs). In both cell types, progression of a promoter-proximal, paused RNA polymerase II (Pol II) into productive elongation is a rate-limiting step in transcription of ∼40% of mRNA-encoding genes. Importantly, quantitative comparisons between cell types reveal that transcription is controlled frequently at paused Pol II's entry into elongation. Furthermore, “bivalent” ESC genes (exhibiting both active and repressive histone modifications) bound by Polycomb group complexes PRC1 (Polycomb-repressive complex 1) and PRC2 show dramatically reduced levels of paused Pol II at promoters relative to an average gene. In contrast, bivalent promoters bound by only PRC2 allow Pol II pausing, but it is confined to extremely 5′ proximal regions. Altogether, these findings identify rate-limiting targets for transcription regulation during cell differentiation. PMID:21460038

Usherin expression is highly conserved in mouse and human tissues.

PubMed

Pearsall, Nicole; Bhattacharya, Gautam; Wisecarver, Jim; Adams, Joe; Cosgrove, Dominic; Kimberling, William

2002-12-01

Usher syndrome is an autosomal recessive disease that results in varying degrees of hearing loss and retinitis pigmentosa. Three types of Usher syndrome (I, II, and III) have been identified clinically with Usher type II being the most common of the three types. Usher type II has been localized to three different chromosomes 1q41, 3p, and 5q, corresponding to Usher type 2A, 2B, and 2C respectively. Usherin is a basement membrane protein encoded by the USH2A gene. Expression of usherin has been localized in the basement membrane of several tissues, however it is not ubiquitous. Immunohistochemistry detected usherin in the following human tissues: retina, cochlea, small and large intestine, pancreas, bladder, prostate, esophagus, trachea, thymus, salivary glands, placenta, ovary, fallopian tube, uterus, and testis. Usherin was absent in many other tissues such as heart, lung, liver, kidney, and brain. This distribution is consistent with the usherin distribution seen in the mouse. Conservation of usherin is also seen at the nucleotide and amino acid level when comparing the mouse and human gene sequences. Evolutionary conservation of usherin expression at the molecular level and in tissues unaffected by Usher 2a supports the important structural and functional role this protein plays in the human. In addition, we believe that these results could lead to a diagnostic procedure for the detection of Usher syndrome and those who carry an USH2A mutation.

Type 1 angiotensin II receptor-associated protein ARAP1 binds and recycles the receptor to the plasma membrane.

PubMed

Guo, Deng-Fu; Chenier, Isabelle; Tardif, Valerie; Orlov, Sergei N; Inagami, Tadashi

2003-10-31

The carboxyl terminus of the type 1 angiotensin II receptor (AT(1)) plays an important role in receptor phosphorylation, desensitization, and internalization. The yeast two-hybrid system was employed to isolate proteins associated with the carboxyl terminal region of the AT(1A) receptor. In the present study, we report the isolation of a novel protein, ARAP1, which promotes recycling of AT(1A) to the plasma membrane in HEK-293 cells. ARAP1 cDNA encodes a 493-amino-acid protein and its mRNA is ubiquitously expressed in rat tissues. A complex of ARAP1 and AT(1A) was observed by immunoprecipitation and Western blotting in HEK-293 cells. In the presence of ARAP1, recycled AT(1A) showed a significant Ca(2+) release response to a second stimulation by Ang II 30 min after the first treatment. Immunocytochemical analysis revealed co-localization of recycled AT(1A) and ARAP1 in the plasma membrane 45 min after the initial exposure to Ang II. Taken together, these results indicate a role for ARAP1 in the recycling of the AT(1) receptor to the plasma membrane with presumable concomitant recovery of receptor signal functions.

KSHV LANA inhibits TGF-β signaling through epigenetic silencing of the TGF-β type II receptor

PubMed Central

Di Bartolo, Daniel L.; Cannon, Mark; Liu, Yi-Fang; Renne, Rolf; Chadburn, Amy; Boshoff, Chris

2008-01-01

Signaling through the transforming growth factor–β (TGF-β) pathway results in growth inhibition and induction of apoptosis in various cell types. We show that this pathway is blocked in Kaposi sarcoma herpesvirus (KSHV)–infected primary effusion lymphoma through down-regulation of the TGF-β type II receptor (TβRII) by epigenetic mechanisms. Our data also suggest that KSHV infection may result in lower expression of TβRII in Kaposi sarcoma and multicentric Castleman disease. KSHV-encoded LANA associates with the promoter of TβRII and leads to its methylation and to the deacetylation of proximal histones. Reestablishment of signaling through this pathway reduces viability of these cells, inferring that KSHV-mediated blockage of TGF-β signaling plays a role in the establishment and progression of KSHV-associated neoplasia. These data suggest a mechanism whereby KSHV evades both the antiproliferative effects of TGF-β signaling by silencing TβRII gene expression and immune recognition by suppressing TGF-β–responsive immune cells through the elevated secretion of TGF-β1. PMID:18199825

Evidence for regulation of columnar habit in apple by a putative 2OG-Fe(II) oxygenase.

PubMed

Wolters, Pieter J; Schouten, Henk J; Velasco, Riccardo; Si-Ammour, Azeddine; Baldi, Paolo

2013-12-01

Understanding the genetic mechanisms controlling columnar-type growth in the apple mutant 'Wijcik' will provide insights on how tree architecture and growth are regulated in fruit trees. In apple, columnar-type growth is controlled by a single major gene at the Columnar (Co) locus. By comparing the genomic sequence of the Co region of 'Wijcik' with its wild-type 'McIntosh', a novel non-coding DNA element of 1956 bp specific to Pyreae was found to be inserted in an intergenic region of 'Wijcik'. Expression analysis of selected genes located in the vicinity of the insertion revealed the upregulation of the MdCo31 gene encoding a putative 2OG-Fe(II) oxygenase in axillary buds of 'Wijcik'. Constitutive expression of MdCo31 in Arabidopsis thaliana resulted in compact plants with shortened floral internodes, a phenotype reminiscent of the one observed in columnar apple trees. We conclude that MdCo31 is a strong candidate gene for the control of columnar growth in 'Wijcik'. No claim to original European Union works. New Phytologist © 2013 New Phytologist Trust.

Members of the amylovora group of Erwinia are cellulolytic and possess genes homologous to the type II secretion pathway.

PubMed

Riekki, R; Palomäki, T; Virtaharju, O; Kokko, H; Romantschuk, M; Saarilahti, H T

2000-07-01

A cellulase-producing clone was isolated from a genomic library of the Erwinia rhapontici (Millard) Burkholder strain NCPPB2989. The corresponding gene, named celA, encodes an endoglucanase (EC 3.2.1.4) with the extremely low pH optimum of 3.4 and a temperature optimum between 40 and 50 degrees C. A single ORF of 999 nt was found to be responsible for the Cel activity. The corresponding protein, named CelA, showed 67% identity to the endoglucanase Y of E. chrysanthemi and 51.5% identity to the endoglucanase of Cellulomonas uda, and thus belongs to the glycosyl hydrolase family 8. The celA gene, or its homologue, was found to be present in all E. rhapontici isolates analysed, in E. chrysanthemi, and in E. amylovora. The presence of plant cell wall-degrading enzymes in the amylovora group of Erwinia spp. had not previously been established. Furthermore, the DNA of both E. rhapontici and E. amylovora was found to exhibit homology to genes encoding the type II (GSP) secretion pathway, which is known to be responsible for extracellular targeting of cellulases and pectinases in Erwinia spp. that cause soft rotting, such as E. carotovora and E. chrysanthemi. Secretion of the CelA protein by E. rhapontici could not be verified. However, the CelA protein itself was found to include the information necessary for heterologous secretion by E. chrysanthemi.

Developmental Loss of Photosystem II Activity and Structure in a Chloroplast-Encoded Tobacco Mutant, Lutescens-11

PubMed Central

Chia, Catherine P.; Duesing, John H.; Arntzen, Charles J.

1986-01-01

Lutescens-1, a tobacco mutant with a maternally inherited dysfunction, displayed an unusual developmental phenotype. In vivo measurement of chlorophyll fluorescence revealed deterioration in photosystem II (PSII) function as leaves expanded. Analysis of thylakoid membrane proteins by polyacrylamide gel electrophoresis indicated the physical loss of nuclear- and chloroplast-encoded polypeptides comprising the PSII core complex concomitant with loss of activity. Freeze fracture electron micrographs of mutant thylakoids showed a reduced density, compared to wild type, of the EFs particles which have been shown previously to be the structural entity containing PSII core complexes and associated pigment-proteins. The selective loss of PSII cores from thylakoids resulted in a higher ratio of antenna chlorophyll to reaction centers and an altered 77 K chlorophyll fluorescence emission spectra; these data are interpreted to indicate functional isolation of light-harvesting chlorophyll a/b complexes in the absence of PSII centers. Examination of PSII reaction centers (which were present at lower levels in mutant membranes) by monitoring the light-dependent phosphorylation of PSII polypeptides and flash-induced O2 evolution patterns demonstrated that the PSII cores which were assembled in mutant thylakoids were functionally identical to those of wild type. We conclude that the lutescens-1 mutation affected the correct stoichiometry of PSII centers, in relation to other membrane constituents, by disrupting the proper assembly and maintenance of PSII complexes in lutescens-1 thylakoid membranes. Images Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:16664990

Functional Angucycline-Like Antibiotic Gene Cluster in the Terminal Inverted Repeats of the Streptomyces ambofaciens Linear Chromosome

PubMed Central

Pang, Xiuhua; Aigle, Bertrand; Girardet, Jean-Michel; Mangenot, Sophie; Pernodet, Jean-Luc; Decaris, Bernard; Leblond, Pierre

2004-01-01

Streptomyces ambofaciens has an 8-Mb linear chromosome ending in 200-kb terminal inverted repeats. Analysis of the F6 cosmid overlapping the terminal inverted repeats revealed a locus similar to type II polyketide synthase (PKS) gene clusters. Sequence analysis identified 26 open reading frames, including genes encoding the β-ketoacyl synthase (KS), chain length factor (CLF), and acyl carrier protein (ACP) that make up the minimal PKS. These KS, CLF, and ACP subunits are highly homologous to minimal PKS subunits involved in the biosynthesis of angucycline antibiotics. The genes encoding the KS and ACP subunits are transcribed constitutively but show a remarkable increase in expression after entering transition phase. Five genes, including those encoding the minimal PKS, were replaced by resistance markers to generate single and double mutants (replacement in one and both terminal inverted repeats). Double mutants were unable to produce either diffusible orange pigment or antibacterial activity against Bacillus subtilis. Single mutants showed an intermediate phenotype, suggesting that each copy of the cluster was functional. Transformation of double mutants with a conjugative and integrative form of F6 partially restored both phenotypes. The pigmented and antibacterial compounds were shown to be two distinct molecules produced from the same biosynthetic pathway. High-pressure liquid chromatography analysis of culture extracts from wild-type and double mutants revealed a peak with an associated bioactivity that was absent from the mutants. Two additional genes encoding KS and CLF were present in the cluster. However, disruption of the second KS gene had no effect on either pigment or antibiotic production. PMID:14742212

In vivo inhibition of CC and CX3C chemokine-induced leukocyte infiltration and attenuation of glomerulonephritis in Wistar-Kyoto (WKY) rats by vMIP-II.

PubMed

Chen, S; Bacon, K B; Li, L; Garcia, G E; Xia, Y; Lo, D; Thompson, D A; Siani, M A; Yamamoto, T; Harrison, J K; Feng, L

1998-07-06

Chemokines play a central role in immune and inflammatory responses. It has been observed recently that certain viruses have evolved molecular piracy and mimicry mechanisms by encoding and synthesizing proteins that interfere with the normal host defense response. One such viral protein, vMIP-II, encoded by human herpesvirus 8, has been identified with in vitro antagonistic activities against CC and CXC chemokine receptors. We report here that vMIP-II has additional antagonistic activity against CX3CR1, the receptor for fractalkine. To investigate the potential therapeutic effect of this broad-spectrum chemokine antagonist, we studied the antiinflammatory activity of vMIP-II in a rat model of experimental glomerulonephritis induced by an antiglomerular basement membrane antibody. vMIP-II potently inhibited monocyte chemoattractant protein 1-, macrophage inflammatory protein 1beta-, RANTES (regulated on activation, normal T cell expressed and secreted)-, and fractalkine-induced chemotaxis of activated leukocytes isolated from nephritic glomeruli, significantly reduced leukocyte infiltration to the glomeruli, and markedly attenuated proteinuria. These results suggest that molecules encoded by some viruses may serve as useful templates for the development of antiinflammatory compounds.

Recombinant plasmids for encoding restriction enzymes DpnI and DpnII of streptococcus pneumontae

DOEpatents

Lacks, Sanford A.

1990-01-01

Chromosomal DNA cassettes containing genes encoding either the DpnI or DpnII restriction endonucleases from Streptococcus pneumoniae are cloned into a streptococcal vector, pLS101. Large amounts of the restriction enzymes are produced by cells containing the multicopy plasmids, pLS202 and pLS207, and their derivatives pLS201, pLS211, pLS217, pLS251 and pLS252.

Recombinant plasmids for encoding restriction enzymes DpnI and DpnII of Streptococcus pneumontae

DOEpatents

Lacks, S.A.

1990-10-02

Chromosomal DNA cassettes containing genes encoding either the DpnI or DpnII restriction endonucleases from Streptococcus pneumoniae are cloned into a streptococcal vector, pLS101. Large amounts of the restriction enzymes are produced by cells containing the multicopy plasmids, pLS202 and pLS207, and their derivatives pLS201, pLS211, pLS217, pLS251 and pLS252. 9 figs.

Cloning, Sequencing, and Functional Analysis of an Iterative Type I Polyketide Synthase Gene Cluster for Biosynthesis of the Antitumor Chlorinated Polyenone Neocarzilin in “Streptomyces carzinostaticus”

PubMed Central

Otsuka, Miyuki; Ichinose, Koji; Fujii, Isao; Ebizuka, Yutaka

2004-01-01

Neocarzilins (NCZs) are antitumor chlorinated polyenones produced by “Streptomyces carzinostaticus” var. F-41. The gene cluster responsible for the biosynthesis of NCZs was cloned and characterized. DNA sequence analysis of a 33-kb region revealed a cluster of 14 open reading frames (ORFs), three of which (ORF4, ORF5, and ORF6) encode type I polyketide synthase (PKS), which consists of four modules. Unusual features of the modular organization is the lack of an obvious acyltransferase domain on modules 2 and 4 and the presence of longer interdomain regions more than 200 amino acids in length on each module. Involvement of the PKS genes in NCZ biosynthesis was demonstrated by heterologous expression of the cluster in Streptomyces coelicolor CH999, which produced the apparent NCZ biosynthetic intermediates dechloroneocarzillin A and dechloroneocarzilin B. Disruption of ORF5 resulted in a failure of NCZ production, providing further evidence that the cluster is essential for NCZ biosynthesis. Mechanistic consideration of NCZ formation indicates the iterative use of at least one module of the PKS, which subsequently releases its product by decarboxylation to generate an NCZ skeleton, possibly catalyzed by a type II thioesterase encoded by ORF7. This is a novel type I PKS system of bacterial origin for the biosynthesis of a reduced polyketide chain. Additionally, the protein encoded by ORF3, located upstream of the PKS genes, closely resembles the FADH2-dependent halogenases involved in the formation of halometabolites. The ORF3 protein could be responsible for the halogenation of NCZs, presenting a unique example of a halogenase involved in the biosynthesis of an aliphatic halometabolite. PMID:15328113

Sulfide oxidation, nitrate respiration, carbon acquisition, and electron transport pathways suggested by the draft genome of a single orange Guaymas Basin Beggiatoa (Cand. Maribeggiatoa) sp. filament.

PubMed

MacGregor, Barbara J; Biddle, Jennifer F; Harbort, Christopher; Matthysse, Ann G; Teske, Andreas

2013-09-01

A near-complete draft genome has been obtained for a single vacuolated orange Beggiatoa (Cand. Maribeggiatoa) filament from a Guaymas Basin seafloor microbial mat, the third relatively complete sequence for the Beggiatoaceae. Possible pathways for sulfide oxidation; nitrate respiration; inorganic carbon fixation by both Type II RuBisCO and the reductive tricarboxylic acid cycle; acetate and possibly formate uptake; and energy-generating electron transport via both oxidative phosphorylation and the Rnf complex are discussed here. A role in nitrite reduction is suggested for an abundant orange cytochrome produced by the Guaymas strain; this has a possible homolog in Beggiatoa (Cand. Isobeggiatoa) sp. PS, isolated from marine harbor sediment, but not Beggiatoa alba B18LD, isolated from a freshwater rice field ditch. Inferred phylogenies for the Calvin-Benson-Bassham (CBB) cycle and the reductive (rTCA) and oxidative (TCA) tricarboxylic acid cycles suggest that genes encoding succinate dehydrogenase and enzymes for carboxylation and/or decarboxylation steps (including RuBisCO) may have been introduced to (or exported from) one or more of the three genomes by horizontal transfer, sometimes by different routes. Sequences from the two marine strains are generally more similar to each other than to sequences from the freshwater strain, except in the case of RuBisCO: only the Guaymas strain encodes a Type II enzyme, which (where studied) discriminates less against oxygen than do Type I RuBisCOs. Genes subject to horizontal transfer may represent key steps for adaptation to factors such as oxygen and carbon dioxide concentration, organic carbon availability, and environmental variability. © 2013.

Characterization of BcaA, a Putative Classical Autotransporter Protein in Burkholderia pseudomallei

PubMed Central

Campos, Cristine G.; Borst, Luke

2013-01-01

Burkholderia pseudomallei is a tier 1 select agent, and the causative agent of melioidosis, a disease with effects ranging from chronic abscesses to fulminant pneumonia and septic shock, which can be rapidly fatal. Autotransporters (ATs) are outer membrane proteins belonging to the type V secretion system family, and many have been shown to play crucial roles in pathogenesis. The open reading frame Bp1026b_II1054 (bcaA) in B. pseudomallei strain 1026b is predicted to encode a classical autotransporter protein with an approximately 80-kDa passenger domain that contains a subtilisin-related domain. Immediately 3′ to bcaA is Bp11026_II1055 (bcaB), which encodes a putative prolyl 4-hydroxylase. To investigate the role of these genes in pathogenesis, large in-frame deletion mutations of bcaA and bcaB were constructed in strain Bp340, an efflux pump mutant derivative of the melioidosis clinical isolate 1026b. Comparison of Bp340ΔbcaA and Bp340ΔbcaB mutants to wild-type B. pseudomallei in vitro demonstrated similar levels of adherence to A549 lung epithelial cells, but the mutant strains were defective in their ability to invade these cells and to form plaques. In a BALB/c mouse model of intranasal infection, similar bacterial burdens were observed after 48 h in the lungs and liver of mice infected with Bp340ΔbcaA, Bp340ΔbcaB, and wild-type bacteria. However, significantly fewer bacteria were recovered from the spleen of Bp340ΔbcaA-infected mice, supporting the idea of a role for this AT in dissemination or in survival in the passage from the site of infection to the spleen. PMID:23340315

Characterization of BcaA, a putative classical autotransporter protein in Burkholderia pseudomallei.

PubMed

Campos, Cristine G; Borst, Luke; Cotter, Peggy A

2013-04-01

Burkholderia pseudomallei is a tier 1 select agent, and the causative agent of melioidosis, a disease with effects ranging from chronic abscesses to fulminant pneumonia and septic shock, which can be rapidly fatal. Autotransporters (ATs) are outer membrane proteins belonging to the type V secretion system family, and many have been shown to play crucial roles in pathogenesis. The open reading frame Bp1026b_II1054 (bcaA) in B. pseudomallei strain 1026b is predicted to encode a classical autotransporter protein with an approximately 80-kDa passenger domain that contains a subtilisin-related domain. Immediately 3' to bcaA is Bp11026_II1055 (bcaB), which encodes a putative prolyl 4-hydroxylase. To investigate the role of these genes in pathogenesis, large in-frame deletion mutations of bcaA and bcaB were constructed in strain Bp340, an efflux pump mutant derivative of the melioidosis clinical isolate 1026b. Comparison of Bp340ΔbcaA and Bp340ΔbcaB mutants to wild-type B. pseudomallei in vitro demonstrated similar levels of adherence to A549 lung epithelial cells, but the mutant strains were defective in their ability to invade these cells and to form plaques. In a BALB/c mouse model of intranasal infection, similar bacterial burdens were observed after 48 h in the lungs and liver of mice infected with Bp340ΔbcaA, Bp340ΔbcaB, and wild-type bacteria. However, significantly fewer bacteria were recovered from the spleen of Bp340ΔbcaA-infected mice, supporting the idea of a role for this AT in dissemination or in survival in the passage from the site of infection to the spleen.

Type 4 pili are dispensable for biofilm development in the cyanobacterium Synechococcus elongatus.

PubMed

Nagar, Elad; Zilberman, Shaul; Sendersky, Eleonora; Simkovsky, Ryan; Shimoni, Eyal; Gershtein, Diana; Herzberg, Moshe; Golden, Susan S; Schwarz, Rakefet

2017-07-01

The hair-like cell appendages denoted as type IV pili are crucial for biofilm formation in diverse eubacteria. The protein complex responsible for type IV pilus assembly is homologous with the type II protein secretion complex. In the cyanobacterium Synechococcus elongatus PCC 7942, the gene Synpcc7942_2071 encodes an ATPase homologue of type II/type IV systems. Here, we report that inactivation of Synpcc7942_2071 strongly affected the suite of proteins present in the extracellular milieu (exo-proteome) and eliminated pili observable by electron microscopy. These results support a role for this gene product in protein secretion as well as in pili formation. As we previously reported, inactivation of Synpcc7942_2071 enables biofilm formation and suppresses the planktonic growth of S. elongatus. Thus, pili are dispensable for biofilm development in this cyanobacterium, in contrast to their biofilm-promoting function in type IV pili-producing heterotrophic bacteria. Nevertheless, pili removal is not required for biofilm formation as evident by a piliated mutant of S. elongatus that develops biofilms. We show that adhesion and timing of biofilm development differ between the piliated and non-piliated strains. The study demonstrates key differences in the process of biofilm formation between cyanobacteria and well-studied type IV pili-producing heterotrophic bacteria. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Structural and Kinetic Properties of Lumazine Synthase Isoenzymes in the Order Rhizobiales

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

Klinke,S.; Zylberman, V.; Bonomi, H.

6, 7-Dimethyl-8-ribityllumazine synthase (lumazine synthase; LS) catalyzes the penultimate step in the biosynthesis of riboflavin in plants and microorganisms. This protein is known to exhibit different quaternary assemblies between species, existing as free pentamers, decamers (dimers of pentamers) and icosahedrally arranged dodecamers of pentamers. A phylogenetic analysis on eubacterial, fungal and plant LSs allowed us to classify them into two categories: Type I LSs (pentameric or icosahedral) and Type II LSs (decameric). The Rhizobiales represent an order of ?-proteobacteria that includes, among others, the genera Mesorhizobium, Agrobacterium and Brucella. Here, we present structural and kinetic studies on several LSs frommore » Rhizobiales. Interestingly, Mesorhizobium and Brucella encode both a Type-I LS and a Type-II LS called RibH1 and RibH2, respectively. We show that Type II LSs appear to be almost inactive, whereas Type I LSs present a highly variable catalytic activity according to the genus. Additionally, we have solved four RibH1/RibH2 crystallographic structures from the genera Mesorhizobium and Brucella. The relationship between the active-site architecture and catalytic properties in these isoenzymes is discussed, and a model that describes the enzymatic behavior is proposed. Furthermore, sequence alignment studies allowed us to extend our results to the genus Agrobacterium. Our results suggest that the selective pressure controlling the riboflavin pathway favored the evolution of catalysts with low reaction rates, since the excess of flavins in the intracellular pool in Rhizobiales could act as a negative factor when these bacteria are exposed to oxidative or nitrosative stress.« less

Molecular analysis of pericentrin gene (PCNT) in a series of 24 Seckel/microcephalic osteodysplastic primordial dwarfism type II (MOPD II) families.

PubMed

Willems, M; Geneviève, D; Borck, G; Baumann, C; Baujat, G; Bieth, E; Edery, P; Farra, C; Gerard, M; Héron, D; Leheup, B; Le Merrer, M; Lyonnet, S; Martin-Coignard, D; Mathieu, M; Thauvin-Robinet, C; Verloes, A; Colleaux, L; Munnich, A; Cormier-Daire, V

2010-12-01

Microcephalic osteodysplastic primordial dwarfism type II (MOPD II, MIM 210720) and Seckel syndrome (SCKL, MIM 210600) belong to the primordial dwarfism group characterised by intrauterine growth retardation, severe proportionate short stature, and pronounced microcephaly. MOPD II is distinct from SCKL by more severe growth retardation, radiological abnormalities, and absent or mild mental retardation. Seckel syndrome is associated with defective ATR dependent DNA damage signalling. In 2008, loss-of-function mutations in the pericentrin gene (PCNT) have been identified in 28 patients, including 3 SCKL and 25 MOPDII cases. This gene encodes a centrosomal protein which plays a key role in the organisation of mitotic spindles. The aim of this study was to analyse PCNT in a large series of SCKL-MOPD II cases to further define the clinical spectrum associated with PCNT mutations. Among 18 consanguineous families (13 SCKL and 5 MOPDII) and 6 isolated cases (3 SCKL and 3 MOPD II), 13 distinct mutations were identified in 5/16 SCKL and 8/8 MOPDII including five stop mutations, five frameshift mutations, two splice site mutations, and one apparent missense mutation affecting the last base of exon 19. Moreover, we demonstrated that this latter mutation leads to an abnormal splicing with a predicted premature termination of translation. The clinical analysis of the 5 SCKL cases with PCNT mutations showed that they all presented minor skeletal changes and clinical features compatible with MOPDII diagnosis. It is therefore concluded that, despite variable severity, MOPDII is a genetically homogeneous condition due to loss-of-function of pericentrin.

Screening of the USH1G gene among Spanish patients with Usher syndrome. Lack of mutations and evidence of a minor role in the pathogenesis of the syndrome.

PubMed

Aller, Elena; Jaijo, Teresa; Beneyto, Magdalena; Nájera, Carmen; Morera, Constantino; Pérez-Garrigues, Herminio; Ayuso, Carmen; Millán, Jose

2007-09-01

The Usher syndrome (USH) is an autosomal recessive hereditary disorder characterized by the association of sensorineural hearing loss, retinitis pigmentosa (RP) and, in some cases, vestibular dysfunction. The USH1G gene, encoding SANS, has been found to cause both Usher syndrome type I and atypical Usher syndrome. 109 Spanish unrelated patients suffering from Usher syndrome type I, type II, type III and unclassified Usher syndrome were screened for mutations in this gene, but only eight different changes without a clear pathogenic effect have been detected. Based on these results as well as previous studies in other populations where mutational analysis of this gene has been carried out, one can conclude that USH1G has a minor involvement in Usher syndrome pathogenesis.

Inactivation of a Pleurotus ostreatus versatile peroxidase-encoding gene (mnp2) results in reduced lignin degradation.

PubMed

Salame, Tomer M; Knop, Doriv; Levinson, Dana; Mabjeesh, Sameer J; Yarden, Oded; Hadar, Yitzhak

2014-01-01

Lignin biodegradation by white-rot fungi is pivotal to the earth's carbon cycle. Manganese peroxidases (MnPs), the most common extracellular ligninolytic peroxidases produced by white-rot fungi, are considered key in ligninolysis. Pleurotus ostreatus, the oyster mushroom, is a preferential lignin degrader occupying niches rich in lignocellulose such as decaying trees. Here, we provide direct, genetically based proof for the functional significance of MnP to P. ostreatus ligninolytic capacity under conditions mimicking its natural habitat. When grown on a natural lignocellulosic substrate of cotton stalks under solid-state culture conditions, gene and isoenzyme expression profiles of its short MnP and versatile peroxidase (VP)-encoding gene family revealed that mnp2 was predominately expressed. mnp2, encoding the versatile short MnP isoenzyme 2 was disrupted. Inactivation of mnp2 resulted in three interrelated phenotypes, relative to the wild-type strain: (i) reduction of 14% and 36% in lignin mineralization of stalks non-amended and amended with Mn(2+), respectively; (ii) marked reduction of the bioconverted lignocellulose sensitivity to subsequent bacterial hydrolyses; and (iii) decrease in fungal respiration rate. These results may serve as the basis to clarify the roles of the various types of fungal MnPs and VPs in their contribution to white-rot decay of wood and lignocellulose in various ecosystems. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

Frequency and expression of mutacin biosynthesis genes in isolates of Streptococcus mutans with different mutacin-producing phenotypes.

PubMed

Kamiya, Regianne Umeko; Höfling, José Francisco; Gonçalves, Reginaldo Bruno

2008-05-01

The aim of this study was to analyse the frequency and expression of biosynthesis genes in 47 Streptococcus mutans isolates with different mutacin-producing phenotypes. Detection of the frequency and expression of genes encoding mutacin types I, II, III and IV were carried out by PCR and semi-quantitative RT-PCR, respectively, using primers specific for each type of biosynthesis gene. In addition, a further eight genes encoding putative bacteriocins, designated bsm 283, bsm 299, bsm 423, bsm 1889c, bsm 1892c, bsm 1896, bsm 1906c and bsm 1914, were also screened. There was a high phenotypic diversity; some Streptococcus mutans isolates presented broad antimicrobial spectra against other Streptococcus mutans clinical isolates, including bacteria resistant to common antibiotics, as well as Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis and Streptococcus pyogenes. The expression frequency of the bsm gene was higher than that of the previously characterized mutacins (I-IV). There was no positive correlation between the number of indicator strains inhibited (antimicrobial spectra) and the number of biosynthesis genes expressed (Spearman correlation test, r=-0.03, P>0.05). In conclusion, the high diversity of mutacin-producing phenotypes, associated with high frequency of expression of the biosynthesis genes screened, reveals a broad repertoire of genetic determinants encoding antimicrobial peptides that can act in different combinations.

Coding of time-dependent stimuli in homogeneous and heterogeneous neural populations.

PubMed

Beiran, Manuel; Kruscha, Alexandra; Benda, Jan; Lindner, Benjamin

2018-04-01

We compare the information transmission of a time-dependent signal by two types of uncoupled neuron populations that differ in their sources of variability: i) a homogeneous population whose units receive independent noise and ii) a deterministic heterogeneous population, where each unit exhibits a different baseline firing rate ('disorder'). Our criterion for making both sources of variability quantitatively comparable is that the interspike-interval distributions are identical for both systems. Numerical simulations using leaky integrate-and-fire neurons unveil that a non-zero amount of both noise or disorder maximizes the encoding efficiency of the homogeneous and heterogeneous system, respectively, as a particular case of suprathreshold stochastic resonance. Our findings thus illustrate that heterogeneity can render similarly profitable effects for neuronal populations as dynamic noise. The optimal noise/disorder depends on the system size and the properties of the stimulus such as its intensity or cutoff frequency. We find that weak stimuli are better encoded by a noiseless heterogeneous population, whereas for strong stimuli a homogeneous population outperforms an equivalent heterogeneous system up to a moderate noise level. Furthermore, we derive analytical expressions of the coherence function for the cases of very strong noise and of vanishing intrinsic noise or heterogeneity, which predict the existence of an optimal noise intensity. Our results show that, depending on the type of signal, noise as well as heterogeneity can enhance the encoding performance of neuronal populations.

A novel p. Gly630Ser mutation of COL2A1 in a Chinese family with presentations of Legg-Calvé-Perthes disease or avascular necrosis of the femoral head.

PubMed

Li, Na; Yu, Jian; Cao, Xiang; Wu, Qiu-Yue; Li, Wei-Wei; Li, Tian-Fu; Zhang, Cui; Cui, Ying-Xia; Li, Xiao-Jun; Yin, Zhi-Min; Xia, Xin-Yi

2014-01-01

Mutations in the type II collagen gene are associated with certain human disorders, collectively termed type II collagenopathies. They include Legg-Calvé-Perthes disease (LCPD) and avascular necrosis of the femoral head (ANFH). These two diseases are skeletal dysplasias, inherited in an autosomal dominant fashion, characterized by groin pain, dislocation of the hip and diminished joint mobility. Coxa vara and elevation of the greater trochanter of the femur comprise the typical phenotype of LCPD, but do not occur in ANFH. Lack of synthesis of type II collagen and structural defects are responsible for the major clinical outcomes, because collagen is the essential matrix protein of all connective tissues. Type II collagen, encoded by the COL2A1 gene, contains N- and C- terminal regions that are cleaved after secretion into the extracellular matrix, and the core area is composed of a triple helical (Gly-X-Y) domain. If the Gly in this specific region is replaced by other amino acids, the structure of type II collagen will be destroyed. Forty-five members of a four-generation family were recruited and investigated. Diagnosis was made by independent orthopedic surgeons and radiologists. A mutation of the COL2A1 gene was detected. In our research, we identify a heterozygous mutation (c.1888 G>A, p. Gly630Ser) in exon 29 of COL2A1 in the Gly-X-Y domain, in a Chinese family affected by LCPD and ANFH. Our findings provide significant clues to the phenotype-genotype relationships in these syndromes and may be helpful in clinical diagnosis. Furthermore, these results should assist further studies of the mechanisms underlying collagen diseases. Our data add new variants to the repertoire of COL2A1 mutation resulting in related collagenopathies.

BioProspecting: novel marker discovery obtained by mining the bibleome.

PubMed

Elkin, Peter L; Tuttle, Mark S; Trusko, Brett E; Brown, Steven H

2009-02-05

BioProspecting is a novel approach that enabled our team to mine data related to genetic markers from the New England Journal of Medicine (NEJM) utilizing SNOMED CT and the Human Gene Onotology (HUGO). The Biomedical Informatics Research Collaborative was able to link genes and disorders using the Multi-threaded Clinical Vocabulary Server (MCVS) and natural language processing engine, whose output creates an ontology-network using the semantic encodings of the literature that is organized by these two terminologies. We identified relationships between (genes or proteins) and (diseases or drugs) as linked by metabolic functions and identified potentially novel functional relationships between, for example, genes and diseases (e.g. Article #1 ([Gene - IL27] = > {Enzyme - Dipeptidyl Carboxypeptidase 1}) and Article #2 ({Enzyme - Dipeptidyl Carboxypeptidase 1} < = [Disorder - Type II DM]) showing a metabolic link between IL27 and Type II DM). In this manuscript we describe our method for developing the database and its content as well as its potential to assist in the discovery of novel markers and drugs.

The oxygenating constituent of 3,6-diketocamphane monooxygenase from the CAM plasmid of Pseudomonas putida: the first crystal structure of a type II Baeyer-Villiger monooxygenase.

PubMed

Isupov, Michail N; Schröder, Ewald; Gibson, Robert P; Beecher, Jean; Donadio, Giuliana; Saneei, Vahid; Dcunha, Stephlina A; McGhie, Emma J; Sayer, Christopher; Davenport, Colin F; Lau, Peter C; Hasegawa, Yoshie; Iwaki, Hiroaki; Kadow, Maria; Balke, Kathleen; Bornscheuer, Uwe T; Bourenkov, Gleb; Littlechild, Jennifer A

2015-11-01

The three-dimensional structures of the native enzyme and the FMN complex of the overexpressed form of the oxygenating component of the type II Baeyer-Villiger 3,6-diketocamphane monooxygenase have been determined to 1.9 Å resolution. The structure of this dimeric FMN-dependent enzyme, which is encoded on the large CAM plasmid of Pseudomonas putida, has been solved by a combination of multiple anomalous dispersion from a bromine crystal soak and molecular replacement using a bacterial luciferase model. The orientation of the isoalloxazine ring of the FMN cofactor in the active site of this TIM-barrel fold enzyme differs significantly from that previously observed in enzymes of the bacterial luciferase-like superfamily. The Ala77 residue is in a cis conformation and forms a β-bulge at the C-terminus of β-strand 3, which is a feature observed in many proteins of this superfamily.

Comparison of the nucleotide and amino acid sequences of the RsrI and EcoRI restriction endonucleases.

PubMed

Stephenson, F H; Ballard, B T; Boyer, H W; Rosenberg, J M; Greene, P J

1989-12-21

The RsrI endonuclease, a type-II restriction endonuclease (ENase) found in Rhodobacter sphaeroides, is an isoschizomer of the EcoRI ENase. A clone containing an 11-kb BamHI fragment was isolated from an R. sphaeroides genomic DNA library by hybridization with synthetic oligodeoxyribonucleotide probes based on the N-terminal amino acid (aa) sequence of RsrI. Extracts of E. coli containing a subclone of the 11-kb fragment display RsrI activity. Nucleotide sequence analysis reveals an 831-bp open reading frame encoding a polypeptide of 277 aa. A 50% identity exists within a 266-aa overlap between the deduced aa sequences of RsrI and EcoRI. Regions of 75-100% aa sequence identity correspond to key structural and functional regions of EcoRI. The type-II ENases have many common properties, and a common origin might have been expected. Nevertheless, this is the first demonstration of aa sequence similarity between ENases produced by different organisms.

Cellobiohydrolase variants and polynucleotides encoding same

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

Wogulis, Mark

The present invention relates to variants of a parent cellobiohydrolase II. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

Genetic and functional properties of uncultivated thermophilic crenarchaeotes from a subsurface gold mine as revealed by analysis of genome fragments.

PubMed

Nunoura, Takuro; Hirayama, Hisako; Takami, Hideto; Oida, Hanako; Nishi, Shinro; Shimamura, Shigeru; Suzuki, Yohey; Inagaki, Fumio; Takai, Ken; Nealson, Kenneth H; Horikoshi, Koki

2005-12-01

Within a phylum Crenarchaeota, only some members of the hyperthermophilic class Thermoprotei, have been cultivated and characterized. In this study, we have constructed a metagenomic library from a microbial mat formation in a subsurface hot water stream of the Hishikari gold mine, Japan, and sequenced genome fragments of two different phylogroups of uncultivated thermophilic Crenarchaeota: (i) hot water crenarchaeotic group (HWCG) I (41.2 kb), and (ii) HWCG III (49.3 kb). The genome fragment of HWCG I contained a 16S rRNA gene, two tRNA genes and 35 genes encoding proteins but no 23S rRNA gene. Among the genes encoding proteins, several genes for putative aerobic-type carbon monoxide dehydrogenase represented a potential clue with regard to the yet unknown metabolism of HWCG I Archaea. The genome fragment of HWCG III contained a 16S/23S rRNA operon and 44 genes encoding proteins. In the 23S rRNA gene, we detected a homing-endonuclease encoding a group I intron similar to those detected in hyperthermophilic Crenarchaeota and Bacteria, as well as eukaryotic organelles. The reconstructed phylogenetic tree based on the 23S rRNA gene sequence reinforced the intermediate phylogenetic affiliation of HWCG III bridging the hyperthermophilic and non-thermophilic uncultivated Crenarchaeota.

WHITE STRIPE LEAF4 Encodes a Novel P-Type PPR Protein Required for Chloroplast Biogenesis during Early Leaf Development

PubMed Central

Wang, Ying; Ren, Yulong; Zhou, Kunneng; Liu, Linglong; Wang, Jiulin; Xu, Yang; Zhang, Huan; Zhang, Long; Feng, Zhiming; Wang, Liwei; Ma, Weiwei; Wang, Yunlong; Guo, Xiuping; Zhang, Xin; Lei, Cailin; Cheng, Zhijun; Wan, Jianmin

2017-01-01

Pentatricopeptide repeat (PPR) proteins comprise a large family in higher plants and perform diverse functions in organellar RNA metabolism. Despite the rice genome encodes 477 PRR proteins, the regulatory effects of PRR proteins on chloroplast development remains unknown. In this study, we report the functional characterization of the rice white stripe leaf4 (wsl4) mutant. The wsl4 mutant develops white-striped leaves during early leaf development, characterized by decreased chlorophyll content and malformed chloroplasts. Positional cloning of the WSL4 gene, together with complementation and RNA-interference tests, reveal that it encodes a novel P-family PPR protein with 12 PPR motifs, and is localized to chloroplast nucleoids. Quantitative RT-PCR analyses demonstrate that WSL4 is a low temperature response gene abundantly expressed in young leaves. Further expression analyses show that many nuclear- and plastid-encoded genes in the wsl4 mutant are significantly affected at the RNA and protein levels. Notably, the wsl4 mutant causes defects in the splicing of atpF, ndhA, rpl2, and rps12. Our findings identify WSL4 as a novel P-family PPR protein essential for chloroplast RNA group II intron splicing during early leaf development in rice. PMID:28694820

Comparative genomic analysis of Acinetobacter strains isolated from murine colonic crypts.

PubMed

Saffarian, Azadeh; Touchon, Marie; Mulet, Céline; Tournebize, Régis; Passet, Virginie; Brisse, Sylvain; Rocha, Eduardo P C; Sansonetti, Philippe J; Pédron, Thierry

2017-07-11

A restricted set of aerobic bacteria dominated by the Acinetobacter genus was identified in murine intestinal colonic crypts. The vicinity of such bacteria with intestinal stem cells could indicate that they protect the crypt against cytotoxic and genotoxic signals. Genome analyses of these bacteria were performed to better appreciate their biodegradative capacities. Two taxonomically different clusters of Acinetobacter were isolated from murine proximal colonic crypts, one was identified as A. modestus and the other as A. radioresistens. Their identification was performed through biochemical parameters and housekeeping gene sequencing. After selection of one strain of each cluster (A. modestus CM11G and A. radioresistens CM38.2), comparative genomic analysis was performed on whole-genome sequencing data. The antibiotic resistance pattern of these two strains is different, in line with the many genes involved in resistance to heavy metals identified in both genomes. Moreover whereas the operon benABCDE involved in benzoate metabolism is encoded by the two genomes, the operon antABC encoding the anthranilate dioxygenase, and the phenol hydroxylase gene cluster are absent in the A. modestus genomic sequence, indicating that the two strains have different capacities to metabolize xenobiotics. A common feature of the two strains is the presence of a type IV pili system, and the presence of genes encoding proteins pertaining to secretion systems such as Type I and Type II secretion systems. Our comparative genomic analysis revealed that different Acinetobacter isolated from the same biological niche, even if they share a large majority of genes, possess unique features that could play a specific role in the protection of the intestinal crypt.

Photocontrol of the expression of genes encoding chlorophyll a/b binding proteins and small subunit of ribulose-1,5-bisphosphate carboxylase in etiolated seedlings of Lycopersicon esculentum (L. ) and Nicotiana tabacum (L. )

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

Wehmeyer, B.; Cashmore, A.R.; Schaefer, E.

Phytochrome and the blue ultraviolet-A photoreceptor control light-induced expression of genes encoding the chlorophyll a/b binding protein of photosystem II and photosystem I and the genes for the small subunit of the ribulose-1,5-bisphosphate carboxylase in etiolated seedlings of Lycopersicon esculentum (tomato) and Nicotiana tabacum (tobacco). A high irradiance response also controls the induction of these genes. Genes encoding photosystem II- and I-associated chlorophyll a/b binding proteins both exhibit a transient rapid increase in expression in response to light pulse or to continuous irradiation. In contrast, genes encoding the small subunit exhibit a continuous increase in expression in response to light.more » These distinct expression characteristics are shown to reflect differences at the level of transcription.« less

OPTIM trial: a Phase III trial of an oncolytic herpes virus encoding GM-CSF for unresectable stage III or IV melanoma.

PubMed

Kaufman, Howard L; Bines, Steven D

2010-06-01

There are few effective treatment options available for patients with advanced melanoma. An oncolytic herpes simplex virus type 1 encoding granulocyte macrophage colony-stimulating factor (GM-CSF; Oncovex(GM-CSF)) for direct injection into accessible melanoma lesions resulted in a 28% objective response rate in a Phase II clinical trial. Responding patients demonstrated regression of both injected and noninjected lesions highlighting the dual mechanism of action of Oncovex(GM-CSF) that includes both a direct oncolytic effect in injected tumors and a secondary immune-mediated anti-tumor effect on noninjected tumors. Based on these preliminary results a prospective, randomized Phase III clinical trial in patients with unresectable Stage IIIb or c and Stage IV melanoma has been initiated. The rationale, study design, end points and future development of the Oncovex(GM-CSF) Pivotal Trial in Melanoma (OPTIM) trial are discussed in this article.

Cloning and characterization of a cDNA encoding topoisomerase II in pea and analysis of its expression in relation to cell proliferation.

PubMed

Reddy, M K; Nair, S; Tewari, K K; Mudgil, Y; Yadav, B S; Sopory, S K

1999-09-01

We have isolated and sequenced four overlapping cDNA clones to identify the full-length cDNA for topoisomerase II (PsTopII) from pea. Using degenerate primers, based on the conserved amino acid sequences of other eukaryotic type II topoisomerases, a 680 bp fragment was PCR-amplified with pea cDNA as template. This fragment was used as a probe to screen an oligo-dT-primed pea cDNA library. A partial cDNA clone was isolated that was truncated at the 3' end. RACE-PCR was employed to isolate the remaining portion of the gene. The total size of PsTopII is 4639 bp with an open reading frame of 4392 bp. The deduced amino acid sequence shows a strong homology to other eukaryotic topoisomerase II (topo II) at the N-terminus end. The topo II transcript was abundant in proliferative tissues. We also show that the level of topo II transcripts could be stimulated by exogenous application of growth factors that induced proliferation in vitro cultures. Light irradiation to etiolated tissue strongly stimulated the expression of topo II. These results suggest that topo II gene expression is up-regulated in response to light and hormones and correlates with cell proliferation. Besides, we have also isolated and analysed the 5'-flanking region of the pea TopII gene. This is first report on the isolation of a putative promoter for topoisomerase II from plants.

A strategy for genetic modification of the spike-encoding segment of human reovirus T3D for reovirus targeting.

PubMed

van den Wollenberg, D J M; van den Hengel, S K; Dautzenberg, I J C; Cramer, S J; Kranenburg, O; Hoeben, R C

2008-12-01

Human Orthoreovirus Type 3 Dearing is not pathogenic to humans and has been evaluated clinically as an oncolytic agent. Its transduction efficiency and the tumor cell selectivity may be enhanced by incorporating ligands for alternative receptors. However, the genetic modification of reoviruses has been difficult, and genetic targeting of reoviruses has not been reported so far. Here we describe a technique for generating genetically targeted reoviruses. The propagation of wild-type reoviruses on cells expressing a modified sigma 1-encoding segment embedded in a conventional RNA polymerase II transcript leads to substitution of the wild-type genome segment by the modified version. This technique was used for generating reoviruses that are genetically targeted to an artificial receptor expressed on U118MG cells. These cells lack the junction adhesion molecule-1 and therefore resist infection by wild-type reoviruses. The targeted reoviruses were engineered to carry the ligand for this receptor at the C terminus of the sigma 1 spike protein. This demonstrates that the C terminus of the sigma 1 protein is a suitable locale for the insertion of oligopeptide ligands and that targeting of reoviruses is feasible. The genetically targeted viruses can be propagated using the modified U118MG cells as helper cells. This technique may be applicable for the improvement of human reoviruses as oncolytic agents.

A new eIF4E1 allele characterized by RNAseq data mining is associated with resistance to potato virus Y in tomato albeit with a low durability.

PubMed

Lebaron, Caroline; Rosado, Aurélie; Sauvage, Christopher; Gauffier, Camille; German-Retana, Sylvie; Moury, Benoît; Gallois, Jean-Luc

2016-11-01

Allele mining on susceptibility factors offers opportunities to find new sources of resistance among crop wild relatives for breeding purposes. As a proof of concept, we used available RNAseq data to investigate polymorphisms among the four tomato genes encoding translation initiation factors [eIF4E1 and eIF4E2, eIFiso4E and the related gene new cap-binding protein(nCBP)] to look for new potential resistance alleles to potyviruses. By analysing polymorphism among RNAseq data obtained for 20 tomato accessions, 10 belonging to the cultivated type Solanum lycopersicum and 10 belonging to the closest related wild species Solanum pimpinellifolium, we isolated one new eIF4E1 allele, in the S. pimpinellifolium LA0411 accession, which encodes a potential new resistance allele, mainly due to a polymorphism associated with an amino acid change within eIF4E1 region II. We confirmed that this new allele, pot12, is indeed associated with resistance to potato virus Y, although with a restricted resistance spectrum and a very low durability potential. This suggests that mutations occurring in eIF4E region II only may not be sufficient to provide efficient and durable resistance in plants. However, our study emphasizes the opportunity brought by RNAseq data to mine for new resistance alleles. Moreover, this approach could be extended to seek for putative new resistance alleles by screening for variant forms of susceptibility genes encoding plant host proteins known to interact with viral proteins.

Glutamate-dependent ectodomain shedding of neuregulin-1 type II precursors in rat forebrain neurons.

PubMed

Iwakura, Yuriko; Wang, Ran; Inamura, Naoko; Araki, Kazuaki; Higashiyama, Shigeki; Takei, Nobuyuki; Nawa, Hiroyuki

2017-01-01

The neurotrophic factor neuregulin 1 (NRG1) regulates neuronal development, glial differentiation, and excitatory synapse maturation. NRG1 is synthesized as a membrane-anchored precursor and is then liberated by proteolytic processing or exocytosis. Mature NRG1 then binds to its receptors expressed by neighboring neurons or glial cells. However, the molecular mechanisms that govern this process in the nervous system are not defined in detail. Here we prepared neuron-enriched and glia-enriched cultures from embryonic rat neocortex to investigate the role of neurotransmitters that regulate the liberation/release of NRG1 from the membrane of neurons or glial cells. Using a two-site enzyme immunoassay to detect soluble NRG1, we show that, of various neurotransmitters, glutamate was the most potent inducer of NRG1 release in neuron-enriched cultures. NRG1 release in glia-enriched cultures was relatively limited. Furthermore, among glutamate receptor agonists, N-Methyl-D-Aspartate (NMDA) and kainate (KA), but not AMPA or tACPD, mimicked the effects of glutamate. Similar findings were acquired from analysis of the hippocampus of rats with KA-induced seizures. To evaluate the contribution of members of a disintegrin and metalloproteinase (ADAM) families to NRG1 release, we transfected primary cultures of neurons with cDNA vectors encoding NRG1 types I, II, or III precursors, each tagged with the alkaline phosphatase reporter. Analysis of alkaline phosphatase activity revealed that the NRG1 type II precursor was subjected to tumor necrosis factor-α-converting enzyme (TACE) / a Disintegrin And Metalloproteinase 17 (ADAM17) -dependent ectodomain shedding in a protein kinase C-dependent manner. These results suggest that glutamatergic neurotransmission positively regulates the ectodomain shedding of NRG1 type II precursors and liberates the active NRG1 domain in an activity-dependent manner.

Biocavity laser spectroscopy of genetically altered yeast cells and isolated yeast mitochondria

NASA Astrophysics Data System (ADS)

Gourley, Paul L.; Hendricks, Judy K.; McDonald, Anthony E.; Copeland, R. Guild; Naviaux, Robert K.; Yaffe, Michael P.

2006-02-01

We report an analysis of 2 yeast cell mutants using biocavity laser spectroscopy. The two yeast strains differed only by the presence or absence of mitochondrial DNA. Strain 104 is a wild-type (ρ +) strain of the baker's yeast, Saccharomyces cerevisiae. Strain 110 was derived from strain 104 by removal of its mitochondrial DNA (mtDNA). Removal of mtDNA causes strain 110 to grow as a "petite" (ρ -), named because it forms small colonies (of fewer cells because it grows more slowly) on agar plates supplemented with a variety of different carbon sources. The absence of mitochondrial DNA results in the complete loss of all the mtDNA-encoded proteins and RNAs, and loss of the pigmented, heme-containing cytochromes a and b. These cells have mitochondria, but the mitochondria lack the normal respiratory chain complexes I, III, IV, and V. Complex II is preserved because its subunits are encoded by genes located in nuclear DNA. The frequency distributions of the peak shifts produced by wild-type and petite cells and mitochondria show striking differences in the symmetry and patterns of the distributions. Wild-type ρ + cells (104) and mitochondria produced nearly symmetric, Gaussian distributions. The ρ - cells (110) and mitochondria showed striking asymmetry and skew that appeared to follow a Poisson distribution.

Gene expression profile of collagen types, osteopontin in the tympanic membrane of patients with tympanosclerosis.

PubMed

Sakowicz-Burkiewicz, Monika; Kuczkowski, Jerzy; Przybyła, Tomasz; Grdeń, Marzena; Starzyńska, Anna; Pawełczyk, Tadeusz

2017-09-01

Tympanosclerosis is a pathological process involving the middle ear. The hallmark of this disease is the formation of calcium deposits. In the submucosal layer, as well as in the right layer of the tympanic membrane, the calcium deposits result in a significant increase in the activity of fibroblasts and deposition of collagen fibers. The aim of our study was to examine the expression level of genes encoding collagen type I, II, III and IV (COL1A1, COL2A1, COL3A1, COL4A1) and osteopontin (SPP1) in the tympanic membrane of patients with tympanosclerosis. The total RNA was isolated from middle ear tissues with tympanosclerosis, received from 25 patients and from 19 normal tympanic membranes. The gene expression level was determined by real-time RT-PCR. The gene expression levels were correlated with clinical Tos classification of tympanosclerosis. We observed that in the tympanic membrane of patients with tympanosclerosis, the expression of type I collagen is decreased, while the expression of type II and IV collagen and osteopontin is increased. Moreover, mRNA levels of the investigated genes strongly correlated with the clinical stages of tympanosclerosis. The strong correlations between the expression of type I, II, IV collagen and osteopontin and the clinical stage of tympanosclerosis indicate the involvement of these proteins in excessive fibrosis and pathological remodeling of the tympanic membrane. In the future, a treatment aiming to modulate these gene expressions and/or regulation of the degradation of their protein products could be used as a new medical approach for patients with tympanosclerosis.

Mycobacterium tuberculosis Rv3406 Is a Type II Alkyl Sulfatase Capable of Sulfate Scavenging

PubMed Central

Sogi, Kimberly M.; Gartner, Zev J.; Breidenbach, Mark A.; Appel, Mason J.; Schelle, Michael W.; Bertozzi, Carolyn R.

2013-01-01

The genome of Mycobacterium tuberculosis (Mtb) encodes nine putative sulfatases, none of which have a known function or substrate. Here, we characterize Mtb’s single putative type II sulfatase, Rv3406, as a non-heme iron (II) and α-ketoglutarate-dependent dioxygenase that catalyzes the oxidation and subsequent cleavage of alkyl sulfate esters. Rv3406 was identified based on its homology to the alkyl sulfatase AtsK from Pseudomonas putida. Using an in vitro biochemical assay, we confirmed that Rv3406 is a sulfatase with a preference for alkyl sulfate substrates similar to those processed by AtsK. We determined the crystal structure of the apo Rv3406 sulfatase at 2.5 Å. The active site residues of Rv3406 and AtsK are essentially superimposable, suggesting that the two sulfatases share the same catalytic mechanism. Finally, we generated an Rv3406 mutant (Δrv3406) in Mtb to study the sulfatase’s role in sulfate scavenging. The Δrv3406 strain did not replicate in minimal media with 2-ethyl hexyl sulfate as the sole sulfur source, in contrast to wild type Mtb or the complemented strain. We conclude that Rv3406 is an iron and α-ketoglutarate-dependent sulfate ester dioxygenase that has unique substrate specificity that is likely distinct from other Mtb sulfatases. PMID:23762287

Characterization of the interferon genes in homozygous rainbow trout reveals two novel genes, alternate splicing and differential regulation of duplicated genes

USGS Publications Warehouse

Purcell, M.K.; Laing, K.J.; Woodson, J.C.; Thorgaard, G.H.; Hansen, J.D.

2009-01-01

The genes encoding the type I and type II interferons (IFNs) have previously been identified in rainbow trout and their proteins partially characterized. These previous studies reported a single type II IFN (rtIFN-??) and three rainbow trout type I IFN genes that are classified into either group I (rtIFN1, rtIFN2) or group II (rtIFN3). In this present study, we report the identification of a novel IFN-?? gene (rtIFN-??2) and a novel type I group II IFN (rtIFN4) in homozygous rainbow trout and predict that additional IFN genes or pseudogenes exist in the rainbow trout genome. Additionally, we provide evidence that short and long forms of rtIFN1 are actively and differentially transcribed in homozygous trout, and likely arose due to alternate splicing of the first exon. Quantitative reverse transcriptase PCR (qRT-PCR) assays were developed to systematically profile all of the rainbow trout IFN transcripts, with high specificity at an individual gene level, in na??ve fish and after stimulation with virus or viral-related molecules. Cloned PCR products were used to ensure the specificity of the qRT-PCR assays and as absolute standards to assess transcript abundance of each gene. All IFN genes were modulated in response to Infectious hematopoietic necrosis virus (IHNV), a DNA vaccine based on the IHNV glycoprotein, and poly I:C. The most inducible of the type I IFN genes, by all stimuli tested, were rtIFN3 and the short transcript form of rtIFN1. Gene expression of rtIFN-??1 and rtIFN-??2 was highly up-regulated by IHNV infection and DNA vaccination but rtIFN-??2 was induced to a greater magnitude. The specificity of the qRT-PCR assays reported here will be useful for future studies aimed at identifying which cells produce IFNs at early time points after infection. ?? 2008 Elsevier Ltd.

Splicing of a group II intron involved in the conjugative transfer of pRS01 in lactococci.

PubMed

Mills, D A; McKay, L L; Dunny, G M

1996-06-01

Analysis of a region involved in the conjugative transfer of the lactococcal conjugative element pRS01 has revealed a bacteria] group II intron. Splicing of this lactococcal intron (designated Ll.ltrB) in vivo resulted in the ligation of two exon messages (ltrBE1 and ltrBE2) which encoded a putative conjugative relaxase essential for the transfer of pRS01. Like many group II introns, the Ll.ltrB intron possessed an open reading frame (ltrA) with homology to reverse transcriptases. Remarkably, sequence analysis of ltrA suggested a greater similarity to open reading frames encoded by eukaryotic mitochondrial group II introns than to those identified to date from other bacteria. Several insertional mutations within ltrA resulted in plasmids exhibiting a conjugative transfer-deficient phenotype. These results provide the first direct evidence for splicing of a prokaryotic group II intron in vivo and suggest that conjugative transfer is a mechanism for group II intron dissemination in bacteria.

Engineering hyperthermophilic archaeon Pyrococcus furiosus to overproduce its cytoplasmic [NiFe]-hydrogenase.

PubMed

Chandrayan, Sanjeev K; McTernan, Patrick M; Hopkins, R Christopher; Sun, Junsong; Jenney, Francis E; Adams, Michael W W

2012-01-27

The cytoplasmic hydrogenase (SHI) of the hyperthermophilic archaeon Pyrococcus furiosus is an NADP(H)-dependent heterotetrameric enzyme that contains a nickel-iron catalytic site, flavin, and six iron-sulfur clusters. It has potential utility in a range of bioenergy systems in vitro, but a major obstacle in its use is generating sufficient amounts. We have engineered P. furiosus to overproduce SHI utilizing a recently developed genetic system. In the overexpression (OE-SHI) strain, transcription of the four-gene SHI operon was under the control of a strong constitutive promoter, and a Strep-tag II was added to the N terminus of one subunit. OE-SHI and wild-type P. furiosus strains had similar rates of growth and H(2) production on maltose. Strain OE-SHI had a 20-fold higher transcription of the polycistronic hydrogenase mRNA encoding SHI, and the specific activity of the cytoplasmic hydrogenase was ∼10-fold higher when compared with the wild-type strain, although the expression levels of genes encoding processing and maturation of SHI were the same in both strains. Overexpressed SHI was purified by a single affinity chromatography step using the Strep-tag II, and it and the native form had comparable activities and physical properties. Based on protein yield per gram of cells (wet weight), the OE-SHI strain yields a 100-fold higher amount of hydrogenase when compared with the highest homologous [NiFe]-hydrogenase system previously reported (from Synechocystis). This new P. furiosus system will allow further engineering of SHI and provide hydrogenase for efficient in vitro biohydrogen production.

Engineering Hyperthermophilic Archaeon Pyrococcus furiosus to Overproduce Its Cytoplasmic [NiFe]-Hydrogenase*

PubMed Central

Chandrayan, Sanjeev K.; McTernan, Patrick M.; Hopkins, R. Christopher; Sun, Junsong; Jenney, Francis E.; Adams, Michael W. W.

2012-01-01

The cytoplasmic hydrogenase (SHI) of the hyperthermophilic archaeon Pyrococcus furiosus is an NADP(H)-dependent heterotetrameric enzyme that contains a nickel-iron catalytic site, flavin, and six iron-sulfur clusters. It has potential utility in a range of bioenergy systems in vitro, but a major obstacle in its use is generating sufficient amounts. We have engineered P. furiosus to overproduce SHI utilizing a recently developed genetic system. In the overexpression (OE-SHI) strain, transcription of the four-gene SHI operon was under the control of a strong constitutive promoter, and a Strep-tag II was added to the N terminus of one subunit. OE-SHI and wild-type P. furiosus strains had similar rates of growth and H2 production on maltose. Strain OE-SHI had a 20-fold higher transcription of the polycistronic hydrogenase mRNA encoding SHI, and the specific activity of the cytoplasmic hydrogenase was ∼10-fold higher when compared with the wild-type strain, although the expression levels of genes encoding processing and maturation of SHI were the same in both strains. Overexpressed SHI was purified by a single affinity chromatography step using the Strep-tag II, and it and the native form had comparable activities and physical properties. Based on protein yield per gram of cells (wet weight), the OE-SHI strain yields a 100-fold higher amount of hydrogenase when compared with the highest homologous [NiFe]-hydrogenase system previously reported (from Synechocystis). This new P. furiosus system will allow further engineering of SHI and provide hydrogenase for efficient in vitro biohydrogen production. PMID:22157005

Contribution of Asparagine Catabolism to Salmonella Virulence

PubMed Central

McLaughlin, Patrick A.; McClelland, Michael; Yang, Hee-Jeong; Porwollik, Steffen; Bogomolnaya, Lydia; Chen, Juei-Suei; Andrews-Polymenis, Helene

2016-01-01

ABSTRACT Salmonellae are pathogenic bacteria that cause significant morbidity and mortality in humans worldwide. Salmonellae establish infection and avoid clearance by the immune system by mechanisms that are not well understood. We previously showed that l-asparaginase II produced by Salmonella enterica serovar Typhimurium (S. Typhimurium) inhibits T cell responses and mediates virulence. In addition, we previously showed that asparagine deprivation such as that mediated by l-asparaginase II of S. Typhimurium causes suppression of activation-induced T cell metabolic reprogramming. Here, we report that STM3997, which encodes a homolog of disulfide bond protein A (dsbA) of Escherichia coli, is required for l-asparaginase II stability and function. Furthermore, we report that l-asparaginase II localizes primarily to the periplasm and acts together with l-asparaginase I to provide S. Typhimurium the ability to catabolize asparagine and assimilate nitrogen. Importantly, we determined that, in a murine model of infection, S. Typhimurium lacking both l-asparaginase I and II genes competes poorly with wild-type S. Typhimurium for colonization of target tissues. Collectively, these results indicate that asparagine catabolism contributes to S. Typhimurium virulence, providing new insights into the competition for nutrients at the host-pathogen interface. PMID:27849183

Contribution of Asparagine Catabolism to Salmonella Virulence.

PubMed

McLaughlin, Patrick A; McClelland, Michael; Yang, Hee-Jeong; Porwollik, Steffen; Bogomolnaya, Lydia; Chen, Juei-Suei; Andrews-Polymenis, Helene; van der Velden, Adrianus W M

2017-02-01

Salmonellae are pathogenic bacteria that cause significant morbidity and mortality in humans worldwide. Salmonellae establish infection and avoid clearance by the immune system by mechanisms that are not well understood. We previously showed that l-asparaginase II produced by Salmonella enterica serovar Typhimurium (S Typhimurium) inhibits T cell responses and mediates virulence. In addition, we previously showed that asparagine deprivation such as that mediated by l-asparaginase II of S Typhimurium causes suppression of activation-induced T cell metabolic reprogramming. Here, we report that STM3997, which encodes a homolog of disulfide bond protein A (dsbA) of Escherichia coli, is required for l-asparaginase II stability and function. Furthermore, we report that l-asparaginase II localizes primarily to the periplasm and acts together with l-asparaginase I to provide S Typhimurium the ability to catabolize asparagine and assimilate nitrogen. Importantly, we determined that, in a murine model of infection, S Typhimurium lacking both l-asparaginase I and II genes competes poorly with wild-type S Typhimurium for colonization of target tissues. Collectively, these results indicate that asparagine catabolism contributes to S Typhimurium virulence, providing new insights into the competition for nutrients at the host-pathogen interface. Copyright © 2017 American Society for Microbiology.

Typing of Panton-Valentine leukocidin-encoding phages carried by methicillin-susceptible and methicillin-resistant Staphylococcus aureus from Italy.

PubMed

Sanchini, A; Del Grosso, M; Villa, L; Ammendolia, M G; Superti, F; Monaco, M; Pantosti, A

2014-11-01

Panton-Valentine leukocidin (PVL) is the hallmark of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) but can also be found in methicillin-susceptible S. aureus (MSSA) sharing pathogenic and epidemiological characteristics of CA-MRSA. PVL is encoded by two co-transcribed genes that are carried by different staphylococcal bacteriophages. We applied an extended PCR-based typing scheme for the identification of two morphological groups (elongated-head group and icosahedral-head group I phages) and specific PVL phage types in S. aureus isolates recovered in Italy. We examined 48 PVL-positive isolates (25 MSSA and 23 MRSA) collected from different hospital laboratories from April 2005 to May 2011. spa typing, multilocus sequence typing and staphylococcal cassette chromosome mec typing were applied to categorize the isolates. Phage typeability was 48.0% in MSSA and 91.3% in MRSA, highlighting the limitation of the PCR typing scheme when applied to PVL-positive MSSA. Five different PVL phages and two variants of a known phage were detected, the most prevalent being ΦSa2usa, recovered in 15 out of 48 (31.2%) isolates, and carried by both MSSA and MRSA belonging to CC8 and CC5. The recently described ΦTCH60 was recovered in four isolates. A PVL phage (ΦSa119) from an ST772 MRSA, that was not detected using the previous typing scheme, was sequenced, and new primers were designed for the identification of the icosahedral-head group II PVL phages present in ST772 and ST59 MRSA. A comprehensive PVL-phage typing can contribute to the understanding of the epidemiology and evolution of PVL-positive MSSA and MRSA. © 2014 The Authors Clinical Microbiology and Infection © 2014 European Society of Clinical Microbiology and Infectious Diseases.

Evolutionary Divergence of Plant Borate Exporters and Critical Amino Acid Residues for the Polar Localization and Boron-Dependent Vacuolar Sorting of AtBOR1.

PubMed

Wakuta, Shinji; Mineta, Katsuhiko; Amano, Taro; Toyoda, Atsushi; Fujiwara, Toru; Naito, Satoshi; Takano, Junpei

2015-05-01

Boron (B) is an essential micronutrient for plants but is toxic when accumulated in excess. The plant BOR family encodes plasma membrane-localized borate exporters (BORs) that control translocation and homeostasis of B under a wide range of conditions. In this study, we examined the evolutionary divergence of BORs among terrestrial plants and showed that the lycophyte Selaginella moellendorffii and angiosperms have evolved two types of BOR (clades I and II). Clade I includes AtBOR1 and homologs previously shown to be involved in efficient transport of B under conditions of limited B availability. AtBOR1 shows polar localization in the plasma membrane and high-B-induced vacuolar sorting, important features for efficient B transport under low-B conditions, and rapid down-regulation to avoid B toxicity. Clade II includes AtBOR4 and barley Bot1 involved in B exclusion for high-B tolerance. We showed, using yeast complementation and B transport assays, that three genes in S. moellendorffii, SmBOR1 in clade I and SmBOR3 and SmBOR4 in clade II, encode functional BORs. Furthermore, amino acid sequence alignments identified an acidic di-leucine motif unique in clade I BORs. Mutational analysis of AtBOR1 revealed that the acidic di-leucine motif is required for the polarity and high-B-induced vacuolar sorting of AtBOR1. Our data clearly indicated that the common ancestor of vascular plants had already acquired two types of BOR for low- and high-B tolerance, and that the BOR family evolved to establish B tolerance in each lineage by adapting to their environments. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Structural Insight into How Bacteria Prevent Interference between Multiple Divergent Type IV Secretion Systems

PubMed Central

Phan, Isabelle Q. H.; Scheib, Holger; Subramanian, Sandhya; Edwards, Thomas E.; Lehman, Stephanie S.; Piitulainen, Hanna; Sayeedur Rahman, M.; Rennoll-Bankert, Kristen E.; Staker, Bart L.; Taira, Suvi; Stacy, Robin; Myler, Peter J.; Azad, Abdu F.

2015-01-01

ABSTRACT Prokaryotes use type IV secretion systems (T4SSs) to translocate substrates (e.g., nucleoprotein, DNA, and protein) and/or elaborate surface structures (i.e., pili or adhesins). Bacterial genomes may encode multiple T4SSs, e.g., there are three functionally divergent T4SSs in some Bartonella species (vir, vbh, and trw). In a unique case, most rickettsial species encode a T4SS (rvh) enriched with gene duplication. Within single genomes, the evolutionary and functional implications of cross-system interchangeability of analogous T4SS protein components remains poorly understood. To lend insight into cross-system interchangeability, we analyzed the VirB8 family of T4SS channel proteins. Crystal structures of three VirB8 and two TrwG Bartonella proteins revealed highly conserved C-terminal periplasmic domain folds and dimerization interfaces, despite tremendous sequence divergence. This implies remarkable structural constraints for VirB8 components in the assembly of a functional T4SS. VirB8/TrwG heterodimers, determined via bacterial two-hybrid assays and molecular modeling, indicate that differential expression of trw and vir systems is the likely barrier to VirB8-TrwG interchangeability. We also determined the crystal structure of Rickettsia typhi RvhB8-II and modeled its coexpressed divergent paralog RvhB8-I. Remarkably, while RvhB8-I dimerizes and is structurally similar to other VirB8 proteins, the RvhB8-II dimer interface deviates substantially from other VirB8 structures, potentially preventing RvhB8-I/RvhB8-II heterodimerization. For the rvh T4SS, the evolution of divergent VirB8 paralogs implies a functional diversification that is unknown in other T4SSs. Collectively, our data identify two different constraints (spatiotemporal for Bartonella trw and vir T4SSs and structural for rvh T4SSs) that mediate the functionality of multiple divergent T4SSs within a single bacterium. PMID:26646013

The Function of Herpes Simplex Virus Genes: A Primer for Genetic Engineering of Novel Vectors

NASA Astrophysics Data System (ADS)

Roizman, Bernard

1996-10-01

Herpes simplex virus vectors are being developed for delivery and expression of human genes to the central nervous system, selective destruction of cancer cells, and as carriers for genes encoding antigens that induce protective immunity against infectious agents. Vectors constructed to meet these objectives must differ from wild-type virus with respect to host range, reactivation from latency, and expression of viral genes. The vectors currently being developed are (i) helper free amplicons, (ii) replication defective viruses, and (iii) genetically engineered replication competent viruses with restricted host range. Whereas the former two types of vectors require stable, continuous cell lines expressing viral genes for their replication, the replication competent viruses will replicate on approved primary human cell strains.

Early-onset progressive ataxia associated with the first CACNA1A mutation identified within the I-II loop.

PubMed

Cricchi, F; Di Lorenzo, C; Grieco, G S; Rengo, C; Cardinale, A; Racaniello, M; Santorelli, F M; Nappi, G; Pierelli, F; Casali, C

2007-03-15

Familial hemiplegic migraine type 1, spinocerebellar ataxia type 6 (SCA6) and episodic ataxia type 2 (EA2) are allelic disorders associated with mutations in the CACNA1A gene, which encodes the alpha1 subunit of the P/Q-type calcium channel (Ca(V)2.1). SCA6 and EA2 share a number of clinical features, such as prominent cerebellar involvement and good response to acetazolamide therapy. However, while SCA6 develops as a late-onset, progressive ataxia, EA2 has an earlier, and episodic, onset. We report on two sisters with a heterogeneous clinical phenotype. The first developed progressive cerebellar ataxia after age 30, without noticeable episodes of vertigo or headache. A 1 year trial with acetazolamide did not produce significant results. The other reported episodes of vertigo, headache and gait imbalance since late childhood, with good response to acetazolamide, before developing moderate chronic cerebellar ataxia. Brain MRI showed cerebellar atrophy, especially in the vermis, in both patients. Direct sequencing of CACNA1A identified a heterozygous 1360G>A mutation in exon 11 resulting in the substitution of alanine for threonine at residue 454 (p.Ala454Thr). This is the first description of a change residing in the cytoplasmic I-II loop associated with a clinical phenotype.

Whole genome sequence analysis of Geitlerinema sp. FC II unveils competitive edge of the strain in marine cultivation system for biofuel production.

PubMed

Batchu, Navish Kumar; Khater, Shradha; Patil, Sonal; Nagle, Vinod; Das, Gautam; Bhadra, Bhaskar; Sapre, Ajit; Dasgupta, Santanu

2018-03-05

A filamentous cyanobacteria, Geitlerinema sp. FC II, was isolated from marine algae culture pond at Reliance Industries Limited (RIL), India. The 6.7 Mb draft genome of FC II encodes for 6697 protein coding genes. Analysis of the whole genome sequence revealed presence of nif gene cluster, supporting its capability to fix atmospheric nitrogen. FC II genome contains two variants of sulfide:quinone oxidoreductases (SQR), which is a crucial elector donor in cyanobacterial metabolic processes. FC II is characterized by the presence of multiple CRISPR- Cas (Clustered Regularly Interspaced Short Palindrome Repeats - CRISPR associated proteins) clusters, multiple variants of genes encoding photosystem reaction centres, biosynthetic gene clusters of alkane, polyketides and non-ribosomal peptides. Presence of these pathways will help FC II in gaining an ecological advantage over other strains for biomass production in large scale cultivation system. Hence, FC II may be used for production of biofuel and other industrially important metabolites. Copyright © 2018 Elsevier Inc. All rights reserved.

Characterization of a major late herpes simplex virus type 1 mRNA.

PubMed

Costa, R H; Devi, B G; Anderson, K P; Gaylord, B H; Wagner, E K

1981-05-01

A major, late 6-kilobase (6-kb) mRNa mapping in the large unique region of herpes simplex virus type 1 (HSV-1) was characterized by using two recombinant DNA clones, one containing EcoRI fragment G (0.190 to 0.30 map units) in lambda. WES.B (L. Enquist, M. Madden, P. Schiop-Stansly, and G. Vandl Woude, Science 203:541-544, 1979) and one containing HindIII fragment J (0.181 to 0.259 map units) in pBR322. This 6-kb mRNA had its 3' end to the left of 0.231 on the prototypical arrangement of the HSV-1 genome and was transcribed from right to left. It was bounded on both sides by regions containing a large number of distinct mRNA species, and its 3' end was partially colinear with a 1.5-kb mRNA which encoded a 35,000-dalton polypeptide. The 6-kb mRNA encoded a 155,000-dalton polypeptide which was shown to be the only one of this size detectable by hybrid-arrested translation encoded by late polyadenylated polyribosomal RNA. The S1 nuclease mapping experiments indicated that there were no introns in the coding sequence for this mRNA and that its 3' end mapped approximately 800 nucleotides to the left of the BglII site at 0.231, whereas its 5' end extended very close to the BamHI site at 0.266.

Recovery of episodic memory subprocesses in mild and complicated mild traumatic brain injury at 1 and 12 months post injury.

PubMed

Tayim, Fadi M; Flashman, Laura A; Wright, Matthew J; Roth, Robert M; McAllister, Thomas W

2016-11-01

Episodic memory complaints are commonly reported after traumatic brain injury (TBI). The contributions of specific memory subprocesses (encoding, consolidation, and retrieval), however, are not well understood in mild TBI (mTBI). In the present study, we evaluated subprocesses of episodic memory in patients with mTBI using the item-specific deficit approach (ISDA), which analyzes responses on list learning tasks at an item level. We also conducted exploratory analyses to evaluate the effects of complicated mTBI (comp-mTBI) on memory. We compared episodic verbal memory performance in mTBI (n = 92) at approximately 1 and 12 months post TBI, as well as in a healthy comparison (HC) group (n = 40) at equivalent time points. Episodic memory was assessed using the California Verbal Learning Test-2nd Edition (CVLT-II), and both standard CVLT-II scores and ISDA indices were evaluated. Compared to the HC group, the mTBI group showed significantly poorer encoding and learning across time, as measured by ISDA and CVLT-II. Further analyses of these mTBI subgroups [(noncomplicated mTBI (NC-mTBI, n = 77) and comp-mTBI (n = 15)], indicated that it was the comp-mTBI group who continued to demonstrate poorer encoding ability than the HC group. When the patient groups were directly compared, the NC-mTBI group improved slightly on the ISDA Encoding Deficit Index. While the comp-mTBI group worsened slightly over time, their poorer encoding ability was not likely clinically meaningful. These findings indicate that, while the NC-mTBI and HC groups' performances were comparable by 12 months, a primary, long-term deficit in encoding of auditory verbal information remained problematic in the comp-mTBI group.

A cysteine protease encoded by the baculovirus Bombyx mori nuclear polyhedrosis virus.

PubMed Central

Ohkawa, T; Majima, K; Maeda, S

1994-01-01

Sequence analysis of the BamHI F fragment of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) revealed an open reading frame whose deduced amino acid sequence had homology to those of cysteine proteases of the papain superfamily. The putative cysteine protease sequence (BmNPV-CP) was 323 amino acids long and showed 35% identity to a cysteine proteinase precursor from Trypanosoma brucei. Of 36 residues conserved among cathepsins B, H, L, and S and papain, 31 were identical in BmNPV-CP. In order to determine the activity and function of the putative cysteine protease, a BmNPV mutant (BmCysPD) was constructed by homologous recombination of the protease gene with a beta-galactosidase gene cassette. BmCysPD-infected BmN cell extracts were significantly reduced in acid protease activity compared with wild-type virus-infected cell extracts. The cysteine protease inhibitor E-64 [trans-epoxysuccinylleucylamido-(4-guanidino)butane] inhibited wild-type virus-expressed protease activity. Deletion of the cysteine protease gene had no significant effect on viral growth or polyhedron production in BmN cells, indicating that the cysteine protease was not essential for viral replication in vitro. However, B. mori larvae infected with BmCysPD showed symptoms different from those of wild-type BmNPV-infected larvae, e.g., less degradation of the body, including fat body cells, white body surface color due presumably to undegraded epidermal cells, and an increase in the number of polyhedra released into the hemolymph. This is the first report of (i) a virus-encoded protease with activity on general substrates and (ii) evidence that a virus-encoded protease may play a role in degradation of infected larvae to facilitate horizontal transmission of the virus. Images PMID:8083997

Cognitive training of self-initiation of semantic encoding strategies in schizophrenia: A pilot study.

PubMed

Guimond, Synthia; Lepage, Martin

2016-01-01

Available cognitive remediation interventions have a significant but relatively small to moderate impact on episodic memory in schizophrenia. The present study aimed to evaluate the efficacy and feasibility of a brief novel episodic memory training targeting the self-initiation of semantic encoding strategies. To select patients with such deficits, 28 participants with schizophrenia performed our Semantic Encoding Memory Task (SEMT) that provides a measure of self-initiated semantic encoding strategies. This task identified a deficit in 13 participants who were then offered two 60-minute training sessions one week apart. After the training, patients performed an alternate version of the SEMT. The CVLT-II (a standardised measure of semantic encoding strategies) and the BVMT-R (a control spatial memory task) were used to quantify memory pre- and post-training. After the training, participants were significantly better at self-initiating semantic encoding strategies in the SEMT (p = .004) and in the CVLT-II (p = .002). No significant differences were found in the BVMT-R. The current study demonstrates that a brief and specific training in memory strategies can help patients to improve a deficient memory process in schizophrenia. Future studies will need to test this intervention further using a randomised controlled trial, and to explore its functional impact.

The tad locus: postcards from the widespread colonization island.

PubMed

Tomich, Mladen; Planet, Paul J; Figurski, David H

2007-05-01

The Tad (tight adherence) macromolecular transport system, which is present in many bacterial and archaeal species, represents an ancient and major new subtype of type II secretion. The tad genes are present on a genomic island named the widespread colonization island (WCI), and encode the machinery that is required for the assembly of adhesive Flp (fimbrial low-molecular-weight protein) pili. The tad genes are essential for biofilm formation, colonization and pathogenesis in the genera Aggregatibacter (Actinobacillus), Haemophilus, Pasteurella, Pseudomonas, Yersinia, Caulobacter and perhaps others. Here we review the structure, function and evolution of the Tad secretion system.

Bacterial Group II Introns: Identification and Mobility Assay.

PubMed

Toro, Nicolás; Molina-Sánchez, María Dolores; Nisa-Martínez, Rafael; Martínez-Abarca, Francisco; García-Rodríguez, Fernando Manuel

2016-01-01

Group II introns are large catalytic RNAs and mobile retroelements that encode a reverse transcriptase. Here, we provide methods for their identification in bacterial genomes and further analysis of their splicing and mobility capacities.

Population genomic analysis of strain variation in Leptospirillum group II bacteria involved in acid mine drainage formation.

PubMed

Simmons, Sheri L; Dibartolo, Genevieve; Denef, Vincent J; Goltsman, Daniela S Aliaga; Thelen, Michael P; Banfield, Jillian F

2008-07-22

Deeply sampled community genomic (metagenomic) datasets enable comprehensive analysis of heterogeneity in natural microbial populations. In this study, we used sequence data obtained from the dominant member of a low-diversity natural chemoautotrophic microbial community to determine how coexisting closely related individuals differ from each other in terms of gene sequence and gene content, and to uncover evidence of evolutionary processes that occur over short timescales. DNA sequence obtained from an acid mine drainage biofilm was reconstructed, taking into account the effects of strain variation, to generate a nearly complete genome tiling path for a Leptospirillum group II species closely related to L. ferriphilum (sampling depth approximately 20x). The population is dominated by one sequence type, yet we detected evidence for relatively abundant variants (>99.5% sequence identity to the dominant type) at multiple loci, and a few rare variants. Blocks of other Leptospirillum group II types ( approximately 94% sequence identity) have recombined into one or more variants. Variant blocks of both types are more numerous near the origin of replication. Heterogeneity in genetic potential within the population arises from localized variation in gene content, typically focused in integrated plasmid/phage-like regions. Some laterally transferred gene blocks encode physiologically important genes, including quorum-sensing genes of the LuxIR system. Overall, results suggest inter- and intrapopulation genetic exchange involving distinct parental genome types and implicate gain and loss of phage and plasmid genes in recent evolution of this Leptospirillum group II population. Population genetic analyses of single nucleotide polymorphisms indicate variation between closely related strains is not maintained by positive selection, suggesting that these regions do not represent adaptive differences between strains. Thus, the most likely explanation for the observed patterns of polymorphism is divergence of ancestral strains due to geographic isolation, followed by mixing and subsequent recombination.

Population Genomic Analysis of Strain Variation in Leptospirillum Group II Bacteria Involved in Acid Mine Drainage Formation

PubMed Central

Denef, Vincent J; Goltsman, Daniela S. Aliaga; Thelen, Michael P; Banfield, Jillian F

2008-01-01

Deeply sampled community genomic (metagenomic) datasets enable comprehensive analysis of heterogeneity in natural microbial populations. In this study, we used sequence data obtained from the dominant member of a low-diversity natural chemoautotrophic microbial community to determine how coexisting closely related individuals differ from each other in terms of gene sequence and gene content, and to uncover evidence of evolutionary processes that occur over short timescales. DNA sequence obtained from an acid mine drainage biofilm was reconstructed, taking into account the effects of strain variation, to generate a nearly complete genome tiling path for a Leptospirillum group II species closely related to L. ferriphilum (sampling depth ∼20×). The population is dominated by one sequence type, yet we detected evidence for relatively abundant variants (>99.5% sequence identity to the dominant type) at multiple loci, and a few rare variants. Blocks of other Leptospirillum group II types (∼94% sequence identity) have recombined into one or more variants. Variant blocks of both types are more numerous near the origin of replication. Heterogeneity in genetic potential within the population arises from localized variation in gene content, typically focused in integrated plasmid/phage-like regions. Some laterally transferred gene blocks encode physiologically important genes, including quorum-sensing genes of the LuxIR system. Overall, results suggest inter- and intrapopulation genetic exchange involving distinct parental genome types and implicate gain and loss of phage and plasmid genes in recent evolution of this Leptospirillum group II population. Population genetic analyses of single nucleotide polymorphisms indicate variation between closely related strains is not maintained by positive selection, suggesting that these regions do not represent adaptive differences between strains. Thus, the most likely explanation for the observed patterns of polymorphism is divergence of ancestral strains due to geographic isolation, followed by mixing and subsequent recombination. PMID:18651792

Escape of X-linked miRNA genes from meiotic sex chromosome inactivation

PubMed Central

Sosa, Enrique; Flores, Luis; Yan, Wei; McCarrey, John R.

2015-01-01

Past studies have indicated that transcription of all X-linked genes is repressed by meiotic sex chromosome inactivation (MSCI) during the meiotic phase of spermatogenesis in mammals. However, more recent studies have shown an increase in steady-state levels of certain X-linked miRNAs in pachytene spermatocytes, suggesting that either synthesis of these miRNAs increases or that degradation of these miRNAs decreases dramatically in these cells. To distinguish between these possibilities, we performed RNA-FISH to detect nascent transcripts from multiple miRNA genes in various spermatogenic cell types. Our results show definitively that Type I X-linked miRNA genes are subject to MSCI, as are all or most X-linked mRNA genes, whereas Type II and III X-linked miRNA genes escape MSCI by continuing ongoing, active transcription in primary spermatocytes. We corroborated these results by co-localization of RNA-FISH signals with both a corresponding DNA-FISH signal and an immunofluorescence signal for RNA polymerase II. We also found that X-linked miRNA genes that escape MSCI locate non-randomly to the periphery of the XY body, whereas genes that are subject to MSCI remain located within the XY body in pachytene spermatocytes, suggesting that the mechanism of escape of X-linked miRNA genes from MSCI involves their relocation to a position outside of the repressive chromatin domain associated with the XY body. The fact that Type II and III X-linked miRNA genes escape MSCI suggests an immediacy of function of the encoded miRNAs specifically required during the meiotic stages of spermatogenesis. PMID:26395485

Cloning, overexpression, purification and preliminary crystallographic studies of a mitochondrial type II peroxiredoxin from Pisum sativum.

PubMed

Barranco-Medina, Sergio; López-Jaramillo, Francisco Javier; Bernier-Villamor, Laura; Sevilla, Francisca; Lázaro, Juan José

2006-07-01

A cDNA encoding an open reading frame of 199 amino acids corresponding to a type II peroxiredoxin from Pisum sativum with its transit peptide was isolated by RT-PCR. The 171-amino-acid mature protein (estimated molecular weight 18.6 kDa) was cloned into the pET3d vector and overexpressed in Escherichia coli. The recombinant protein was purified and crystallized by the hanging-drop vapour-diffusion technique. A full data set (98.2% completeness) was collected using a rotating-anode generator to a resolution of 2.8 angstroms from a single crystal flash-cooled at 100 K. X-ray data revealed that the protein crystallizes in space group P1, with unit-cell parameters a = 61.88, b = 66.40, c = 77.23 angstroms, alpha = 102.90, beta = 104.40, gamma = 99.07 degrees, and molecular replacement using a theoretical model predicted from the primary structure as a search model confirmed the presence of six molecules in the unit cell as expected from the Matthews coefficient. Refinement of the structure is in progress.

Type II protein arginine methyltransferase 5 (PRMT5) is required for circadian period determination in Arabidopsis thaliana.

PubMed

Hong, Sunghyun; Song, Hae-Ryong; Lutz, Kerry; Kerstetter, Randall A; Michael, Todd P; McClung, C Robertson

2010-12-07

Posttranslational modification is an important element in circadian clock function from cyanobacteria through plants and mammals. For example, a number of key clock components are phosphorylated and thereby marked for subsequent ubiquitination and degradation. Through forward genetic analysis we demonstrate that protein arginine methyltransferase 5 (PRMT5; At4g31120) is a critical determinant of circadian period in Arabidopsis. PRMT5 is coregulated with a set of 1,253 genes that shows alterations in phase of expression in response to entrainment to thermocycles versus photocycles in constant temperature. PRMT5 encodes a type II protein arginine methyltransferase that catalyzes the symmetric dimethylation of arginine residues (Rsme2). Rsme2 modification has been observed in many taxa, and targets include histones, components of the transcription complex, and components of the spliceosome. Neither arginine methylation nor PRMT5 has been implicated previously in circadian clock function, but the period lengthening associated with mutational disruption of prmt5 indicates that Rsme2 is a decoration important for the Arabidopsis clock and possibly for clocks in general.

Construction and properties of a temperature-sensitive mutation in the gene for the bacteriophage SPO1 DNA-binding protein TF1.

PubMed

Sayre, M H; Geiduschek, E P

1990-08-01

The Bacillus subtilis bacteriophage SPO1 encodes the DNA-binding protein TF1, a homolog of the ubiquitous type II DNA-binding proteins that are components of bacterial chromatin. The known three-dimensional structure of a related protein was used in devising a scheme of site-directed mutagenesis that led to the creation of a temperature-sensitive mutation in the TF1 gene. At the nonpermissive temperature, this mutation disrupted the temporal regulation of viral protein synthesis and processing, altered the kinetics of accumulation of at least one viral transcript, and prohibited the production of infective progeny phage. We suggest that TF1 function is required to shut off the expression of several early-middle and middle viral genes and that TF1 plays a role in phage head morphogenesis. Spontaneous second-site mutations of the temperature-sensitive mutant TF1 allele that suppressed its associated phenotypes were analyzed. These suppressor mutations conferred greater amino acid sequence homology with the type II DNA-binding protein from the thermophile Bacillus stearothermophilus.

Type II protein arginine methyltransferase 5 (PRMT5) is required for circadian period determination in Arabidopsis thaliana

PubMed Central

Hong, Sunghyun; Lutz, Kerry; Kerstetter, Randall A.; Michael, Todd P.; McClung, C. Robertson

2010-01-01

Posttranslational modification is an important element in circadian clock function from cyanobacteria through plants and mammals. For example, a number of key clock components are phosphorylated and thereby marked for subsequent ubiquitination and degradation. Through forward genetic analysis we demonstrate that protein arginine methyltransferase 5 (PRMT5; At4g31120) is a critical determinant of circadian period in Arabidopsis. PRMT5 is coregulated with a set of 1,253 genes that shows alterations in phase of expression in response to entrainment to thermocycles versus photocycles in constant temperature. PRMT5 encodes a type II protein arginine methyltransferase that catalyzes the symmetric dimethylation of arginine residues (Rsme2). Rsme2 modification has been observed in many taxa, and targets include histones, components of the transcription complex, and components of the spliceosome. Neither arginine methylation nor PRMT5 has been implicated previously in circadian clock function, but the period lengthening associated with mutational disruption of prmt5 indicates that Rsme2 is a decoration important for the Arabidopsis clock and possibly for clocks in general. PMID:21097700

Vaccinia Virus Blocks Stat1-Dependent and Stat1-Independent Gene Expression Induced by Type I and Type II Interferons

PubMed Central

Mann, Brandon A.; Huang, Julia He; Li, Ping; Chang, Hua-Chen; Slee, Roger B.; O'Sullivan, Audrey; Mathur, Anita; Yeh, Norman; Klemsz, Michael J.; Brutkiewicz, Randy R.; Blum, Janice S.

2008-01-01

Blocking the function of Stat (signal transducer and activator of transcription) proteins, which are critical for antiviral responses, has evolved as a common mechanism for pathogen immune evasion. The poxvirus-encoded phosphatase H1 is critical for viral replication, and may play an additional role in the evasion of host defense by dephosphorylating Stat1 and blocking interferon (IFN)-stimulated innate immune responses. Vaccinia virus (VACV) H1 can inhibit the phosphorylation of the transcription factor Stat1 after IFN-γ stimulation of epithelial cells, greatly attenuating IFN-induced biological functions. In this study, we demonstrate that VACV infection is capable of inhibiting the phosphorylation of Stat1 and Stat2 after stimulation of fibroblasts or bone marrow-derived macrophages with either type I or type II IFNs, but did not inhibit the activation of Stat3 or Stat5 in either cell type. By using recombinant proteins for in vitro assays, we observe that variola virus H1 is more active than VACV H1, although it has similar selectivity for Stat targets. Differential effects of VACV infection were observed on the induction of IFN-stimulated genes, with complete inhibition of some genes by VACV infection, while others were less affected. Despite the IFN-γ-induced expression of some genes in VACV-infected cells, IFN-γ was unable to rescue the VACV-mediated inhibition of MHC class II antigen presentation. Moreover, VACV infection can affect the IFN-induced expression of Stat1-dependent and Stat1-independent genes, suggesting that the virus may target additional IFN-activated pathways. Thus, VACV targets multiple signaling pathways in the evasion of antiviral immune responses. PMID:18593332

Recombination and mutation of class II histocompatibility genes in wild mice.

PubMed

Wakeland, E K; Darby, B R

1983-12-01

We have compared the tryptic peptide fingerprints of the A alpha, A beta, E alpha, and E beta subunits encoded by four wild-derived H-2 complexes expressing A molecules closely related to Ak. The A molecules encoded by these Ak-related mice have A alpha and A beta subunits that differ from A alpha k and A beta k by less than 10% of their tryptic peptides. Comparisons among the four wild-derived A molecules suggested that these contemporary A alpha and A beta alleles arose by sequential mutational events from common ancestor A alpha and A beta alleles. These results suggest that A alpha and A beta may co-evolve as an A beta A alpha gene duplex in wild mice. Tryptic peptide fingerprint comparisons of the E beta gene linked to these Ak-related A beta A alpha gene duplexes indicate that two encode E beta d-like subunits, whereas another encodes an E beta s-like subunit. These results strongly suggest that the A beta A alpha duplex and E beta recombine in wild mouse populations. The significantly different evolutionary patterns exhibited by the class II genes encoding A vs E molecules are discussed.

The Genome Sequence of the Obligately Chemolithoautotrophic, Facultatively Anaerobic Bacterium Thiobacillus denitrificans

PubMed Central

Beller, Harry R.; Chain, Patrick S. G.; Letain, Tracy E.; Chakicherla, Anu; Larimer, Frank W.; Richardson, Paul M.; Coleman, Matthew A.; Wood, Ann P.; Kelly, Donovan P.

2006-01-01

The complete genome sequence of Thiobacillus denitrificans ATCC 25259 is the first to become available for an obligately chemolithoautotrophic, sulfur-compound-oxidizing, β-proteobacterium. Analysis of the 2,909,809-bp genome will facilitate our molecular and biochemical understanding of the unusual metabolic repertoire of this bacterium, including its ability to couple denitrification to sulfur-compound oxidation, to catalyze anaerobic, nitrate-dependent oxidation of Fe(II) and U(IV), and to oxidize mineral electron donors. Notable genomic features include (i) genes encoding c-type cytochromes totaling 1 to 2 percent of the genome, which is a proportion greater than for almost all bacterial and archaeal species sequenced to date, (ii) genes encoding two [NiFe]hydrogenases, which is particularly significant because no information on hydrogenases has previously been reported for T. denitrificans and hydrogen oxidation appears to be critical for anaerobic U(IV) oxidation by this species, (iii) a diverse complement of more than 50 genes associated with sulfur-compound oxidation (including sox genes, dsr genes, and genes associated with the AMP-dependent oxidation of sulfite to sulfate), some of which occur in multiple (up to eight) copies, (iv) a relatively large number of genes associated with inorganic ion transport and heavy metal resistance, and (v) a paucity of genes encoding organic-compound transporters, commensurate with obligate chemolithoautotrophy. Ultimately, the genome sequence of T. denitrificans will enable elucidation of the mechanisms of aerobic and anaerobic sulfur-compound oxidation by β-proteobacteria and will help reveal the molecular basis of this organism's role in major biogeochemical cycles (i.e., those involving sulfur, nitrogen, and carbon) and groundwater restoration. PMID:16452431

Direct bone morphogenetic protein 2 and Indian hedgehog gene transfer for articular cartilage repair using bone marrow coagulates.

PubMed

Sieker, J T; Kunz, M; Weißenberger, M; Gilbert, F; Frey, S; Rudert, M; Steinert, A F

2015-03-01

Bone morphogenetic protein 2 (BMP-2, encoded by BMP2) and Indian hedgehog protein (IHH, encoded by IHH) are well known regulators of chondrogenesis and chondrogenic hypertrophy. Despite being a potent chondrogenic factor BMP-2 was observed to induce chondrocyte hypertrophy in osteoarthritis (OA), growth plate cartilage and adult mesenchymal stem cells (MSCs). IHH might induce chondrogenic differentiation through different intracellular signalling pathways without inducing subsequent chondrocyte hypertrophy. The primary objective of this study is to test the efficacy of direct BMP2 and IHH gene delivery via bone marrow coagulates to influence histological repair cartilage quality in vivo. Vector-laden autologous bone marrow coagulates with 10(11) adenoviral vector particles encoding BMP2, IHH or the Green fluorescent protein (GFP) were delivered to 3.2 mm osteochondral defects in the trochlea of rabbit knees. After 13 weeks the histological repair cartilage quality was assessed using the ICRS II scoring system and the type II collagen positive area. IHH treatment resulted in superior histological repair cartilage quality than GFP controls in all of the assessed parameters (with P < 0.05 in five of 14 assessed parameters). Results of BMP2 treatment varied substantially, including severe intralesional bone formation in two of six joints after 13 weeks. IHH gene transfer is effective to improve repair cartilage quality in vivo, whereas BMP2 treatment, carried the risk intralesional bone formation. Therefore IHH protein can be considered as an attractive alternative candidate growth factor for further preclinical research and development towards improved treatments for articular cartilage defects. Copyright © 2014 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Transformation of Acinetobacter sp. Strain BD413(pFG4ΔnptII) with Transgenic Plant DNA in Soil Microcosms and Effects of Kanamycin on Selection of Transformants

PubMed Central

Nielsen, Kaare M.; van Elsas, Jan D.; Smalla, Kornelia

2000-01-01

Here we show that horizontal transfer of DNA, extracted from transgenic sugar beets, to bacteria, based on homologous recombination, can occur in soil. Restoration of a 317-bp-deleted nptII gene in Acinetobacter sp. strain BD413(pFG4) cells incubated in sterile soil microcosms was detected after addition of nutrients and transgenic plant DNA encoding a functional nptII gene conferring bacterial kanamycin resistance. Selective effects of the addition of kanamycin on the population dynamics of Acinetobacter sp. cells in soil were found, and high concentrations of kanamycin reduced the CFU of Acinetobacter sp. cells from 109 CFU/g of soil to below detection. In contrast to a chromosomal nptII-encoded kanamycin resistance, the pFG4-generated resistance was found to be unstable over a 31-day incubation period in vitro. PMID:10698801

The group II intron maturase: a reverse transcriptase and splicing factor go hand in hand.

PubMed

Zhao, Chen; Pyle, Anna Marie

2017-12-01

The splicing of group II introns in vivo requires the assistance of a multifunctional intron encoded protein (IEP, or maturase). Each IEP is also a reverse-transcriptase enzyme that enables group II introns to behave as mobile genetic elements. During splicing or retro-transposition, each group II intron forms a tight, specific complex with its own encoded IEP, resulting in a highly reactive holoenzyme. This review focuses on the structural basis for IEP function, as revealed by recent crystal structures of an IEP reverse transcriptase domain and cryo-EM structures of an IEP-intron complex. These structures explain how the same IEP scaffold is utilized for intron recognition, splicing and reverse transcription, while providing a physical basis for understanding the evolutionary transformation of the IEP into the eukaryotic splicing factor Prp8. Copyright © 2017 Elsevier Ltd. All rights reserved.

iso-Migrastatin, migrastatin, and dorrigocin production in Streptomyces platensis NRRL 18993 is governed by a single biosynthetic machinery featuring an acyltransferase-less type I polyketide synthase.

PubMed

Lim, Si-Kyu; Ju, Jianhua; Zazopoulos, Emmanuel; Jiang, Hui; Seo, Jeong-Woo; Chen, Yihua; Feng, Zhiyang; Rajski, Scott R; Farnet, Chris M; Shen, Ben

2009-10-23

iso-Migrastatin and related glutarimide-containing polyketides are potent inhibitors of tumor cell migration and their implied potential as antimetastatic agents for human cancers has garnered significant attention. Genome scanning of Streptomyces platensis NRRL 18993 unveiled two candidate gene clusters (088D and mgs); each encodes acyltransferase-less type I polyketide synthases commensurate with iso-migrastatin biosynthesis. Both clusters were inactivated by lambda-RED-mediated PCR-targeting mutagenesis in S. platensis; iso-migrastatin production was completely abolished in the DeltamgsF mutant SB11012 strain, whereas inactivation of 088D-orf7 yielded the SB11006 strain that exhibited no discernible change in iso-migrastatin biosynthesis. These data indicate that iso-migrastatin production is governed by the mgs cluster. Systematic gene inactivation allowed determination of the precise boundaries of the mgs cluster and the essentiality of the genes within the mgs cluster in iso-migrastatin production. The mgs cluster consists of 11 open reading frames that encode three acyltransferase-less type I polyketide synthases (MgsEFG), one discrete acyltransferase (MgsH), a type II thioesterase (MgsB), three post-PKS tailoring enzymes (MgsIJK), two glutarimide biosynthesis enzymes (MgsCD), and one regulatory protein (MgsA). A model for iso-migrastatin biosynthesis is proposed based on functional assignments derived from bioinformatics and is further supported by the results of in vivo gene inactivation experiments.

iso-Migrastatin, Migrastatin, and Dorrigocin Production in Streptomyces platensis NRRL 18993 Is Governed by a Single Biosynthetic Machinery Featuring an Acyltransferase-less Type I Polyketide Synthase*

PubMed Central

Lim, Si-Kyu; Ju, Jianhua; Zazopoulos, Emmanuel; Jiang, Hui; Seo, Jeong-Woo; Chen, Yihua; Feng, Zhiyang; Rajski, Scott R.; Farnet, Chris M.; Shen, Ben

2009-01-01

iso-Migrastatin and related glutarimide-containing polyketides are potent inhibitors of tumor cell migration and their implied potential as antimetastatic agents for human cancers has garnered significant attention. Genome scanning of Streptomyces platensis NRRL 18993 unveiled two candidate gene clusters (088D and mgs); each encodes acyltransferase-less type I polyketide synthases commensurate with iso-migrastatin biosynthesis. Both clusters were inactivated by λ-RED-mediated PCR-targeting mutagenesis in S. platensis; iso-migrastatin production was completely abolished in the ΔmgsF mutant SB11012 strain, whereas inactivation of 088D-orf7 yielded the SB11006 strain that exhibited no discernible change in iso-migrastatin biosynthesis. These data indicate that iso-migrastatin production is governed by the mgs cluster. Systematic gene inactivation allowed determination of the precise boundaries of the mgs cluster and the essentiality of the genes within the mgs cluster in iso-migrastatin production. The mgs cluster consists of 11 open reading frames that encode three acyltransferase-less type I polyketide synthases (MgsEFG), one discrete acyltransferase (MgsH), a type II thioesterase (MgsB), three post-PKS tailoring enzymes (MgsIJK), two glutarimide biosynthesis enzymes (MgsCD), and one regulatory protein (MgsA). A model for iso-migrastatin biosynthesis is proposed based on functional assignments derived from bioinformatics and is further supported by the results of in vivo gene inactivation experiments. PMID:19726666

Regulation of glutamine synthetase II activity in Rhizobium meliloti 104A14.

PubMed Central

Shatters, R G; Somerville, J E; Kahn, M L

1989-01-01

Most rhizobia contain two glutamine synthetase (GS) enzymes: GSI, encoded by glnA, and GSII, encoded by glnII. We have found that WSU414, a Rhizobium meliloti 104A14 glutamine auxotroph derived from a glnA parental strain, is an ntrA mutant. The R. meliloti glnII promoter region contains DNA sequences similar to those found in front of other genes that require ntrA for their transcription. No GSII was found in the glnA ntrA mutant, and when a translational fusion of glnII to the Escherichia coli lacZ gene was introduced into WSU414, no beta-galactosidase was expressed. These results indicate that ntrA is required for glnII expression. The ntrA mutation did not prevent the expression of GSI. In free-living culture, the level of GSII and of the glnII-lacZ fusion protein was regulated by altering transcription in response to available nitrogen. No GSII protein was detected in alfalfa, pea, or soybean nodules when anti-GSII-specific antiserum was used. Images PMID:2570059

Recent mobility of plastid encoded group II introns and twintrons in five strains of the unicellular red alga Porphyridium

PubMed Central

Perrineau, Marie-Mathilde; Price, Dana C.; Mohr, Georg

2015-01-01

Group II introns are closely linked to eukaryote evolution because nuclear spliceosomal introns and the small RNAs associated with the spliceosome are thought to trace their ancient origins to these mobile elements. Therefore, elucidating how group II introns move, and how they lose mobility can potentially shed light on fundamental aspects of eukaryote biology. To this end, we studied five strains of the unicellular red alga Porphyridium purpureum that surprisingly contain 42 group II introns in their plastid genomes. We focused on a subset of these introns that encode mobility-conferring intron-encoded proteins (IEPs) and found them to be distributed among the strains in a lineage-specific manner. The reverse transcriptase and maturase domains were present in all lineages but the DNA endonuclease domain was deleted in vertically inherited introns, demonstrating a key step in the loss of mobility. P. purpureum plastid intron RNAs had a classic group IIB secondary structure despite variability in the DIII and DVI domains. We report for the first time the presence of twintrons (introns-within-introns, derived from the same mobile element) in Rhodophyta. The P. purpureum IEPs and their mobile introns provide a valuable model for the study of mobile retroelements in eukaryotes and offer promise for biotechnological applications. PMID:26157604

Analysis of a MULE-cyanide hydratase gene fusion in Verticillium dahliae

USDA-ARS?s Scientific Manuscript database

The genome of the phytopathogenic fungus Verticillium dahliae encodes numerous Class II “cut-and-paste” transposable elements, including those of a small group of MULE transposons. We have previously identified a fusion event between a MULE transposon sequence and sequence encoding a cyanide hydrata...

Multi-protocol header generation system

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

Roberts, David A.; Ignatowski, Michael; Jayasena, Nuwan

A communication device includes a data source that generates data for transmission over a bus, and a data encoder that receives and encodes outgoing data. An encoder system receives outgoing data from a data source and stores the outgoing data in a first queue. An encoder encodes outgoing data with a header type that is based upon a header type indication from a controller and stores the encoded data that may be a packet or a data word with at least one layered header in a second queue for transmission. The device is configured to receive at a payload extractor,more » a packet protocol change command from the controller and to remove the encoded data and to re-encode the data to create a re-encoded data packet and placing the re-encoded data packet in the second queue for transmission.« less

Identification of a Third Mn(II) Oxidase Enzyme in Pseudomonas putida GB-1

PubMed Central

Smesrud, Logan; Tebo, Bradley M.

2016-01-01

ABSTRACT The oxidation of soluble Mn(II) to insoluble Mn(IV) is a widespread bacterial activity found in a diverse array of microbes. In the Mn(II)-oxidizing bacterium Pseudomonas putida GB-1, two Mn(II) oxidase genes, named mnxG and mcoA, were previously identified; each encodes a multicopper oxidase (MCO)-type enzyme. Expression of these two genes is positively regulated by the response regulator MnxR. Preliminary investigation into putative additional regulatory pathways suggested that the flagellar regulators FleN and FleQ also regulate Mn(II) oxidase activity; however, it also revealed the presence of a third, previously uncharacterized Mn(II) oxidase activity in P. putida GB-1. A strain from which both of the Mn(II) oxidase genes and fleQ were deleted exhibited low levels of Mn(II) oxidase activity. The enzyme responsible was genetically and biochemically identified as an animal heme peroxidase (AHP) with domain and sequence similarity to the previously identified Mn(II) oxidase MopA. In the ΔfleQ strain, P. putida GB-1 MopA is overexpressed and secreted from the cell, where it actively oxidizes Mn. Thus, deletion of fleQ unmasked a third Mn(II) oxidase activity in this strain. These results provide an example of an Mn(II)-oxidizing bacterium utilizing both MCO and AHP enzymes. IMPORTANCE The identity of the Mn(II) oxidase enzyme in Pseudomonas putida GB-1 has been a long-standing question in the field of bacterial Mn(II) oxidation. In the current work, we demonstrate that P. putida GB-1 employs both the multicopper oxidase- and animal heme peroxidase-mediated pathways for the oxidation of Mn(II), rendering this model organism relevant to the study of both types of Mn(II) oxidase enzymes. The presence of three oxidase enzymes in P. putida GB-1 deepens the mystery of why microorganisms oxidize Mn(II) while providing the field with the tools necessary to address this question. The initial identification of MopA as a Mn(II) oxidase in this strain required the deletion of FleQ, a regulator involved in both flagellum synthesis and biofilm synthesis in Pseudomonas aeruginosa. Therefore, these results are also an important step toward understanding the regulation of Mn(II) oxidation. PMID:27084014

Synapsin-dependent development of glutamatergic synaptic vesicles and presynaptic plasticity in postnatal mouse brain.

PubMed

Bogen, I L; Jensen, V; Hvalby, O; Walaas, S I

2009-01-12

Inactivation of the genes encoding the neuronal, synaptic vesicle-associated proteins synapsin I and II leads to severe reductions in the number of synaptic vesicles in the CNS. We here define the postnatal developmental period during which the synapsin I and/or II proteins modulate synaptic vesicle number and function in excitatory glutamatergic synapses in mouse brain. In wild-type mice, brain levels of both synapsin I and synapsin IIb showed developmental increases during synaptogenesis from postnatal days 5-20, while synapsin IIa showed a protracted increase during postnatal days 20-30. The vesicular glutamate transporters (VGLUT) 1 and VGLUT2 showed synapsin-independent development during postnatal days 5-10, following which significant reductions were seen when synapsin-deficient brains were compared with wild-type brains following postnatal day 20. A similar, synapsin-dependent developmental profile of vesicular glutamate uptake occurred during the same age periods. Physiological analysis of the development of excitatory glutamatergic synapses, performed in the CA1 stratum radiatum of the hippocampus from the two genotypes, showed that both the synapsin-dependent part of the frequency facilitation and the synapsin-dependent delayed response enhancement were restricted to the period after postnatal day 10. Our data demonstrate that while both synaptic vesicle number and presynaptic short-term plasticity are essentially independent of synapsin I and II prior to postnatal day 10, maturation and function of excitatory synapses appear to be strongly dependent on synapsin I and II from postnatal day 20.

Various mutations compensate for a deleterious lacZα insert in the replication enhancer of M13 bacteriophage

PubMed Central

Zygiel, Emily M.; Noren, Karen A.; Adamkiewicz, Marta A.; Aprile, Richard J.; Bowditch, Heather K.; Carroll, Christine L.; Cerezo, Maria Abigail S.; Dagher, Adelle M.; Hebert, Courtney R.; Hebert, Lauren E.; Mahame, Gloria M.; Milne, Stephanie C.; Silvestri, Kelly M.; Sutherland, Sara E.; Sylvia, Alexandria M.; Taveira, Caitlyn N.; VanValkenburgh, David J.; Noren, Christopher J.

2017-01-01

M13 and other members of the Ff class of filamentous bacteriophages have been extensively employed in myriad applications. The Ph.D. series of phage-displayed peptide libraries were constructed from the M13-based vector M13KE. As a direct descendent of M13mp19, M13KE contains the lacZα insert in the intergenic region between genes IV and II, where it interrupts the replication enhancer of the (+) strand origin. Phage carrying this 816-nucleotide insert are viable, but propagate in E. coli at a reduced rate compared to wild-type M13 phage, presumably due to a replication defect caused by the insert. We have previously reported thirteen compensatory mutations in the 5’-untranslated region of gene II, which encodes the replication initiator protein gIIp. Here we report several additional mutations in M13KE that restore a wild-type propagation rate. Several clones from constrained-loop variable peptide libraries were found to have ejected the majority of lacZα gene in order to reconstruct the replication enhancer, albeit with a small scar. In addition, new point mutations in the gene II 5’-untranslated region or the gene IV coding sequence have been spontaneously observed or synthetically engineered. Through phage propagation assays, we demonstrate that all these genetic modifications compensate for the replication defect in M13KE and restore the wild-type propagation rate. We discuss the mechanisms by which the insertion and ejection of the lacZα gene, as well as the mutations in the regulatory region of gene II, influence the efficiency of replication initiation at the (+) strand origin. We also examine the presence and relevance of fast-propagating mutants in phage-displayed peptide libraries. PMID:28445507

Various mutations compensate for a deleterious lacZα insert in the replication enhancer of M13 bacteriophage.

PubMed

Zygiel, Emily M; Noren, Karen A; Adamkiewicz, Marta A; Aprile, Richard J; Bowditch, Heather K; Carroll, Christine L; Cerezo, Maria Abigail S; Dagher, Adelle M; Hebert, Courtney R; Hebert, Lauren E; Mahame, Gloria M; Milne, Stephanie C; Silvestri, Kelly M; Sutherland, Sara E; Sylvia, Alexandria M; Taveira, Caitlyn N; VanValkenburgh, David J; Noren, Christopher J; Hall, Marilena Fitzsimons

2017-01-01

M13 and other members of the Ff class of filamentous bacteriophages have been extensively employed in myriad applications. The Ph.D. series of phage-displayed peptide libraries were constructed from the M13-based vector M13KE. As a direct descendent of M13mp19, M13KE contains the lacZα insert in the intergenic region between genes IV and II, where it interrupts the replication enhancer of the (+) strand origin. Phage carrying this 816-nucleotide insert are viable, but propagate in E. coli at a reduced rate compared to wild-type M13 phage, presumably due to a replication defect caused by the insert. We have previously reported thirteen compensatory mutations in the 5'-untranslated region of gene II, which encodes the replication initiator protein gIIp. Here we report several additional mutations in M13KE that restore a wild-type propagation rate. Several clones from constrained-loop variable peptide libraries were found to have ejected the majority of lacZα gene in order to reconstruct the replication enhancer, albeit with a small scar. In addition, new point mutations in the gene II 5'-untranslated region or the gene IV coding sequence have been spontaneously observed or synthetically engineered. Through phage propagation assays, we demonstrate that all these genetic modifications compensate for the replication defect in M13KE and restore the wild-type propagation rate. We discuss the mechanisms by which the insertion and ejection of the lacZα gene, as well as the mutations in the regulatory region of gene II, influence the efficiency of replication initiation at the (+) strand origin. We also examine the presence and relevance of fast-propagating mutants in phage-displayed peptide libraries.

Genetic abnormalities in leukemia secondary to treatment in patients with Hodgkin's disease.

PubMed

Salas, Consuelo; Pérez-Vera, Patricia; Frías, Sara

2011-01-01

Hodgkin's disease has been treated mainly with two chemotherapy schedules, MOPP (nitrogen mustard, Oncovin, procarbazine and prednisone), which includes alkylating agents, and ABVD (adriamycin, bleomycin, vinblastine and dacarbazine), which includes topoisomerase II inhibitors, either with or without radiation therapy. Due to the types of agents used, patients with Hodgkin's disease often develop secondary leukemias. The alkylating agents included in the MOPP scheme were the first drugs associated with the development of therapy-related myelodysplastic syndrome (t-MDS) and acute myeloid leukemia (t-AML); both entities are the result of the clonal selection of cells with accumulated genomic lesions induced by antineoplastic therapy. In patients who developed t-MDS and t-AML, eight alternative routes with specific cytogenetic and molecular changes have been identified, and the routes are related to the type of therapy, alkylating agents or DNA topoisomerase II inhibitors. At the cytogenetic level, patients treated with alkylating agents show deletion 5q/monosomy 5 and deletion 7q/monosomy 7; in contrast, those who were treated with topoisomerase II inhibitors show 11q23 translocations involving the MLL gene. At the molecular level, there are two types of mutations: Class I, which alter the RAS-BRAF signal transduction pathways and increase cell proliferation; Class II, which disrupt genes that encode transcription factors and NPM1 that are involved in cell differentiation, and the inactivation of p53 tumor suppressor gene. Knowledge of the genetic alterations in these conditions is important for the classification, treatment and prognosis of patients as well as essential for increasing the knowledge of the biology of these diseases, which leads to identifying potential therapeutic targets.

Modified Dynamic Decode-and-Forward Relaying Protocol for Type II Relay in LTE-Advanced and Beyond

PubMed Central

Nam, Sung Sik; Alouini, Mohamed-Slim; Choi, Seyeong

2016-01-01

In this paper, we propose a modified dynamic decode-and-forward (MoDDF) relaying protocol to meet the critical requirements for user equipment (UE) relays in next-generation cellular systems (e.g., LTE-Advanced and beyond). The proposed MoDDF realizes the fast jump-in relaying and the sequential decoding with an application of random codeset to encoding and re-encoding process at the source and the multiple UE relays, respectively. A subframe-by-subframe decoding based on the accumulated (or buffered) messages is employed to achieve energy, information, or mixed combining. Finally, possible early termination of decoding at the end user can lead to the higher spectral efficiency and more energy saving by reducing the frequency of redundant subframe transmission and decoding. These attractive features eliminate the need of directly exchanging control messages between multiple UE relays and the end user, which is an important prerequisite for the practical UE relay deployment. PMID:27898712

Modified Dynamic Decode-and-Forward Relaying Protocol for Type II Relay in LTE-Advanced and Beyond.

PubMed

Nam, Sung Sik; Alouini, Mohamed-Slim; Choi, Seyeong

2016-01-01

In this paper, we propose a modified dynamic decode-and-forward (MoDDF) relaying protocol to meet the critical requirements for user equipment (UE) relays in next-generation cellular systems (e.g., LTE-Advanced and beyond). The proposed MoDDF realizes the fast jump-in relaying and the sequential decoding with an application of random codeset to encoding and re-encoding process at the source and the multiple UE relays, respectively. A subframe-by-subframe decoding based on the accumulated (or buffered) messages is employed to achieve energy, information, or mixed combining. Finally, possible early termination of decoding at the end user can lead to the higher spectral efficiency and more energy saving by reducing the frequency of redundant subframe transmission and decoding. These attractive features eliminate the need of directly exchanging control messages between multiple UE relays and the end user, which is an important prerequisite for the practical UE relay deployment.

Canonical and Non-Canonical Autophagy in HIV-1 Replication Cycle

PubMed Central

Leymarie, Olivier; Lepont, Leslie; Berlioz-Torrent, Clarisse

2017-01-01

Autophagy is a lysosomal-dependent degradative process essential for maintaining cellular homeostasis, and is a key player in innate and adaptive immune responses to intracellular pathogens such as human immunodeficiency virus type 1 (HIV-1). In HIV-1 target cells, autophagy mechanisms can (i) selectively direct viral proteins and viruses for degradation; (ii) participate in the processing and presentation of viral-derived antigens through major histocompatibility complexes; and (iii) contribute to interferon production in response to HIV-1 infection. As a consequence, HIV-1 has evolved different strategies to finely regulate the autophagy pathway to favor its replication and dissemination. HIV-1 notably encodes accessory genes encoding Tat, Nef and Vpu proteins, which are able to perturb and hijack canonical and non-canonical autophagy mechanisms. This review outlines the current knowledge on the complex interplay between autophagy and HIV-1 replication cycle, providing an overview of the autophagy-mediated molecular processes deployed both by infected cells to combat the virus and by HIV-1 to evade antiviral response. PMID:28946621

Pea chloroplast DNA encodes homologues of Escherichia coli ribosomal subunit S2 and the beta'-subunit of RNA polymerase.

PubMed Central

Cozens, A L; Walker, J E

1986-01-01

The nucleotide sequence has been determined of a segment of 4680 bases of the pea chloroplast genome. It adjoins a sequence described elsewhere that encodes subunits of the F0 membrane domain of the ATP-synthase complex. The sequence contains a potential gene encoding a protein which is strongly related to the S2 polypeptide of Escherichia coli ribosomes. It also encodes an incomplete protein which contains segments that are homologous to the beta'-subunit of E. coli RNA polymerase and to yeast RNA polymerases II and III. PMID:3530249

Variability in the impairment of recognition memory in patients with frontal lobe lesions.

PubMed

Bastin, Christine; Van der Linden, Martial; Lekeu, Françoise; Andrés, Pilar; Salmon, Eric

2006-10-01

Fourteen patients with frontal lobe lesions and 14 normal subjects were tested on a recognition memory task that required discriminating between target words, new words that are synonyms of the targets and unrelated distractors. A deficit was found in 12 of the patients. Moreover, three different patterns of recognition impairment were identified: (I) poor memory for targets, (II) normal hits but increased false recognitions for both types of distractors, (III) normal hit rates, but increased false recognitions for synonyms only. Differences in terms of location of the damage and behavioral characteristics between these subgroups were examined. An encoding deficit was proposed to explain the performance of patients in subgroup I. The behavioral patterns of the patients in subgroups II and III could be interpreted as deficient post-retrieval verification processes and an inability to recollect item-specific information, respectively.

Topological and organizational properties of the products of house-keeping and tissue-specific genes in protein-protein interaction networks.

PubMed

Lin, Wen-Hsien; Liu, Wei-Chung; Hwang, Ming-Jing

2009-03-11

Human cells of various tissue types differ greatly in morphology despite having the same set of genetic information. Some genes are expressed in all cell types to perform house-keeping functions, while some are selectively expressed to perform tissue-specific functions. In this study, we wished to elucidate how proteins encoded by human house-keeping genes and tissue-specific genes are organized in human protein-protein interaction networks. We constructed protein-protein interaction networks for different tissue types using two gene expression datasets and one protein-protein interaction database. We then calculated three network indices of topological importance, the degree, closeness, and betweenness centralities, to measure the network position of proteins encoded by house-keeping and tissue-specific genes, and quantified their local connectivity structure. Compared to a random selection of proteins, house-keeping gene-encoded proteins tended to have a greater number of directly interacting neighbors and occupy network positions in several shortest paths of interaction between protein pairs, whereas tissue-specific gene-encoded proteins did not. In addition, house-keeping gene-encoded proteins tended to connect with other house-keeping gene-encoded proteins in all tissue types, whereas tissue-specific gene-encoded proteins also tended to connect with other tissue-specific gene-encoded proteins, but only in approximately half of the tissue types examined. Our analysis showed that house-keeping gene-encoded proteins tend to occupy important network positions, while those encoded by tissue-specific genes do not. The biological implications of our findings were discussed and we proposed a hypothesis regarding how cells organize their protein tools in protein-protein interaction networks. Our results led us to speculate that house-keeping gene-encoded proteins might form a core in human protein-protein interaction networks, while clusters of tissue-specific gene-encoded proteins are attached to the core at more peripheral positions of the networks.

The cell wall pectic polymer rhamnogalacturonan-II is required for proper pollen tube elongation: implications of a putative sialyltransferase-like protein.

PubMed

Dumont, Marie; Lehner, Arnaud; Bouton, Sophie; Kiefer-Meyer, Marie Christine; Voxeur, Aline; Pelloux, Jérôme; Lerouge, Patrice; Mollet, Jean-Claude

2014-10-01

Rhamnogalacturonan-II (RG-II) is one of the pectin motifs found in the cell wall of all land plants. It contains sugars such as 2-keto-3-deoxy-d-lyxo-heptulosaric acid (Dha) and 2-keto-3-deoxy-d-manno-octulosonic acid (Kdo), and within the wall RG-II is mostly found as a dimer via a borate diester cross-link. To date, little is known regarding the biosynthesis of this motif. Here, after a brief review of our current knowledge on RG-II structure, biosynthesis and function in plants, this study explores the implications of the presence of a Golgi-localized sialyltransferase-like 2 (SIA2) protein that is possibly involved in the transfer of Dha or Kdo in the RG-II of Arabidopsis thaliana pollen tubes, a fast-growing cell type used as a model for the study of cell elongation. Two heterozygous mutant lines of arabidopsis (sia2-1+/- and qrt1 × sia2-2+/-) were investigated. sia2-2+/- was in a quartet1 background and the inserted T-DNA contained the reporter gene β-glucuronidase (GUS) under the pollen-specific promoter LAT52. Pollen germination and pollen tube phenotype and growth were analysed both in vitro and in vivo by microscopy. Self-pollination of heterozygous lines produced no homozygous plants in the progeny, which may suggest that the mutation could be lethal. Heterozygous mutants displayed a much lower germination rate overall and exhibited a substantial delay in germination (20 h of delay to reach 30 % of pollen grain germination compared with the wild type). In both lines, mutant pollen grains that were able to produce a tube had tubes that were either bursting, abnormal (swollen or dichotomous branching tip) or much shorter compared with wild-type pollen tubes. In vivo, mutant pollen tubes were restricted to the style, whereas the wild-type pollen tubes were detected at the base of the ovary. This study highlights that the mutation in arabidopsis SIA2 encoding a sialyltransferase-like protein that may transfer Dha or Kdo on the RG-II motif has a dramatic effect on the stability of the pollen tube cell wall. © The Author 2014. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

A second gene for type I signal peptidase in Bradyrhizobium japonicum, sipF, is located near genes involved in RNA processing and cell division.

PubMed

Bairl, A; Müller, P

1998-11-01

The TnphoA-induced Bradyrhizobium japonicum mutant 184 shows slow growth and aberrant colonization of soybean nodules. Using a DNA fragment adjacent to the transposon insertion site as a probe, a 3.4-kb BglII fragment of B. japonicum 110spc4 DNA was identified and cloned. Sequence analysis indicated that two truncated ORFs and three complete ORFs were encoded on this fragment. A database search revealed homologies to several other prokaryotic proteins: PdxJ (an enzyme involved in vitamin B6 biosynthesis), AcpS (acyl carrier protein synthase), Lep or Sip (prokaryotic type I signal peptidase), RNase III (an endoribonuclease which processes double-stranded rRNA precursors and mRNA) and Era (a GTP-binding protein required for cell division). The mutation in strain 184 was found to lie within the signal peptidase gene, which was designated sipF. Therefore, sipF is located in a region that encodes gene products involved in posttranscriptional and posttranslational processing processes. By complementation of the lep(ts) E. coli mutant strain IT41 it was demonstrated that sipF indeed encodes a functional signal peptidase, and genetic complementation of B. japonicum mutant 184 by a 2.8-kb SalI fragment indicated that sipF is expressed from a promoter located directly upstream of sipF. Using a non-polar kanamycin resistance cassette, a specific sipF mutant was constructed which exhibited defects in symbiosis similar to those of the original mutant 184.

Transgenic rice seed synthesizing diverse flavonoids at high levels: a new platform for flavonoid production with associated health benefits.

PubMed

Ogo, Yuko; Ozawa, Kenjiro; Ishimaru, Tsutomu; Murayama, Tsugiya; Takaiwa, Fumio

2013-08-01

Flavonoids possess diverse health-promoting benefits but are nearly absent from rice, because most of the genes encoding enzymes for flavonoid biosynthesis are not expressed in rice seeds. In the present study, a transgenic rice plant producing several classes of flavonoids in seeds was developed by introducing multiple genes encoding enzymes involved in flavonoid synthesis, from phenylalanine to the target flavonoids, into rice. Rice accumulating naringenin was developed by introducing phenylalanine ammonia lyase (PAL) and chalcone synthase (CHS) genes. Rice producing other classes of flavonoids, kaempferol, genistein, and apigenin, was developed by introducing, together with PAL and CHS, genes encoding flavonol synthase/flavanone-3-hydroxylase, isoflavone synthase, and flavone synthases, respectively. The endosperm-specific GluB-1 promoter or embryo- and aleurone-specific 18-kDa oleosin promoters were used to express these biosynthetic genes in seed. The target flavonoids of naringenin, kaempferol, genistein, and apigenin were highly accumulated in each transgenic rice, respectively. Furthermore, tricin was accumulated by introducing hydroxylase and methyltransferase, demonstrating that modification to flavonoid backbones can be also well manipulated in rice seeds. The flavonoids accumulated as both aglycones and several types of glycosides, and flavonoids in the endosperm were deposited into PB-II-type protein bodies. Therefore, these rice seeds provide an ideal platform for the production of particular flavonoids due to efficient glycosylation, the presence of appropriate organelles for flavonoid accumulation, and the small effect of endogenous enzymes on the production of flavonoids by exogenous enzymes. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Functional substitution for TAF(II)250 by a retroposed homolog that is expressed in human spermatogenesis.

PubMed

Wang, P Jeremy; Page, David C

2002-09-15

TAF(II)250, the largest subunit of the general transcription factor TFIID, is expressed from the human X chromosome, at least in somatic cells. In male meiosis, however, the sex chromosomes are transcriptionally silenced, while the autosomes remain active. How then are protein-encoding genes transcribed during human male meiosis? Here we present a novel autosomal human gene, TAF1L, which is homologous to TAF(II)250 and is expressed specifically in the testis, apparently in germ cells. We hypothesize that during male meiosis, transcription of protein-encoding genes relies upon TAF1L as a functional substitute for TAF(II)250. Like TAF(II)250, the human TAF1L protein can bind directly to TATA-binding protein, an essential component of TFIID. Most importantly, transfection with human TAF1L rescued the temperature-sensitive lethality of a hamster cell line mutant in TAF(II)250. TAF1L lacks introns and evidently arose by retroposition of a processed TAF(II)250 mRNA during primate evolution. The observation that TAF1L can functionally replace TAF(II)250 provides experimental support for the hypothesis that during male meiosis, autosomes provide cellular functions usually supplied by the X chromosome in somatic cells.

The role of BST2/tetherin in infection with the feline retroviruses

PubMed Central

Dietrich, Isabelle; Hosie, Margaret J.; Willett, Brian J.

2014-01-01

The recently identified host restriction factor tetherin (BST-2, CD317) potently inhibits the release of nascent retrovirus particles from infected cells. Recently, we reported the identification and characterization of tetherin as a novel feline retroviral restriction factor. Based on homology to human tetherin we identified a putative tetherin gene in the genome of the domestic cat (Felis catus) which was found to be expressed in different feline cell lines both prior to and post treatment with either type I or type II interferon (IFN). The predicted structure of feline tetherin (feTHN) was that of a type II single-pass transmembrane protein encoding an N-terminal transmembrane anchor, central predicted coiled-coil bearing extracellular domain to promote dimerization, and a C-terminal GPI-anchor, consistent with conservation of structure between human and feline tetherin. FeTHN displayed potent inhibition of feline immunodeficiency virus (FIV) and human immunodeficiency virus type 1 (HIV-1) particle release in single-cycle replication assays. Notably, feTHN activity was resistant to antagonism by HIV-1 Vpu. However, stable ectopic expression of feTHN mRNA in different feline cell lines had no inhibitory effect on the growth of diverse primary or cell culture-adapted strains of FIV. Hence, whereas feline tetherin efficiently blocks viral particle release in single-cycle replication assays, it might not prevent dissemination of feline retroviruses in vivo. PMID:21715020

Prmt7 is dispensable in tissue culture models for adipogenic differentiation.

PubMed

Hu, Yu-Jie; Sif, Saïd; Imbalzano, Anthony N

2013-01-01

Protein arginine methylation is a common posttranslational modification that has been implicated in numerous biological processes including gene expression. The mammalian genome encodes nine protein arginine methyltransferases (Prmts) that catalyze monomethylation, asymmetric dimethylation, and symmetric dimethylation on arginine residues. Protein arginine methyltransferase 7 (Prmt7) is categorized as a type II and type III enzyme that produces symmetric dimethylated arginine and monomethylated arginine, respectively. However, the biological role of Prmt7 is not well characterized. We previously showed that Prmt5, a type II Prmt that associates with Brg1-based SWI/SNF chromatin remodeling complex, is required for adipocyte differentiation. Since Prmt7 also associates with Brg1-based SWI/SNF complex and modifies core histones, we hypothesized that Prmt7 might play a role in transcriptional regulation of adipogenesis. In the present study, we determined that the expression of Prmt7 did not change throughout adipogenic differentiation of C3H10T1/2 mesenchymal cells. Knockdown or over-expression of Prmt7 had no effect on lipid accumulation or adipogenic gene expression in differentiating C3H10T1/2 cells or in C/EBPα-reprogrammed NIH3T3 fibroblasts. Based on these results, we conclude that Prmt7, unlike Prmt5, is dispensable for adipogenic differentiation in tissue culture models.

Prmt7 is dispensable in tissue culture models for adipogenic differentiation

PubMed Central

Imbalzano, Anthony N.

2013-01-01

Protein arginine methylation is a common posttranslational modification that has been implicated in numerous biological processes including gene expression. The mammalian genome encodes nine protein arginine methyltransferases (Prmts) that catalyze monomethylation, asymmetric dimethylation, and symmetric dimethylation on arginine residues. Protein arginine methyltransferase 7 (Prmt7) is categorized as a type II and type III enzyme that produces symmetric dimethylated arginine and monomethylated arginine, respectively. However, the biological role of Prmt7 is not well characterized. We previously showed that Prmt5, a type II Prmt that associates with Brg1-based SWI/SNF chromatin remodeling complex, is required for adipocyte differentiation. Since Prmt7 also associates with Brg1-based SWI/SNF complex and modifies core histones, we hypothesized that Prmt7 might play a role in transcriptional regulation of adipogenesis. In the present study, we determined that the expression of Prmt7 did not change throughout adipogenic differentiation of C3H10T1/2 mesenchymal cells. Knockdown or over-expression of Prmt7 had no effect on lipid accumulation or adipogenic gene expression in differentiating C3H10T1/2 cells or in C/EBPα-reprogrammed NIH3T3 fibroblasts. Based on these results, we conclude that Prmt7, unlike Prmt5, is dispensable for adipogenic differentiation in tissue culture models. PMID:24715966

Cloning and overexpression of beta-N-acetylglucosaminidase encoding gene nagA from Aspergillus oryzae and enzyme-catalyzed synthesis of human milk oligosaccharide.

PubMed

Matsuo, Ichiro; Kim, Sunhwa; Yamamoto, Yuichi; Ajisaka, Katsumi; Maruyama, Jun-ich; Nakajima, Harushi; Kitamoto, Katsuhiko

2003-03-01

We isolated a beta-N-acetylglucosaminidase encoding gene from the filamentous fungus Aspergillus oryzae, and designated it nagA. The nagA gene encoded a polypeptide of 600 amino acids with significant similarity to glucosaminidases and hexosaminidases of various eukaryotes. A. oryzae strain carrying the nagA gene under the control of the improved glaA promoter produced large amounts of beta-N-acetylglucosaminidase in a wheat bran solid culture. The beta-N-acetylglucosaminidase was purified from crude extracts of the solid culture by column chromatographies on Q-Sepharose and Sephacryl S-200. This enzyme was used for synthesis of lacto-N-triose II, which is contained in human milk. By reverse hydrolysis reaction, lacto-N-triose II and its positional isomer were synthesized from lactose and D-N-acetylglucosamine in 0.21% and 0.15% yield, respectively.

Functional characterizations of malonyl-CoA:acyl carrier protein transacylase (MCAT) in Eimeria tenella.

PubMed

Sun, Mingfei; Zhu, Guan; Qin, Zonghua; Wu, Caiyan; Lv, Minna; Liao, Shenquan; Qi, Nanshan; Xie, Mingquan; Cai, Jianping

2012-07-01

Eimeria tenella, an apicomplexan parasite in chickens, possesses an apicoplast and its associated metabolic pathways including the Type II fatty acid synthesis (FAS II). Malonyl-CoA:acyl-carry protein transacylase (MCAT) encoded by the fabD gene is one of the essential enzymes in the FAS II system. In the present study, the entire E. tenella MCAT gene (EtfabD) was cloned and sequenced. Immunolabeling located this protein in the apicoplast organelle in coccidial sporozoites. Functional replacement of the fabD gene with amber mutation of E. coli temperature-sensitive LA2-89 strain by E. tenella EtMCAT demonstrated that EcFabD and EtMCAT perform the same biochemical function. The recombinant EtMCAT protein was expressed and its general biochemical features were also determined. An alkaloid natural product corytuberine (CAS: 517-56-6) could specifically inhibit the EtMCAT activity (IC(50)=16.47μM), but the inhibition of parasite growth in vitro by corytuberine was very weak (the predicted MIC(50)=0.65mM). Copyright © 2012 Elsevier B.V. All rights reserved.

Is junk DNA bunk? A critique of ENCODE.

PubMed

Doolittle, W Ford

2013-04-02

Do data from the Encyclopedia Of DNA Elements (ENCODE) project render the notion of junk DNA obsolete? Here, I review older arguments for junk grounded in the C-value paradox and propose a thought experiment to challenge ENCODE's ontology. Specifically, what would we expect for the number of functional elements (as ENCODE defines them) in genomes much larger than our own genome? If the number were to stay more or less constant, it would seem sensible to consider the rest of the DNA of larger genomes to be junk or, at least, assign it a different sort of role (structural rather than informational). If, however, the number of functional elements were to rise significantly with C-value then, (i) organisms with genomes larger than our genome are more complex phenotypically than we are, (ii) ENCODE's definition of functional element identifies many sites that would not be considered functional or phenotype-determining by standard uses in biology, or (iii) the same phenotypic functions are often determined in a more diffuse fashion in larger-genomed organisms. Good cases can be made for propositions ii and iii. A larger theoretical framework, embracing informational and structural roles for DNA, neutral as well as adaptive causes of complexity, and selection as a multilevel phenomenon, is needed.

High-quality permanent draft genome sequence of the Bradyrhizobium elkanii type strain USDA 76T, isolated from Glycine max (L.) Merr

DOE PAGES

Reeve, Wayne; van Berkum, Peter; Ardley, Julie; ...

2017-03-04

Bradyrhizobium elkanii USDA 76 T (INSCD = ARAG00000000), the type strain for Bradyrhizobium elkanii, is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from an effective nitrogen-fixing root nodule of Glycine max (L. Merr) grown in the USA. Because of its significance as a microsymbiont of this economically important legume, B. elkanii USDA 76 T was selected as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria sequencing project. Here the symbiotic abilities of B. elkanii USDA 76 T are described, together with its genome sequence information and annotation. The 9,484,767 bpmore » high-quality draft genome is arranged in 2 scaffolds of 25 contigs, containing 9060 protein-coding genes and 91 RNA-only encoding genes. The B. elkanii USDA 76 T genome contains a low GC content region with symbiotic nod and fix genes, indicating the presence of a symbiotic island integration. A comparison of five B. elkanii genomes that formed a clique revealed that 356 of the 9060 protein coding genes of USDA 76 T were unique, including 22 genes of an intact resident prophage. A conserved set of 7556 genes were also identified for this species, including genes encoding a general secretion pathway as well as type II, III, IV and VI secretion system proteins. The type III secretion system has previously been characterized as a host determinant for Rj and/or rj soybean cultivars. Here we show that the USDA 76 T genome contains genes encoding all the type III secretion system components, including a translocon complex protein NopX required for the introduction of effector proteins into host cells. While many bradyrhizobial strains are unable to nodulate the soybean cultivar Clark (rj1), USDA 76 T was able to elicit nodules on Clark (rj1), although in reduced numbers, when plants were grown in Leonard jars containing sand or vermiculite. In these conditions, we postulate that the presence of NopX allows USDA 76 T to introduce various effector molecules into this host to enable nodulation.« less

High-quality permanent draft genome sequence of the Bradyrhizobium elkanii type strain USDA 76T, isolated from Glycine max (L.) Merr

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

Reeve, Wayne; van Berkum, Peter; Ardley, Julie

Bradyrhizobium elkanii USDA 76 T (INSCD = ARAG00000000), the type strain for Bradyrhizobium elkanii, is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from an effective nitrogen-fixing root nodule of Glycine max (L. Merr) grown in the USA. Because of its significance as a microsymbiont of this economically important legume, B. elkanii USDA 76 T was selected as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria sequencing project. Here the symbiotic abilities of B. elkanii USDA 76 T are described, together with its genome sequence information and annotation. The 9,484,767 bpmore » high-quality draft genome is arranged in 2 scaffolds of 25 contigs, containing 9060 protein-coding genes and 91 RNA-only encoding genes. The B. elkanii USDA 76 T genome contains a low GC content region with symbiotic nod and fix genes, indicating the presence of a symbiotic island integration. A comparison of five B. elkanii genomes that formed a clique revealed that 356 of the 9060 protein coding genes of USDA 76 T were unique, including 22 genes of an intact resident prophage. A conserved set of 7556 genes were also identified for this species, including genes encoding a general secretion pathway as well as type II, III, IV and VI secretion system proteins. The type III secretion system has previously been characterized as a host determinant for Rj and/or rj soybean cultivars. Here we show that the USDA 76 T genome contains genes encoding all the type III secretion system components, including a translocon complex protein NopX required for the introduction of effector proteins into host cells. While many bradyrhizobial strains are unable to nodulate the soybean cultivar Clark (rj1), USDA 76 T was able to elicit nodules on Clark (rj1), although in reduced numbers, when plants were grown in Leonard jars containing sand or vermiculite. In these conditions, we postulate that the presence of NopX allows USDA 76 T to introduce various effector molecules into this host to enable nodulation.« less

Wide spread of OXA-23-producing carbapenem-resistant Acinetobacter baumannii belonging to clonal complex II in different hospitals in Lebanon.

PubMed

Al Atrouni, Ahmad; Hamze, Monzer; Jisr, Tamima; Lemarié, Carole; Eveillard, Matthieu; Joly-Guillou, Marie-Laure; Kempf, Marie

2016-11-01

To investigate the molecular epidemiology of Acinetobacter baumannii strains isolated from different hospitals in Lebanon. A total of 119 non-duplicate Acinetobacter strains were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and partial rpoB gene sequencing. Antibiotic susceptibility testing was performed by disc diffusion method and all identified carbapenem-resistant isolates were investigated by PCR assays for the presence of the carbapenemase-encoding genes. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were used for molecular typing. Of the 119 A. baumannii isolates, 76.5% were resistant to carbapenems. The most common carbapenemase was the OXA-23-type, found in 82 isolates. The study of population structure using MLST revealed the presence of 30 sequence types (STs) including 18 new ones, with ST2 being the most commonly detected, accounting for 61% of the isolates typed. PFGE performed on all strains of ST2 identified a major cluster of 53 isolates, in addition to three other minor clusters and ten unique profiles. This study highlights the wide dissemination of highly related OXA-23-producing carbapenem-resistant A. baumannii belonging to the international clone II in Lebanon. Thus, appropriate infection control measures are recommended in order to control the geographical spread of this clone in this country. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Design Strategy of Multi-electron Transfer Catalysts Based on a Bioinformatic Analysis of Oxygen Evolution and Reduction Enzymes.

PubMed

Ooka, Hideshi; Hashimoto, Kazuhito; Nakamura, Ryuhei

2018-05-14

Understanding the design strategy of photosynthetic and respiratory enzymes is important to develop efficient artificial catalysts for oxygen evolution and reduction reactions. Here, based on a bioinformatic analysis of cyanobacterial oxygen evolution and reduction enzymes (photosystem II: PS II and cytochrome c oxidase: COX, respectively), the gene encoding the catalytic D1 subunit of PS II was found to be expressed individually across 38 phylogenetically diverse strains, which is in contrast to the operon structure of the genes encoding major COX subunits. Selective synthesis of the D1 subunit minimizes the repair cost of PS II, which allows compensation for its instability by lowering the turnover number required to generate a net positive energy yield. The different bioenergetics observed between PS II and COX suggest that in addition to the catalytic activity rationalized by the Sabatier principle, stability factors have also provided a major influence on the design strategy of biological multi-electron transfer enzymes. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ubiquitin over-expression phenotypes and ubiquitin gene molecular misreading during aging in Drosophila melanogaster

PubMed Central

Hoe, Nicholas; Huang, Chung M.; Landis, Gary; Verhage, Marian; Ford, Daniel; Yang, Junsheng; van Leeuwen, Fred W.; Tower, John

2011-01-01

Molecular Misreading (MM) is the inaccurate conversion of genomic information into aberrant proteins. For example, when RNA polymerase II transcribes a GAGAG motif it synthesizes at low frequency RNA with a two-base deletion. If the deletion occurs in a coding region, translation will result in production of misframed proteins. During mammalian aging, misframed versions of human amyloid precursor protein (hApp) and ubiquitin (hUbb) accumulate in the aggregates characteristic of neurodegenerative diseases, suggesting dysfunctional degradation or clearance. Here cDNA clones encoding wild-type hUbb and the frame-shifted version hUbb+1 were expressed in transgenic Drosophila using the doxycycline-regulated system. Misframed proteins were abundantly produced, both from the transgenes and from endogenous Drosophila ubiquitin-encoding genes, and their abundance increased during aging in whole-fly extracts. Over-expression of wild-type hUbb, but not hUbb+1, was toxic during fly development. In contrast, when over-expressed specifically in adult flies, hUbb+1 caused small decreases in life span, whereas hUbb was associated with small increases, preferentially in males. The data suggest that MM occurs in Drosophila and that the resultant misframed proteins accumulate with age. MM of the ubiquitin gene can produce alternative ubiquitin gene products with different and sometimes opposing phenotypic effects. PMID:21415465

Expression Analysis of an R3-Type MYB Transcription Factor CPC-LIKE MYB4 (TRICHOMELESS2) and CPL4-Related Transcripts in Arabidopsis

PubMed Central

Tominaga-Wada, Rumi; Nukumizu, Yuka

2012-01-01

The CAPRICE (CPC)-like MYB gene family encodes R3-type MYB transcription factors in Arabidopsis. There are six additional CPC-like MYB sequences in the Arabidopsis genome, including TRYPTICHON (TRY), ENHANCER OF TRY AND CPC1 and 2 (ETC1 and ETC2), ENHANCER OF TRY AND CPC3/CPC-LIKE MYB3 (ETC3/CPL3), and TRICHOMELESS1 and 2 (TCL1 and TCL2). We independently identified CPC-LIKE MYB4 (CPL4), which was found to be identical to TCL2. RT-PCR analysis showed that CPL4 is strongly expressed in shoots, including true leaves, but not in roots. Promoter-GUS analyses indicated that CPL4 is specifically expressed in leaf blades. Although CPC expression was repressed in 35S::ETC1, 35S::ETC2 and 35S::CPL3 backgrounds, CPL4 expression was not affected by ETC1, ETC2 or CPL3 over-expression. Notably, several chimeric transcripts may result from inter-genic alternative splicing of CPL4 and ETC2, two tandemly repeated genes on chromosome II. At least two chimeric transcripts named CPL4-α and CPL4-β are expected to encode complete CPC-like MYB proteins. PMID:22489163

Antimicrobial biosynthetic potential and genetic diversity of endophytic actinomycetes associated with medicinal plants.

PubMed

Gohain, Anwesha; Gogoi, Animesh; Debnath, Rajal; Yadav, Archana; Singh, Bhim P; Gupta, Vijai K; Sharma, Rajeev; Saikia, Ratul

2015-10-01

Endophytic actinomycetes are one of the primary groups that share symbiotic relationships with medicinal plants and are key reservoir of biologically active compounds. In this study, six selective medicinal plants were targeted for the first time for endophytic actinomycetes isolation from Gibbon Wild Life Sanctuary, Assam, India, during winter and summer and 76 isolates were obtained. The isolates were found to be prevalent in roots followed by stem and leaves. 16S rRNA gene sequence analysis revealed 16 genera, including rare genera, Verrucosispora, Isoptericola and Kytococcus, which have never been previously reported as endophytic. The genus Streptomyces (66%) was dominant in both seasons. Shannon's diversity index showed that Azadirachta indica (1.49), Rauwolfia serpentina (1.43) and Emblica officinalis (1.24) were relatively good habitat for endophytic actinomycetes. Antimicrobial strains showed prevalence of polyketide synthase (PKS) type-II (85%) followed by PKS type-I (14%) encoded in the genomes. Expression studies showed 12-fold upregulation of PKSII gene in seventh day of incubation for Streptomyces antibioticus (EAAG90). Our results emphasize that the actinomycetes assemblages within plant tissue exhibited biosynthetic systems encoding for important biologically active compounds. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Osteogenesis imperfecta: recent findings shed new light on this once well-understood condition.

PubMed

Basel, Donald; Steiner, Robert D

2009-06-01

Osteogenesis imperfecta is a systemic heritable disorder of connective tissue whose cardinal manifestation is bone fragility. In approximately 90% of individuals with osteogenesis imperfecta, mutations in either of the genes encoding the pro-alpha1 or pro-alpha2 chains of type I collagen (COL1A1 or COL1A2) can be identified. Of those without collagen mutations, a number of them will have mutations involving the enzyme complex responsible for posttranslational hydroxylation of the position 3 proline residue of COL1A1. Two of the genes encoding proteins involved in that enzyme complex, LEPRE1 and cartilage-associated protein, when mutated have been shown to cause autosomal recessive osteogenesis imperfecta, which has a moderate to severe clinical phenotype, often indistinguishable from osteogenesis imperfecta types II or III. Mutations in COL1A1 or COL1A2 which result in an abnormal protein still capable of forming a triple helix cause a more severe phenotype than mutations that lead to decreased collagen production as a result of the dominant negative effect mediated by continuous protein turnover. The current standard of care includes a multidisciplinary approach with surgical intervention when necessary, proactive physiotherapy, and consideration for the use of bisphosphonates all in attempts to improve quality of life.

Incidence of antimicrobial-resistance genes and integrons in antibiotic-resistant bacteria isolated from eels and aquaculture ponds.

PubMed

Lin, Mao; Wu, Xiaomei; Yan, Qingpi; Ma, Ying; Huang, Lixing; Qin, Yingxue; Xu, Xiaojin

2016-07-07

The overuse of antimicrobials in aquaculture has promoted the selection of antimicrobial-resistant bacteria. Here we investigated the abundance of antimicrobial-resistance genes and integrons in 108 strains of antibiotic-resistant bacteria isolated from eels and aquaculture ponds in China. Conventional PCR was implemented to examine common antibiotic-resistance genes, integrons, and their gene cassette arrays. The results showed that the antibiotic-resistance genes blaTEM, tetC, sulI, aadA, floR, and qnrB were detected at high percentages, as were a number of other resistance genes. Class I integrons were present in 79.63% of the strains, and 10 out of 108 isolates carried class II integrons. Class III integrons were not detected. Three strains carried both class I and class II integrons, and 73.26% of the class I integron-positive isolates contained the qacEΔ1/sul1 gene. Fourteen types of integron cassette arrays were found among class I integron-positive isolates. A new array, dfrB4-catB3-blaOXA-10-aadA1, was discovered in this study. The gene cassette array dfrA12-orfF-aadA2 was the most widely distributed. In summary, 23 different gene cassettes encoding resistance to 8 classes of antibiotics were identified in the class I integrons, and the main cassettes contained genes encoding resistance to aminoglycosides (aad) and trimethoprim (dfr). All class II integron-positive strains had only a single gene cassette array, viz. dfrA1-catB2-sat2-aadA1. High levels of antimicrobial-resistance genes and integrons in eels and auqauculture ponds suggest that the overuse of antimicrobials should be strictly controlled and that the levels of bacterial antimicrobial-resistance genes in aquaculture should be monitored.

CD70 encoded by modified vaccinia virus Ankara enhances CD8 T-cell-dependent protective immunity in MHC class II-deficient mice.

PubMed

Bathke, Barbara; Pätzold, Juliane; Kassub, Ronny; Giessel, Raphael; Lämmermann, Kerstin; Hinterberger, Maria; Brinkmann, Kay; Chaplin, Paul; Suter, Mark; Hochrein, Hubertus; Lauterbach, Henning

2017-12-27

The immunological outcome of infections and vaccinations is largely determined during the initial first days in which antigen-presenting cells instruct T cells to expand and differentiate into effector and memory cells. Besides the essential stimulation of the T-cell receptor complex a plethora of co-stimulatory signals not only ensures a proper T-cell activation but also instils phenotypic and functional characteristics in the T cells appropriate to fight off the invading pathogen. The tumour necrosis factor receptor/ligand pair CD27/CD70 gained a lot of attention because of its key role in regulating T-cell activation, survival, differentiation and maintenance, especially in the course of viral infections and cancer. We sought to investigate the role of CD70 co-stimulation for immune responses induced by the vaccine vector modified vaccinia virus Ankara-Bavarian Nordic ® (MVA-BN ® ). Short-term blockade of CD70 diminished systemic CD8 T-cell effector and memory responses in mice. The dependence on CD70 became even more apparent in the lungs of MHC class II-deficient mice. Importantly, genetically encoded CD70 in MVA-BN ® not only increased CD8 T-cell responses in wild-type mice but also substituted for CD4 T-cell help. MHC class II-deficient mice that were immunized with recombinant MVA-CD70 were fully protected against a lethal virus infection, whereas MVA-BN ® -immunized mice failed to control the virus. These data are in line with CD70 playing an important role for vaccine-induced CD8 T-cell responses and prove the potency of integrating co-stimulatory molecules into the MVA-BN ® backbone. © 2017 The Authors. Immunology Published by John Wiley & Sons Ltd.

Functional Expression of a Bacterial Heavy Metal Transporter in Arabidopsis Enhances Resistance to and Decreases Uptake of Heavy Metals1[w

PubMed Central

Lee, Joohyun; Bae, Hyunju; Jeong, Jeeyon; Lee, Jae-Yun; Yang, Young-Yell; Hwang, Inhwan; Martinoia, Enrico; Lee, Youngsook

2003-01-01

Large parts of agricultural soil are contaminated with lead (Pb) and cadmium (Cd). Although most environments are not heavily contaminated, the low levels observed nonetheless pose a high risk of heavy metal accumulation in the food chain. Therefore, approaches to develop plants with reduced heavy metal uptake are important. Recently, many transgenic plants with increased heavy metal resistance and uptake of heavy metals were developed for the purpose of phytoremediation. However, to reduce heavy metal in the food chain, plants that transfer less heavy metals to the shoot are required. We tested whether an Escherichia coli gene, ZntA, which encodes a Pb(II)/Cd(II)/Zn(II) pump, could be useful for developing plants with reduced heavy metal content. Yeast cells transformed with this gene had improved resistance to Pb(II) and Cd(II). In Arabidopsis plants transformed with ZntA, ZntA was localized at the plasma membrane and improved the resistance of the plants to Pb(II) and Cd(II). The shoots of the transgenic plants had decreased Pb and Cd content. Moreover, the transgenic protoplasts showed lower accumulation of Cd and faster release of preloaded Cd than wild-type protoplasts. These results show that a bacterial transporter gene, ZntA, can be functionally expressed in plant cells, and that that it may be useful for the development of crop plants that are safe from heavy metal contamination. PMID:14512517

Optimising operational amplifiers by evolutionary algorithms and gm/Id method

NASA Astrophysics Data System (ADS)

Tlelo-Cuautle, E.; Sanabria-Borbon, A. C.

2016-10-01

The evolutionary algorithm called non-dominated sorting genetic algorithm (NSGA-II) is applied herein in the optimisation of operational transconductance amplifiers. NSGA-II is accelerated by applying the gm/Id method to estimate reduced search spaces associated to widths (W) and lengths (L) of the metal-oxide-semiconductor field-effect-transistor (MOSFETs), and to guarantee their appropriate bias levels conditions. In addition, we introduce an integer encoding for the W/L sizes of the MOSFETs to avoid a post-processing step for rounding-off their values to be multiples of the integrated circuit fabrication technology. Finally, from the feasible solutions generated by NSGA-II, we introduce a second optimisation stage to guarantee that the final feasible W/L sizes solutions support process, voltage and temperature (PVT) variations. The optimisation results lead us to conclude that the gm/Id method and integer encoding are quite useful to accelerate the convergence of the evolutionary algorithm NSGA-II, while the second optimisation stage guarantees robustness of the feasible solutions to PVT variations.

Dominant-Negative Mutations in α-II Spectrin Cause West Syndrome with Severe Cerebral Hypomyelination, Spastic Quadriplegia, and Developmental Delay

PubMed Central

Saitsu, Hirotomo; Tohyama, Jun; Kumada, Tatsuro; Egawa, Kiyoshi; Hamada, Keisuke; Okada, Ippei; Mizuguchi, Takeshi; Osaka, Hitoshi; Miyata, Rie; Furukawa, Tomonori; Haginoya, Kazuhiro; Hoshino, Hideki; Goto, Tomohide; Hachiya, Yasuo; Yamagata, Takanori; Saitoh, Shinji; Nagai, Toshiro; Nishiyama, Kiyomi; Nishimura, Akira; Miyake, Noriko; Komada, Masayuki; Hayashi, Kenji; Hirai, Syu-ichi; Ogata, Kazuhiro; Kato, Mitsuhiro; Fukuda, Atsuo; Matsumoto, Naomichi

2010-01-01

A de novo 9q33.3-q34.11 microdeletion involving STXBP1 has been found in one of four individuals (group A) with early-onset West syndrome, severe hypomyelination, poor visual attention, and developmental delay. Although haploinsufficiency of STXBP1 was involved in early infantile epileptic encephalopathy in a previous different cohort study (group B), no mutations of STXBP1 were found in two of the remaining three subjects of group A (one was unavailable). We assumed that another gene within the deletion might contribute to the phenotype of group A. SPTAN1 encoding α-II spectrin, which is essential for proper myelination in zebrafish, turned out to be deleted. In two subjects, an in-frame 3 bp deletion and a 6 bp duplication in SPTAN1 were found at the initial nucleation site of the α/β spectrin heterodimer. SPTAN1 was further screened in six unrelated individuals with WS and hypomyelination, but no mutations were found. Recombinant mutant (mut) and wild-type (WT) α-II spectrin could assemble heterodimers with β-II spectrin, but α-II (mut)/β-II spectrin heterodimers were thermolabile compared with the α-II (WT)/β-II heterodimers. Transient expression in mouse cortical neurons revealed aggregation of α-II (mut)/β-II and α-II (mut)/β-III spectrin heterodimers, which was also observed in lymphoblastoid cells from two subjects with in-frame mutations. Clustering of ankyrinG and voltage-gated sodium channels at axon initial segment (AIS) was disturbed in relation to the aggregates, together with an elevated action potential threshold. These findings suggest that pathological aggregation of α/β spectrin heterodimers and abnormal AIS integrity resulting from SPTAN1 mutations were involved in pathogenesis of infantile epilepsy. PMID:20493457

Tetrahymena thermophila acidic ribosomal protein L37 contains an archaebacterial type of C-terminus.

PubMed

Hansen, T S; Andreasen, P H; Dreisig, H; Højrup, P; Nielsen, H; Engberg, J; Kristiansen, K

1991-09-15

We have cloned and characterized a Tetrahymena thermophila macronuclear gene (L37) encoding the acidic ribosomal protein (A-protein) L37. The gene contains a single intron located in the 3'-part of the coding region. Two major and three minor transcription start points (tsp) were mapped 39 to 63 nucleotides upstream from the translational start codon. The uppermost tsp mapped to the first T in a putative T. thermophila RNA polymerase II initiator element, TATAA. The coding region of L37 predicts a protein of 109 amino acid (aa) residues. A substantial part of the deduced aa sequence was verified by protein sequencing. The T. thermophila L37 clearly belongs to the P1-type family of eukaryotic A-proteins, but the C-terminal region has the hallmarks of archaebacterial A-proteins.

Effect of ginseng extract on the TGF-β1 signaling pathway in CCl4-induced liver fibrosis in rats.

PubMed

Hafez, Mohamed M; Hamed, Sherifa S; El-Khadragy, Manal F; Hassan, Zeinab K; Al Rejaie, Salim S; Sayed-Ahmed, Mohamed M; Al-Harbi, Naif O; Al-Hosaini, Khalid A; Al-Harbi, Mohamed M; Alhoshani, Ali R; Al-Shabanah, Othman A; Alsharari, Shakir Dekhal

2017-01-13

Liver diseases are major global health problems. Ginseng extract has antioxidant, immune-modulatory and anti-inflammatory activities. This study investigated the effect of ginseng extract on carbon tetrachloride (CCl4)-induced liver fibrosis in rats. Male Wistar rats were divided into four groups: control group, ginseng group, CCl 4 group and CCl 4  + ginseng group. Liver injury was induced by the intraperitoneal (I.P) injection of 3 ml/kg CCl 4 (30% in olive oil) weekly for 8 weeks. The control group was I.P injected with olive oil. The expression of genes encoding transforming growth factor beta (TGF-β), type I TGF-β receptor (TβR-1), type II TGF-β receptor (TβR-II), mothers against decapentaplegic homolog 2 (Smad2), Smad3, Smad4, matrix metalloproteinase 2 (MMP2), MMP9, tissue inhibitor matrix metalloproteinase-1 (TIMP-1), Collagen 1a2 (Col1a2), Collagen 3a1 (Col3a1), interleukin-8 (IL-8) and interleukin -10 (IL-10) were measured by real-time PCR. Treatment with ginseng extract decreased hepatic fat deposition and lowered hepatic reticular fiber accumulation compared with the CCl 4 group. The CCl 4 group showed a significant increase in hepatotoxicity biomarkers and up-regulation of the expression of genes encoding TGF-β, TβR-I, TβR-II, MMP2, MMP9, Smad-2,-3, -4, and IL-8 compared with the control group. However, CCl 4 administration resulted in the significant down-regulation of IL-10 mRNA expression compared with the control group. Interestingly, ginseng extract supplementation completely reversed the biochemical markers of hepatotoxicity and the gene expression alterations induced by CCl 4 . ginseng extract had an anti-fibrosis effect via the regulation of the TGF-β1/Smad signaling pathway in the CCl 4 -induced liver fibrosis model. The major target was the inhibition of the expression of TGF-β1, Smad2, and Smad3.

A hitchhiker's guide to the MADS world of plants.

PubMed

Gramzow, Lydia; Theissen, Guenter

2010-01-01

Plant life critically depends on the function of MADS-box genes encoding MADS-domain transcription factors, which are present to a limited extent in nearly all major eukaryotic groups, but constitute a large gene family in land plants. There are two types of MADS-box genes, termed type I and type II, and in plants these groups are distinguished by exon-intron and domain structure, rates of evolution, developmental function and degree of functional redundancy. The type I genes are further subdivided into three groups - M alpha, M beta and M gamma - while the type II genes are subdivided into the MIKCC and MIKC* groups. The functional diversification of MIKCC genes is closely linked to the origin of developmental and morphological novelties in the sporophytic (usually diploid) generation of seed plants, most spectacularly the floral organs and fruits of angiosperms. Functional studies suggest different specializations for the different classes of genes; whereas type I genes may preferentially contribute to female gametophyte, embryo and seed development and MIKC*-group genes to male gametophyte development, the MIKCC-group genes became essential for diverse aspects of sporophyte development. Beyond the usual transcriptional regulation, including feedback and feed-forward loops, various specialized mechanisms have evolved to control the expression of MADS-box genes, such as epigenetic control and regulation by small RNAs. In future, more data from genome projects and reverse genetic studies will allow us to understand the birth, functional diversification and death of members of this dynamic and important family of transcription factors in much more detail.

Regulation of voltage-gated Ca(2+) currents by Ca(2+)/calmodulin-dependent protein kinase II in resting sensory neurons.

PubMed

Kostic, Sandra; Pan, Bin; Guo, Yuan; Yu, Hongwei; Sapunar, Damir; Kwok, Wai-Meng; Hudmon, Andy; Wu, Hsiang-En; Hogan, Quinn H

2014-09-01

Calcium/calmodulin-dependent protein kinase II (CaMKII) is recognized as a key element in encoding depolarization activity of excitable cells into facilitated voltage-gated Ca(2+) channel (VGCC) function. Less is known about the participation of CaMKII in regulating VGCCs in resting cells. We examined constitutive CaMKII control of Ca(2+) currents in peripheral sensory neurons acutely isolated from dorsal root ganglia (DRGs) of adult rats. The small molecule CaMKII inhibitor KN-93 (1.0μM) reduced depolarization-induced ICa by 16-30% in excess of the effects produced by the inactive homolog KN-92. The specificity of CaMKII inhibition on VGCC function was shown by the efficacy of the selective CaMKII blocking peptide autocamtide-2-related inhibitory peptide in a membrane-permeable myristoylated form, which also reduced VGCC current in resting neurons. Loss of VGCC currents is primarily due to reduced N-type current, as application of mAIP selectively reduced N-type current by approximately 30%, and prior N-type current inhibition eliminated the effect of mAIP on VGCCs, while prior block of L-type channels did not reduce the effect of mAIP on total ICa. T-type currents were not affected by mAIP in resting DRG neurons. Transduction of sensory neurons in vivo by DRG injection of an adeno-associated virus expressing AIP also resulted in a loss of N-type currents. Together, these findings reveal a novel molecular adaptation whereby sensory neurons retain CaMKII support of VGCCs despite remaining quiescent. Published by Elsevier Inc.

Roles of type II thioesterases and their application for secondary metabolite yield improvement.

PubMed

Kotowska, Magdalena; Pawlik, Krzysztof

2014-09-01

A large number of antibiotics and other industrially important microbial secondary metabolites are synthesized by polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). These multienzymatic complexes provide an enormous flexibility in formation of diverse chemical structures from simple substrates, such as carboxylic acids and amino acids. Modular PKSs and NRPSs, often referred to as megasynthases, have brought about a special interest due to the colinearity between enzymatic domains in the proteins working as an "assembly line" and the chain elongation and modification steps. Extensive efforts toward modified compound biosynthesis by changing organization of PKS and NRPS domains in a combinatorial manner laid good grounds for rational design of new structures and their controllable biosynthesis as proposed by the synthetic biology approach. Despite undeniable progress made in this field, the yield of such "unnatural" natural products is often not satisfactory. Here, we focus on type II thioesterases (TEIIs)--discrete hydrolytic enzymes often encoded within PKS and NRPS gene clusters which can be used to enhance product yield. We review diverse roles of TEIIs (removal of aberrant residues blocking the megasynthase, participation in substrate selection, intermediate, and product release) and discuss their application in new biosynthetic systems utilizing PKS and NRPS parts.

Cloning-independent markerless gene editing in Streptococcus sanguinis: novel insights in type IV pilus biology.

PubMed

Gurung, Ishwori; Berry, Jamie-Lee; Hall, Alexander M J; Pelicic, Vladimir

2017-04-07

Streptococcus sanguinis, a naturally competent opportunistic human pathogen, is a Gram-positive workhorse for genomics. It has recently emerged as a model for the study of type IV pili (Tfp)-exceptionally widespread and important prokaryotic filaments. To enhance genetic manipulation of Streptococcus sanguinis, we have developed a cloning-independent methodology, which uses a counterselectable marker and allows sophisticated markerless gene editing in situ. We illustrate the utility of this methodology by answering several questions regarding Tfp biology by (i) deleting single or mutiple genes, (ii) altering specific bases in genes of interest, and (iii) engineering genes to encode proteins with appended affinity tags. We show that (i) the last six genes in the pil locus harbouring all the genes dedicated to Tfp biology play no role in piliation or Tfp-mediated motility, (ii) two highly conserved Asp residues are crucial for enzymatic activity of the prepilin peptidase PilD and (iii) that pilin subunits with a C-terminally appended hexa-histidine (6His) tag are still assembled into functional Tfp. The methodology for genetic manipulation we describe here should be broadly applicable. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Stimulation of the cell cycle and maize transformation by disruption of the plant retinoblastoma pathway

PubMed Central

Gordon-Kamm, William; Dilkes, Brian P.; Lowe, Keith; Hoerster, George; Sun, Xifan; Ross, Margit; Church, Laura; Bunde, Chris; Farrell, Jeff; Hill, Patrea; Maddock, Sheila; Snyder, Jane; Sykes, Louisa; Li, Zhongsen; Woo, Young-min; Bidney, Dennis; Larkins, Brian A.

2002-01-01

The genome of the Mastreviruses encodes a replication-associated protein (RepA) that interacts with members of the plant retinoblastoma-related protein family, which are putative cell cycle regulators. Expression of ZmRb1, a maize retinoblastoma-related gene, and RepA inhibited and stimulated, respectively, cell division in tobacco cell cultures. The effect of RepA was mitigated by over-expression of ZmRb1. RepA increased transformation frequency and callus growth rate of high type II maize germplasm. RepA-containing transgenic maize calli remained embryogenic, were readily regenerable, and produced fertile plants that transmitted transgene expression in a Mendelian fashion. In high type II, transformation frequency increased with the strength of the promoter driving RepA expression. When a construct in which RepA was expressed behind its native LIR promoter was used, primary transformation frequencies did not improve for two elite Pioneer maize inbreds. However, when LIR:RepA-containing transgenic embryos were used in subsequent rounds of transformation, frequencies were higher in the RepA+ embryos. These data demonstrate that RepA can stimulate cell division and callus growth in culture, and improve maize transformation. PMID:12185243

Cloning-independent markerless gene editing in Streptococcus sanguinis: novel insights in type IV pilus biology

PubMed Central

Gurung, Ishwori; Berry, Jamie-Lee; Hall, Alexander M. J.

2017-01-01

Abstract Streptococcus sanguinis, a naturally competent opportunistic human pathogen, is a Gram-positive workhorse for genomics. It has recently emerged as a model for the study of type IV pili (Tfp)—exceptionally widespread and important prokaryotic filaments. To enhance genetic manipulation of Streptococcus sanguinis, we have developed a cloning-independent methodology, which uses a counterselectable marker and allows sophisticated markerless gene editing in situ. We illustrate the utility of this methodology by answering several questions regarding Tfp biology by (i) deleting single or mutiple genes, (ii) altering specific bases in genes of interest, and (iii) engineering genes to encode proteins with appended affinity tags. We show that (i) the last six genes in the pil locus harbouring all the genes dedicated to Tfp biology play no role in piliation or Tfp-mediated motility, (ii) two highly conserved Asp residues are crucial for enzymatic activity of the prepilin peptidase PilD and (iii) that pilin subunits with a C-terminally appended hexa-histidine (6His) tag are still assembled into functional Tfp. The methodology for genetic manipulation we describe here should be broadly applicable. PMID:27903891

Synthetically derived bat influenza A-like viruses reveal a cell type- but not species-specific tropism.

PubMed

Moreira, Étori Aguiar; Locher, Samira; Kolesnikova, Larissa; Bolte, Hardin; Aydillo, Teresa; García-Sastre, Adolfo; Schwemmle, Martin; Zimmer, Gert

2016-10-24

Two novel influenza A-like viral genome sequences have recently been identified in Central and South American fruit bats and provisionally designated "HL17NL10" and "HL18NL11." All efforts to isolate infectious virus from bats or to generate these viruses by reverse genetics have failed to date. Recombinant vesicular stomatitis virus (VSV) encoding the hemagglutinin-like envelope glycoproteins HL17 or HL18 in place of the VSV glycoprotein were generated to identify cell lines that are susceptible to bat influenza A-like virus entry. More than 30 cell lines derived from various species were screened but only a few cell lines were found to be susceptible, including Madin-Darby canine kidney type II (MDCK II) cells. The identification of cell lines susceptible to VSV chimeras allowed us to recover recombinant HL17NL10 and HL18NL11 viruses from synthetic DNA. Both influenza A-like viruses established a productive infection in MDCK II cells; however, HL18NL11 replicated more efficiently than HL17NL10 in this cell line. Unlike conventional influenza A viruses, bat influenza A-like viruses started the infection preferentially at the basolateral membrane of polarized MDCK II cells; however, similar to conventional influenza A viruses, bat influenza A-like viruses were released primarily from the apical site. The ability of HL18NL11 or HL17NL10 viruses to infect canine and human cells might reflect a zoonotic potential of these recently identified bat viruses.

Synthetically derived bat influenza A-like viruses reveal a cell type- but not species-specific tropism

PubMed Central

Moreira, Étori Aguiar; Locher, Samira; Kolesnikova, Larissa; Bolte, Hardin; Aydillo, Teresa; García-Sastre, Adolfo; Schwemmle, Martin; Zimmer, Gert

2016-01-01

Two novel influenza A-like viral genome sequences have recently been identified in Central and South American fruit bats and provisionally designated “HL17NL10” and “HL18NL11.” All efforts to isolate infectious virus from bats or to generate these viruses by reverse genetics have failed to date. Recombinant vesicular stomatitis virus (VSV) encoding the hemagglutinin-like envelope glycoproteins HL17 or HL18 in place of the VSV glycoprotein were generated to identify cell lines that are susceptible to bat influenza A-like virus entry. More than 30 cell lines derived from various species were screened but only a few cell lines were found to be susceptible, including Madin–Darby canine kidney type II (MDCK II) cells. The identification of cell lines susceptible to VSV chimeras allowed us to recover recombinant HL17NL10 and HL18NL11 viruses from synthetic DNA. Both influenza A-like viruses established a productive infection in MDCK II cells; however, HL18NL11 replicated more efficiently than HL17NL10 in this cell line. Unlike conventional influenza A viruses, bat influenza A-like viruses started the infection preferentially at the basolateral membrane of polarized MDCK II cells; however, similar to conventional influenza A viruses, bat influenza A-like viruses were released primarily from the apical site. The ability of HL18NL11 or HL17NL10 viruses to infect canine and human cells might reflect a zoonotic potential of these recently identified bat viruses. PMID:27791106

The Text Encoding Initiative: Flexible and Extensible Document Encoding.

ERIC Educational Resources Information Center

Barnard, David T.; Ide, Nancy M.

1997-01-01

The Text Encoding Initiative (TEI), an international collaboration aimed at producing a common encoding scheme for complex texts, examines the requirement for generality versus the requirement to handle specialized text types. Discusses how documents and users tax the limits of fixed schemes requiring flexible extensible encoding to support…

Encoding Orientation and the Remembering of Schizophrenic Young Adults

ERIC Educational Resources Information Center

Koh, Soon D.; Peterson, Rolf A.

1978-01-01

This research examines different types of encoding strategies, in addition to semantic and organizational encodings, and their effects on schizophrenics' remembering. Based on Craik and Lockhart (1972), i.e., memory performance is a function of depth of encoding processing, this analysis compares schizophrenics' encoding processing with that of…

Expression analysis of two gene subfamilies encoding the plasma membrane H+-ATPase in Nicotiana plumbaginifolia reveals the major transport functions of this enzyme.

PubMed

Moriau, L; Michelet, B; Bogaerts, P; Lambert, L; Michel, A; Oufattole, M; Boutry, M

1999-07-01

The plasma membrane H+-ATPase couples ATP hydrolysis to proton transport, thereby establishing the driving force for solute transport across the plasma membrane. In Nicotiana plumbaginifolia, this enzyme is encoded by at least nine pma (plasma membrane H+-ATPase) genes. Four of these are classified into two gene subfamilies, pma1-2-3 and pma4, which are the most highly expressed in plant species. We have isolated genomic clones for pma2 and pma4. Mapping of their transcript 5' end revealed the presence of a long leader that contained small open reading frames, regulatory features typical of other pma genes. The gusA reporter gene was then used to determine the expression of pma2, pma3 and pma4 in N. tabacum. These data, together with those obtained previously for pma1, led to the following conclusions. (i) The four pma-gusA genes were all expressed in root, stem, leaf and flower organs, but each in a cell-type specific manner. Expression in these organs was confirmed at the protein level, using subfamily-specific antibodies. (ii) pma4-gusA was expressed in many cell types and notably in root hair and epidermis, in companion cells, and in guard cells, indicating that in N. plumbaginifolia the same H+-ATPase isoform might be involved in mineral nutrition, phloem loading and control of stomata aperture. (iii) The second gene subfamily is composed, in N. plumbaginifolia, of a single gene (pma4) with a wide expression pattern and, in Arabidopsis thaliana, of three genes (aha1, aha2, aha3), at least two of them having a more restrictive expression pattern. (iv) Some cell types expressed pma2 and pma4 at the same time, which encode H+-ATPases with different enzymatic properties.

Insights into the strategies used by related group II introns to adapt successfully for the colonisation of a bacterial genome

PubMed Central

Martínez-Rodríguez, Laura; García-Rodríguez, Fernando M; Molina-Sánchez, María Dolores; Toro, Nicolás; Martínez-Abarca, Francisco

2014-01-01

Group II introns are self-splicing RNAs and site-specific mobile retroelements found in bacterial and organellar genomes. The group II intron RmInt1 is present at high copy number in Sinorhizobium meliloti species, and has a multifunctional intron-encoded protein (IEP) with reverse transcriptase/maturase activities, but lacking the DNA-binding and endonuclease domains. We characterized two RmInt1-related group II introns RmInt2 from S. meliloti strain GR4 and Sr.md.I1 from S. medicae strain WSM419 in terms of splicing and mobility activities. We used both wild-type and engineered intron-donor constructs based on ribozyme ΔORF-coding sequence derivatives, and we determined the DNA target requirements for RmInt2, the element most distantly related to RmInt1. The excision and mobility patterns of intron-donor constructs expressing different combinations of IEP and intron RNA provided experimental evidence for the co-operation of IEPs and intron RNAs from related elements in intron splicing and, in some cases, in intron homing. We were also able to identify the DNA target regions recognized by these IEPs lacking the DNA endonuclease domain. Our results provide new insight into the versatility of related group II introns and the possible co-operation between these elements to facilitate the colonization of bacterial genomes. PMID:25482895

Insights into the strategies used by related group II introns to adapt successfully for the colonisation of a bacterial genome.

PubMed

Martínez-Rodríguez, Laura; García-Rodríguez, Fernando M; Molina-Sánchez, María Dolores; Toro, Nicolás; Martínez-Abarca, Francisco

2014-01-01

Group II introns are self-splicing RNAs and site-specific mobile retroelements found in bacterial and organellar genomes. The group II intron RmInt1 is present at high copy number in Sinorhizobium meliloti species, and has a multifunctional intron-encoded protein (IEP) with reverse transcriptase/maturase activities, but lacking the DNA-binding and endonuclease domains. We characterized two RmInt1-related group II introns RmInt2 from S. meliloti strain GR4 and Sr.md.I1 from S. medicae strain WSM419 in terms of splicing and mobility activities. We used both wild-type and engineered intron-donor constructs based on ribozyme ΔORF-coding sequence derivatives, and we determined the DNA target requirements for RmInt2, the element most distantly related to RmInt1. The excision and mobility patterns of intron-donor constructs expressing different combinations of IEP and intron RNA provided experimental evidence for the co-operation of IEPs and intron RNAs from related elements in intron splicing and, in some cases, in intron homing. We were also able to identify the DNA target regions recognized by these IEPs lacking the DNA endonuclease domain. Our results provide new insight into the versatility of related group II introns and the possible co-operation between these elements to facilitate the colonization of bacterial genomes.

Localization of a bacterial group II intron-encoded protein in human cells.

PubMed

Reinoso-Colacio, Mercedes; García-Rodríguez, Fernando Manuel; García-Cañadas, Marta; Amador-Cubero, Suyapa; García Pérez, José Luis; Toro, Nicolás

2015-08-05

Group II introns are mobile retroelements that self-splice from precursor RNAs to form ribonucleoparticles (RNP), which can invade new specific genomic DNA sites. This specificity can be reprogrammed, for insertion into any desired DNA site, making these introns useful tools for bacterial genetic engineering. However, previous studies have suggested that these elements may function inefficiently in eukaryotes. We investigated the subcellular distribution, in cultured human cells, of the protein encoded by the group II intron RmInt1 (IEP) and several mutants. We created fusions with yellow fluorescent protein (YFP) and with a FLAG epitope. We found that the IEP was localized in the nucleus and nucleolus of the cells. Remarkably, it also accumulated at the periphery of the nuclear matrix. We were also able to identify spliced lariat intron RNA, which co-immunoprecipitated with the IEP, suggesting that functional RmInt1 RNPs can be assembled in cultured human cells.

Localization of a bacterial group II intron-encoded protein in human cells

PubMed Central

Reinoso-Colacio, Mercedes; García-Rodríguez, Fernando Manuel; García-Cañadas, Marta; Amador-Cubero, Suyapa; Pérez, José Luis García; Toro, Nicolás

2015-01-01

Group II introns are mobile retroelements that self-splice from precursor RNAs to form ribonucleoparticles (RNP), which can invade new specific genomic DNA sites. This specificity can be reprogrammed, for insertion into any desired DNA site, making these introns useful tools for bacterial genetic engineering. However, previous studies have suggested that these elements may function inefficiently in eukaryotes. We investigated the subcellular distribution, in cultured human cells, of the protein encoded by the group II intron RmInt1 (IEP) and several mutants. We created fusions with yellow fluorescent protein (YFP) and with a FLAG epitope. We found that the IEP was localized in the nucleus and nucleolus of the cells. Remarkably, it also accumulated at the periphery of the nuclear matrix. We were also able to identify spliced lariat intron RNA, which co-immunoprecipitated with the IEP, suggesting that functional RmInt1 RNPs can be assembled in cultured human cells. PMID:26244523

The VBP and a1/EBP leucine zipper factors bind overlapping subsets of avian retroviral long terminal repeat CCAAT/enhancer elements.

PubMed

Smith, C D; Baglia, L A; Curristin, S M; Ruddell, A

1994-10-01

Two long terminal repeat (LTR) enhancer-binding proteins which may regulate high rates of avian leukosis virus (ALV) LTR-enhanced c-myc transcription during bursal lymphomagenesis have been identified (A. Ruddell, M. Linial, and M. Groudine, Mol. Cell. Biol. 9:5660-5668, 1989). The genes encoding the a1/EBP and a3/EBP binding factors were cloned by expression screening of a lambda gt11 cDNA library from chicken bursal lymphoma cells. The a1/EBP cDNA encodes a novel leucine zipper transcription factor (W. Bowers and A. Ruddell, J. Virol. 66:6578-6586, 1992). The partial a3/EBP cDNA clone encodes amino acids 84 to 313 of vitellogenin gene-binding protein (VBP), a leucine zipper factor that binds the avian vitellogenin II gene promoter (S. Iyer, D. Davis, and J. Burch, Mol. Cell. Biol. 11:4863-4875, 1991). Multiple VBP mRNAs are expressed in B cells in a pattern identical to that previously observed for VBP in other cell types. The LTR-binding activities of VBP, a1/EBP, and B-cell nuclear extract protein were compared and mapped by gel shift, DNase I footprinting, and methylation interference assays. The purified VBP and a1/EBP bacterial fusion proteins bind overlapping but distinct subsets of CCAAT/enhancer elements in the closely related ALV and Rous sarcoma virus (RSV) LTR enhancers. Protein binding to these CCAAT/enhancer elements accounts for most of the labile LTR enhancer-binding activity observed in B-cell nuclear extracts. VBP and a1/EBP could mediate the high rates of ALV and RSV LTR-enhanced transcription in bursal lymphoma cells and many other cell types.

CIITA promoter I CARD-deficient mice express functional MHC class II genes in myeloid and lymphoid compartments.

PubMed

Zinzow-Kramer, W M; Long, A B; Youngblood, B A; Rosenthal, K M; Butler, R; Mohammed, A-U-R; Skountzou, I; Ahmed, R; Evavold, B D; Boss, J M

2012-06-01

Three distinct promoters control the master regulator of major histocompatibility complex (MHC) class II expression, class II transactivator (CIITA), in a cell type-specific manner. Promoter I (pI) CIITA, expressed primarily by dendritic cells (DCs) and macrophages, expresses a unique isoform that contains a caspase-recruitment domain (CARD). The activity and function of this isoform are not understood, but are believed to enhance the function of CIITA in antigen-presenting cells. To determine whether isoform I of CIITA has specific functions, CIITA mutant mice were created in which isoform I was replaced with isoform III sequences. Mice in which pI and the CARD-encoding exon were deleted were also created. No defect in the formation of CD4 T cells, the ability to respond to a model antigen or bacterial or viral challenge was observed in mice lacking CIITA isoform I. Although CIITA and MHC-II expression was decreased in splenic DCs, pI knockout animals expressed CIITA from downstream promoters, suggesting that control of pI activity is mediated by unknown distal elements that could act at pIII, the B-cell promoter. Thus, no critical function is linked to the CARD domain of CIITA isoform I with respect to basic immune system development, function and challenge.

An approach to instrument qualified visual range

NASA Astrophysics Data System (ADS)

Courtade, Benoît; Bonnet, Jordan; Woodruff, Chris; Larson, Josiah; Giles, Andrew; Sonde, Nikhil; Moore, C. J.; Schimon, David; Harris, David Money; Pond, Duane; Way, Scott

2008-04-01

This paper describes a system that calculates aircraft visual range with instrumentation alone. A unique message is encoded using modified binary phase shift keying and continuously flashed at high speed by ALSF-II runway approach lights. The message is sampled at 400 frames per second by an aircraft borne high-speed camera. The encoding is designed to avoid visible flicker and minimize frame rate. Instrument qualified visual range is identified as the largest distance at which the aircraft system can acquire and verify the correct, runway-specific signal. Scaled testing indicates that if the system were implemented on one full ALSF-II fixture, instrument qualified range could be established at 5 miles in clear weather conditions.

Deep and shallow encoding effects on face recognition: an ERP study.

PubMed

Marzi, Tessa; Viggiano, Maria Pia

2010-12-01

Event related potentials (ERPs) were employed to investigate whether and when brain activity related to face recognition varies according to the processing level undertaken at encoding. Recognition was assessed when preceded by a "shallow" (orientation judgement) or by a "deep" study task (occupation judgement). Moreover, we included a further manipulation by presenting at encoding faces either in the upright or inverted orientation. As expected, deeply encoded faces were recognized more accurately and more quickly with respect to shallowly encoded faces. The ERP showed three main findings: i) as witnessed by more positive-going potentials for deeply encoded faces, at early and later processing stage, face recognition was influenced by the processing strategy adopted during encoding; ii) structural encoding, indexed by the N170, turned out to be "cognitively penetrable" showing repetition priming effects for deeply encoded faces; iii) face inversion, by disrupting configural processing during encoding, influenced memory related processes for deeply encoded faces and impaired the recognition of faces shallowly processed. The present study adds weight to the concept that the depth of processing during memory encoding affects retrieval. We found that successful retrieval following deep encoding involved both familiarity- and recollection-related processes showing from 500 ms a fronto-parietal distribution, whereas shallow encoding affected only earlier processing stages reflecting perceptual priming. Copyright © 2010 Elsevier B.V. All rights reserved.

The WNKs: atypical protein kinases with pleiotropic actions

PubMed Central

McCormick, James A.; Ellison, David H.

2011-01-01

WNKs are serine/threonine kinases that comprise a unique branch of the kinome. They are so-named owing to the unusual placement of an essential catalytic lysine. WNKs have now been identified in diverse organisms. In humans and other mammals, four genes encoding WNKs. WNKs are widely expressed at the message level, although data on protein expression is more limited. Soon after the WNKs were identified, mutations in genes encoding WNK 1 and 4 were determined to cause the human disease, Familial Hyperkalemic Hypertension (also known as pseudohypoaldosteronism II, or Gordon’s Syndrome). For this reason, a major focus of investigation has been to dissect the role of WNK kinases in renal regulation of ion transport. More recently, a different mutation in WNK1 was identified as the cause of hereditary sensory and autonomic neuropathy type II (HSANII), an early-onset autosomal disease of peripheral sensory nerves. Thus, the WNKs represent an important family of potential targets for the treatment of human disease, and further elucidation of their physiological actions outside of the kidney and brain is necessary. In this review, we describe the gene structure and mechanisms regulating expression and activity of the WNKs. Subsequently, we outline substrates and targets of WNKs, and effects of WNKs on cellular physiology, both in the kidney and elsewhere. Next, consequences of these effects on integrated physiological function are outlined. Finally, we discuss the known and putative pathophysiological relevance of the WNKs. PMID:21248166

The brown algae Pl.LSU/2 group II intron-encoded protein has functional reverse transcriptase and maturase activities.

PubMed

Zerbato, Madeleine; Holic, Nathalie; Moniot-Frin, Sophie; Ingrao, Dina; Galy, Anne; Perea, Javier

2013-01-01

Group II introns are self-splicing mobile elements found in prokaryotes and eukaryotic organelles. These introns propagate by homing into precise genomic locations, following assembly of a ribonucleoprotein complex containing the intron-encoded protein (IEP) and the spliced intron RNA. Engineered group II introns are now commonly used tools for targeted genomic modifications in prokaryotes but not in eukaryotes. We speculate that the catalytic activation of currently known group II introns is limited in eukaryotic cells. The brown algae Pylaiella littoralis Pl.LSU/2 group II intron is uniquely capable of in vitro ribozyme activity at physiological level of magnesium but this intron remains poorly characterized. We purified and characterized recombinant Pl.LSU/2 IEP. Unlike most IEPs, Pl.LSU/2 IEP displayed a reverse transcriptase activity without intronic RNA. The Pl.LSU/2 intron could be engineered to splice accurately in Saccharomyces cerevisiae and splicing efficiency was increased by the maturase activity of the IEP. However, spliced transcripts were not expressed. Furthermore, intron splicing was not detected in human cells. While further tool development is needed, these data provide the first functional characterization of the PI.LSU/2 IEP and the first evidence that the Pl.LSU/2 group II intron splicing occurs in vivo in eukaryotes in an IEP-dependent manner.

Comprehensive search for accessory proteins encoded with archaeal and bacterial type III CRISPR-cas gene cassettes reveals 39 new cas gene families.

PubMed

Shah, Shiraz A; Alkhnbashi, Omer S; Behler, Juliane; Han, Wenyuan; She, Qunxin; Hess, Wolfgang R; Garrett, Roger A; Backofen, Rolf

2018-06-19

A study was undertaken to identify conserved proteins that are encoded adjacent to cas gene cassettes of Type III CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats - CRISPR associated) interference modules. Type III modules have been shown to target and degrade dsDNA, ssDNA and ssRNA and are frequently intertwined with cofunctional accessory genes, including genes encoding CRISPR-associated Rossman Fold (CARF) domains. Using a comparative genomics approach, and defining a Type III association score accounting for coevolution and specificity of flanking genes, we identified and classified 39 new Type III associated gene families. Most archaeal and bacterial Type III modules were seen to be flanked by several accessory genes, around half of which did not encode CARF domains and remain of unknown function. Northern blotting and interference assays in Synechocystis confirmed that one particular non-CARF accessory protein family was involved in crRNA maturation. Non-CARF accessory genes were generally diverse, encoding nuclease, helicase, protease, ATPase, transporter and transmembrane domains with some encoding no known domains. We infer that additional families of non-CARF accessory proteins remain to be found. The method employed is scalable for potential application to metagenomic data once automated pipelines for annotation of CRISPR-Cas systems have been developed. All accessory genes found in this study are presented online in a readily accessible and searchable format for researchers to audit their model organism of choice: http://accessory.crispr.dk .

Truncated hemoglobins in actinorhizal nodules of Datisca glomerata.

PubMed

Pawlowski, K; Jacobsen, K R; Alloisio, N; Ford Denison, R; Klein, M; Tjepkema, J D; Winzer, T; Sirrenberg, A; Guan, C; Berry, A M

2007-11-01

Three types of hemoglobins exist in higher plants, symbiotic, non-symbiotic, and truncated hemoglobins. Symbiotic (class II) hemoglobins play a role in oxygen supply to intracellular nitrogen-fixing symbionts in legume root nodules, and in one case ( Parasponia Sp.), a non-symbiotic (class I) hemoglobin has been recruited for this function. Here we report the induction of a host gene, dgtrHB1, encoding a truncated hemoglobin in Frankia-induced nodules of the actinorhizal plant Datisca glomerata. Induction takes place specifically in cells infected by the microsymbiont, prior to the onset of bacterial nitrogen fixation. A bacterial gene (Frankia trHBO) encoding a truncated hemoglobin with O (2)-binding kinetics suitable for the facilitation of O (2) diffusion ( ) is also expressed in symbiosis. Nodule oximetry confirms the presence of a molecule that binds oxygen reversibly in D. glomerata nodules, but indicates a low overall hemoglobin concentration suggesting a local function. Frankia trHbO is likely to be responsible for this activity. The function of the D. glomerata truncated hemoglobin is unknown; a possible role in nitric oxide detoxification is suggested.

Inhibition of CRISPR-Cas9 with Bacteriophage Proteins.

PubMed

Rauch, Benjamin J; Silvis, Melanie R; Hultquist, Judd F; Waters, Christopher S; McGregor, Michael J; Krogan, Nevan J; Bondy-Denomy, Joseph

2017-01-12

Bacterial CRISPR-Cas systems utilize sequence-specific RNA-guided nucleases to defend against bacteriophage infection. As a countermeasure, numerous phages are known that produce proteins to block the function of class 1 CRISPR-Cas systems. However, currently no proteins are known to inhibit the widely used class 2 CRISPR-Cas9 system. To find these inhibitors, we searched cas9-containing bacterial genomes for the co-existence of a CRISPR spacer and its target, a potential indicator for CRISPR inhibition. This analysis led to the discovery of four unique type II-A CRISPR-Cas9 inhibitor proteins encoded by Listeria monocytogenes prophages. More than half of L. monocytogenes strains with cas9 contain at least one prophage-encoded inhibitor, suggesting widespread CRISPR-Cas9 inactivation. Two of these inhibitors also blocked the widely used Streptococcus pyogenes Cas9 when assayed in Escherichia coli and human cells. These natural Cas9-specific "anti-CRISPRs" present tools that can be used to regulate the genome engineering activities of CRISPR-Cas9. Copyright © 2017 Elsevier Inc. All rights reserved.

Cloning, sequencing, and analysis of the griseusin polyketide synthase gene cluster from Streptomyces griseus.

PubMed Central

Yu, T W; Bibb, M J; Revill, W P; Hopwood, D A

1994-01-01

A fragment of DNA was cloned from the Streptomyces griseus K-63 genome by using genes (act) for the actinorhodin polyketide synthase (PKS) of Streptomyces coelicolor as a probe. Sequencing of a 5.4-kb segment of the cloned DNA revealed a set of five gris open reading frames (ORFs), corresponding to the act PKS genes, in the following order: ORF1 for a ketosynthase, ORF2 for a chain length-determining factor, ORF3 for an acyl carrier protein, ORF5 for a ketoreductase, and ORF4 for a cyclase-dehydrase. Replacement of the gris genes with a marker gene in the S. griseus genome by using a single-stranded suicide vector propagated in Escherichia coli resulted in loss of the ability to produce griseusins A and B, showing that the five gris genes do indeed encode the type II griseusin PKS. These genes, encoding a PKS that is programmed differently from those for other aromatic PKSs so far available, will provide further valuable material for analysis of the programming mechanism by the construction and analysis of strains carrying hybrid PKS. Images PMID:8169211

Mutations in ORC1, encoding the largest subunit of the origin recognition complex, cause microcephalic primordial dwarfism resembling Meier-Gorlin syndrome.

PubMed

Bicknell, Louise S; Walker, Sarah; Klingseisen, Anna; Stiff, Tom; Leitch, Andrea; Kerzendorfer, Claudia; Martin, Carol-Anne; Yeyati, Patricia; Al Sanna, Nouriya; Bober, Michael; Johnson, Diana; Wise, Carol; Jackson, Andrew P; O'Driscoll, Mark; Jeggo, Penny A

2011-02-27

Studies into disorders of extreme growth failure (for example, Seckel syndrome and Majewski osteodysplastic primordial dwarfism type II) have implicated fundamental cellular processes of DNA damage response signaling and centrosome function in the regulation of human growth. Here we report that mutations in ORC1, encoding a subunit of the origin recognition complex, cause microcephalic primordial dwarfism resembling Meier-Gorlin syndrome. We establish that these mutations disrupt known ORC1 functions including pre-replicative complex formation and origin activation. ORC1 deficiency perturbs S-phase entry and S-phase progression. Additionally, we show that Orc1 depletion in zebrafish is sufficient to markedly reduce body size during rapid embryonic growth. Our data suggest a model in which ORC1 mutations impair replication licensing, slowing cell cycle progression and consequently impeding growth during development, particularly at times of rapid proliferation. These findings establish a novel mechanism for the pathogenesis of microcephalic dwarfism and show a surprising but important developmental impact of impaired origin licensing.

Characterisation and expression analysis of B-cell activating factor (BAFF) in spiny dogfish (Squalus acanthias): cartilaginous fish BAFF has a unique extra exon that may impact receptor binding.

PubMed

Li, Ronggai; Dooley, Helen; Wang, Tiehui; Secombes, Christopher J; Bird, Steve

2012-04-01

B-cell activating factor (BAFF), also known as tumour necrosis factor (TNF) ligand superfamily member 13B, is an important immune regulator with critical roles in B-cell survival, proliferation, differentiation and immunoglobulin secretion. A BAFF gene has been cloned from spiny dogfish (Squalus acanthias) and its expression studied. The dogfish BAFF encodes for an anchored type-II transmembrane protein of 288 aa with a putative furin protease cleavage site and TNF family signature as seen in BAFFs from other species. The identity of dogfish BAFF has also been confirmed by conserved cysteine residues, and phylogenetic tree analysis. The dogfish BAFF gene has an extra exon not seen in teleost fish, birds and mammals that encodes for 29 aa and may impact on receptor binding. The dogfish BAFF is highly expressed in immune tissues, such as spleen, and is up-regulated by PWM in peripheral blood leucocytes, suggesting a potentially important role in the immune system. Copyright © 2011 Elsevier Ltd. All rights reserved.

SAR202 Genomes from the Dark Ocean Predict Pathways for the Oxidation of Recalcitrant Dissolved Organic Matter

PubMed Central

Landry, Zachary; Swan, Brandon K.; Herndl, Gerhard J.; Stepanauskas, Ramunas

2017-01-01

ABSTRACT Deep-ocean regions beyond the reach of sunlight contain an estimated 615 Pg of dissolved organic matter (DOM), much of which persists for thousands of years. It is thought that bacteria oxidize DOM until it is too dilute or refractory to support microbial activity. We analyzed five single-amplified genomes (SAGs) from the abundant SAR202 clade of dark-ocean bacterioplankton and found they encode multiple families of paralogous enzymes involved in carbon catabolism, including several families of oxidative enzymes that we hypothesize participate in the degradation of cyclic alkanes. The five partial genomes encoded 152 flavin mononucleotide/F420-dependent monooxygenases (FMNOs), many of which are predicted to be type II Baeyer-Villiger monooxygenases (BVMOs) that catalyze oxygen insertion into semilabile alicyclic alkanes. The large number of oxidative enzymes, as well as other families of enzymes that appear to play complementary roles in catabolic pathways, suggests that SAR202 might catalyze final steps in the biological oxidation of relatively recalcitrant organic compounds to refractory compounds that persist. PMID:28420738

Overexpression, purification, crystallization and preliminary X-ray studies of Vibrio cholerae EpsG

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

Jens, Jason; Raghunathan, Kannan; Vago, Frank

2010-01-12

EpsG is the major pseudopilin protein of the Vibrio cholerae type II secretion system. An expression plasmid that encodes an N-terminally truncated form of EpsG with a C-terminal noncleavable His tag was constructed. Recombinant EpsG was expressed in Escherichia coli; the truncated protein was purified and crystallized by hanging-drop vapor diffusion against a reservoir containing 6 mM zinc sulfate, 60 mM MES pH 6.5, 15% PEG MME 550. The crystals diffracted X-rays to a resolution of 2.26 {angstrom} and belonged to space group P2{sub 1}, with unit-cell parameters a = 88.61, b = 70.02, c = 131.54 {angstrom}.

Phylogeography and pigment type diversity of Synechococcus cyanobacteria in surface waters of the northwestern pacific ocean.

PubMed

Xia, Xiaomin; Partensky, Frédéric; Garczarek, Laurence; Suzuki, Koji; Guo, Cui; Yan Cheung, Shun; Liu, Hongbin

2017-01-01

The widespread unicellular cyanobacteria Synechococcus are major contributors to global marine primary production. Here, we report their abundance, phylogenetic diversity (as assessed using the RNA polymerase gamma subunit gene rpoC1) and pigment diversity (as indirectly assessed using the laterally transferred cpeBA genes, encoding phycoerythrin-I) in surface waters of the northwestern Pacific Ocean, sampled over nine distinct cruises (2008-2015). Abundance of Synechococcus was low in the subarctic ocean and South China Sea, intermediate in the western subtropical Pacific Ocean, and the highest in the Japan and East China seas. Clades I and II were by far the most abundant Synechococcus lineages, the former dominating in temperate cold waters and the latter in (sub)tropical waters. Clades III and VI were also fairly abundant in warm waters, but with a narrower distribution than clade II. One type of chromatic acclimater (3dA) largely dominated the Synechococcus communities in the subarctic ocean, while another (3dB) and/or cells with a fixed high phycourobilin to phycoerythrobilin ratio (pigment type 3c) predominated at mid and low latitudes. Altogether, our results suggest that the variety of pigment content found in most Synechococcus clades considerably extends the niches that they can colonize and therefore the whole genus habitat. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

GS6, a member of the GRAS gene family, negatively regulates grain size in rice.

PubMed

Sun, Lianjun; Li, Xiaojiao; Fu, Yongcai; Zhu, Zuofeng; Tan, Lubin; Liu, Fengxia; Sun, Xianyou; Sun, Xuewen; Sun, Chuanqing

2013-10-01

Grain size is an important yield-related trait in rice. Intensive artificial selection for grain size during domestication is evidenced by the larger grains of most of today's cultivars compared with their wild relatives. However, the molecular genetic control of rice grain size is still not well characterized. Here, we report the identification and cloning of Grain Size 6 (GS6), which plays an important role in reducing grain size in rice. A premature stop at the +348 position in the coding sequence (CDS) of GS6 increased grain width and weight significantly. Alignment of the CDS regions of GS6 in 90 rice materials revealed three GS6 alleles. Most japonica varieties (95%) harbor the Type I haplotype, and 62.9% of indica varieties harbor the Type II haplotype. Association analysis revealed that the Type I haplotype tends to increase the width and weight of grains more than either of the Type II or Type III haplotypes. Further investigation of genetic diversity and the evolutionary mechanisms of GS6 showed that the GS6 gene was strongly selected in japonica cultivars. In addition, a "ggc" repeat region identified in the region that encodes the GRAS domain of GS6 played an important historic role in the domestication of grain size in rice. Knowledge of the function of GS6 might aid efforts to elucidate the molecular mechanisms that control grain development and evolution in rice plants, and could facilitate the genetic improvement of rice yield. © 2013 Institute of Botany, Chinese Academy of Sciences.

Discrimination of taste qualities among mouse fungiform taste bud cells.

PubMed

Yoshida, Ryusuke; Miyauchi, Aya; Yasuo, Toshiaki; Jyotaki, Masafumi; Murata, Yoshihiro; Yasumatsu, Keiko; Shigemura, Noriatsu; Yanagawa, Yuchio; Obata, Kunihiko; Ueno, Hiroshi; Margolskee, Robert F; Ninomiya, Yuzo

2009-09-15

Multiple lines of evidence from molecular studies indicate that individual taste qualities are encoded by distinct taste receptor cells. In contrast, many physiological studies have found that a significant proportion of taste cells respond to multiple taste qualities. To reconcile this apparent discrepancy and to identify taste cells that underlie each taste quality, we investigated taste responses of individual mouse fungiform taste cells that express gustducin or GAD67, markers for specific types of taste cells. Type II taste cells respond to sweet, bitter or umami tastants, express taste receptors, gustducin and other transduction components. Type III cells possess putative sour taste receptors, and have well elaborated conventional synapses. Consistent with these findings we found that gustducin-expressing Type II taste cells responded best to sweet (25/49), bitter (20/49) or umami (4/49) stimuli, while all GAD67 (Type III) taste cells examined (44/44) responded to sour stimuli and a portion of them showed multiple taste sensitivities, suggesting discrimination of each taste quality among taste bud cells. These results were largely consistent with those previously reported with circumvallate papillae taste cells. Bitter-best taste cells responded to multiple bitter compounds such as quinine, denatonium and cyclohexamide. Three sour compounds, HCl, acetic acid and citric acid, elicited responses in sour-best taste cells. These results suggest that taste cells may be capable of recognizing multiple taste compounds that elicit similar taste sensation. We did not find any NaCl-best cells among the gustducin and GAD67 taste cells, raising the possibility that salt sensitive taste cells comprise a different population.

Gene expansion shapes genome architecture in the human pathogen Lichtheimia corymbifera: an evolutionary genomics analysis in the ancient terrestrial mucorales (Mucoromycotina).

PubMed

Schwartze, Volker U; Winter, Sascha; Shelest, Ekaterina; Marcet-Houben, Marina; Horn, Fabian; Wehner, Stefanie; Linde, Jörg; Valiante, Vito; Sammeth, Michael; Riege, Konstantin; Nowrousian, Minou; Kaerger, Kerstin; Jacobsen, Ilse D; Marz, Manja; Brakhage, Axel A; Gabaldón, Toni; Böcker, Sebastian; Voigt, Kerstin

2014-08-01

Lichtheimia species are the second most important cause of mucormycosis in Europe. To provide broader insights into the molecular basis of the pathogenicity-associated traits of the basal Mucorales, we report the full genome sequence of L. corymbifera and compared it to the genome of Rhizopus oryzae, the most common cause of mucormycosis worldwide. The genome assembly encompasses 33.6 MB and 12,379 protein-coding genes. This study reveals four major differences of the L. corymbifera genome to R. oryzae: (i) the presence of an highly elevated number of gene duplications which are unlike R. oryzae not due to whole genome duplication (WGD), (ii) despite the relatively high incidence of introns, alternative splicing (AS) is not frequently observed for the generation of paralogs and in response to stress, (iii) the content of repetitive elements is strikingly low (<5%), (iv) L. corymbifera is typically haploid. Novel virulence factors were identified which may be involved in the regulation of the adaptation to iron-limitation, e.g. LCor01340.1 encoding a putative siderophore transporter and LCor00410.1 involved in the siderophore metabolism. Genes encoding the transcription factors LCor08192.1 and LCor01236.1, which are similar to GATA type regulators and to calcineurin regulated CRZ1, respectively, indicating an involvement of the calcineurin pathway in the adaption to iron limitation. Genes encoding MADS-box transcription factors are elevated up to 11 copies compared to the 1-4 copies usually found in other fungi. More findings are: (i) lower content of tRNAs, but unique codons in L. corymbifera, (ii) Over 25% of the proteins are apparently specific for L. corymbifera. (iii) L. corymbifera contains only 2/3 of the proteases (known to be essential virulence factors) in comparison to R. oryzae. On the other hand, the number of secreted proteases, however, is roughly twice as high as in R. oryzae.

Gene Expansion Shapes Genome Architecture in the Human Pathogen Lichtheimia corymbifera: An Evolutionary Genomics Analysis in the Ancient Terrestrial Mucorales (Mucoromycotina)

PubMed Central

Wehner, Stefanie; Linde, Jörg; Valiante, Vito; Sammeth, Michael; Riege, Konstantin; Nowrousian, Minou; Kaerger, Kerstin; Jacobsen, Ilse D.; Marz, Manja; Brakhage, Axel A.; Gabaldón, Toni; Böcker, Sebastian; Voigt, Kerstin

2014-01-01

Lichtheimia species are the second most important cause of mucormycosis in Europe. To provide broader insights into the molecular basis of the pathogenicity-associated traits of the basal Mucorales, we report the full genome sequence of L. corymbifera and compared it to the genome of Rhizopus oryzae, the most common cause of mucormycosis worldwide. The genome assembly encompasses 33.6 MB and 12,379 protein-coding genes. This study reveals four major differences of the L. corymbifera genome to R. oryzae: (i) the presence of an highly elevated number of gene duplications which are unlike R. oryzae not due to whole genome duplication (WGD), (ii) despite the relatively high incidence of introns, alternative splicing (AS) is not frequently observed for the generation of paralogs and in response to stress, (iii) the content of repetitive elements is strikingly low (<5%), (iv) L. corymbifera is typically haploid. Novel virulence factors were identified which may be involved in the regulation of the adaptation to iron-limitation, e.g. LCor01340.1 encoding a putative siderophore transporter and LCor00410.1 involved in the siderophore metabolism. Genes encoding the transcription factors LCor08192.1 and LCor01236.1, which are similar to GATA type regulators and to calcineurin regulated CRZ1, respectively, indicating an involvement of the calcineurin pathway in the adaption to iron limitation. Genes encoding MADS-box transcription factors are elevated up to 11 copies compared to the 1–4 copies usually found in other fungi. More findings are: (i) lower content of tRNAs, but unique codons in L. corymbifera, (ii) Over 25% of the proteins are apparently specific for L. corymbifera. (iii) L. corymbifera contains only 2/3 of the proteases (known to be essential virulence factors) in comparision to R. oryzae. On the other hand, the number of secreted proteases, however, is roughly twice as high as in R. oryzae. PMID:25121733

Bacterial Superantigens Promote Acute Nasopharyngeal Infection by Streptococcus pyogenes in a Human MHC Class II-Dependent Manner

PubMed Central

Kasper, Katherine J.; Zeppa, Joseph J.; Wakabayashi, Adrienne T.; Xu, Stacey X.; Mazzuca, Delfina M.; Welch, Ian; Baroja, Miren L.; Kotb, Malak; Cairns, Ewa; Cleary, P. Patrick; Haeryfar, S. M. Mansour; McCormick, John K.

2014-01-01

Establishing the genetic determinants of niche adaptation by microbial pathogens to specific hosts is important for the management and control of infectious disease. Streptococcus pyogenes is a globally prominent human-specific bacterial pathogen that secretes superantigens (SAgs) as ‘trademark’ virulence factors. SAgs function to force the activation of T lymphocytes through direct binding to lateral surfaces of T cell receptors and class II major histocompatibility complex (MHC-II) molecules. S. pyogenes invariably encodes multiple SAgs, often within putative mobile genetic elements, and although SAgs are documented virulence factors for diseases such as scarlet fever and the streptococcal toxic shock syndrome (STSS), how these exotoxins contribute to the fitness and evolution of S. pyogenes is unknown. Here we show that acute infection in the nasopharynx is dependent upon both bacterial SAgs and host MHC-II molecules. S. pyogenes was rapidly cleared from the nasal cavity of wild-type C57BL/6 (B6) mice, whereas infection was enhanced up to ∼10,000-fold in B6 mice that express human MHC-II. This phenotype required the SpeA superantigen, and vaccination with an MHC –II binding mutant toxoid of SpeA dramatically inhibited infection. Our findings indicate that streptococcal SAgs are critical for the establishment of nasopharyngeal infection, thus providing an explanation as to why S. pyogenes produces these potent toxins. This work also highlights that SAg redundancy exists to avoid host anti-SAg humoral immune responses and to potentially overcome host MHC-II polymorphisms. PMID:24875883

Circuit variability interacts with excitatory-inhibitory diversity of interneurons to regulate network encoding capacity.

PubMed

Tsai, Kuo-Ting; Hu, Chin-Kun; Li, Kuan-Wei; Hwang, Wen-Liang; Chou, Ya-Hui

2018-05-23

Local interneurons (LNs) in the Drosophila olfactory system exhibit neuronal diversity and variability, yet it is still unknown how these features impact information encoding capacity and reliability in a complex LN network. We employed two strategies to construct a diverse excitatory-inhibitory neural network beginning with a ring network structure and then introduced distinct types of inhibitory interneurons and circuit variability to the simulated network. The continuity of activity within the node ensemble (oscillation pattern) was used as a readout to describe the temporal dynamics of network activity. We found that inhibitory interneurons enhance the encoding capacity by protecting the network from extremely short activation periods when the network wiring complexity is very high. In addition, distinct types of interneurons have differential effects on encoding capacity and reliability. Circuit variability may enhance the encoding reliability, with or without compromising encoding capacity. Therefore, we have described how circuit variability of interneurons may interact with excitatory-inhibitory diversity to enhance the encoding capacity and distinguishability of neural networks. In this work, we evaluate the effects of different types and degrees of connection diversity on a ring model, which may simulate interneuron networks in the Drosophila olfactory system or other biological systems.

Targeted modification of homogalacturonan by transgenic expression of a fungal polygalacturonase alters plant growth.

PubMed

Capodicasa, Cristina; Vairo, Donatella; Zabotina, Olga; McCartney, Lesley; Caprari, Claudio; Mattei, Benedetta; Manfredini, Cinzia; Aracri, Benedetto; Benen, Jacques; Knox, J Paul; De Lorenzo, Giulia; Cervone, Felice

2004-07-01

Pectins are a highly complex family of cell wall polysaccharides comprised of homogalacturonan (HGA), rhamnogalacturonan I and rhamnogalacturonan II. We have specifically modified HGA in both tobacco (Nicotiana tabacum) and Arabidopsis by expressing the endopolygalacturonase II of Aspergillus niger (AnPGII). Cell walls of transgenic tobacco plants showed a 25% reduction in GalUA content as compared with the wild type and a reduced content of deesterified HGA as detected by antibody labeling. Neutral sugars remained unchanged apart from a slight increase of Rha, Ara, and Gal. Both transgenic tobacco and Arabidopsis were dwarfed, indicating that unesterified HGA is a critical factor for plant cell growth. The dwarf phenotypes were associated with AnPGII activity as demonstrated by the observation that the mutant phenotype of tobacco was completely reverted by crossing the dwarfed plants with plants expressing PGIP2, a strong inhibitor of AnPGII. The mutant phenotype in Arabidopsis did not appear when transformation was performed with a gene encoding AnPGII inactivated by site directed mutagenesis.

The ribosome as a missing link in prebiotic evolution II: Ribosomes encode ribosomal proteins that bind to common regions of their own mRNAs and rRNAs.

PubMed

Root-Bernstein, Robert; Root-Bernstein, Meredith

2016-05-21

We have proposed that the ribosome may represent a missing link between prebiotic chemistries and the first cells. One of the predictions that follows from this hypothesis, which we test here, is that ribosomal RNA (rRNA) must have encoded the proteins necessary for ribosomal function. In other words, the rRNA also functioned pre-biotically as mRNA. Since these ribosome-binding proteins (rb-proteins) must bind to the rRNA, but the rRNA also functioned as mRNA, it follows that rb-proteins should bind to their own mRNA as well. This hypothesis can be contrasted to a "null" hypothesis in which rb-proteins evolved independently of the rRNA sequences and therefore there should be no necessary similarity between the rRNA to which rb-proteins bind and the mRNA that encodes the rb-protein. Five types of evidence reported here support the plausibility of the hypothesis that the mRNA encoding rb-proteins evolved from rRNA: (1) the ubiquity of rb-protein binding to their own mRNAs and autogenous control of their own translation; (2) the higher-than-expected incidence of Arginine-rich modules associated with RNA binding that occurs in rRNA-encoded proteins; (3) the fact that rRNA-binding regions of rb-proteins are homologous to their mRNA binding regions; (4) the higher than expected incidence of rb-protein sequences encoded in rRNA that are of a high degree of homology to their mRNA as compared with a random selection of other proteins; and (5) rRNA in modern prokaryotes and eukaryotes encodes functional proteins. None of these results can be explained by the null hypothesis that assumes independent evolution of rRNA and the mRNAs encoding ribosomal proteins. Also noteworthy is that very few proteins bind their own mRNAs that are not associated with ribosome function. Further tests of the hypothesis are suggested: (1) experimental testing of whether rRNA-encoded proteins bind to rRNA at their coding sites; (2) whether tRNA synthetases, which are also known to bind to their own mRNAs, are encoded by the tRNA sequences themselves; (3) and the prediction that archaeal and prokaryotic (DNA-based) genomes were built around rRNA "genes" so that rRNA-related sequences will be found to make up an unexpectedly high proportion of these genomes. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

C-terminal domain deletion enhances the protective activity of cpa/cpb loaded solid lipid nanoparticles against Leishmania major in BALB/c mice.

PubMed

Doroud, Delaram; Zahedifard, Farnaz; Vatanara, Alireza; Taslimi, Yasaman; Vahabpour, Rouholah; Torkashvand, Fatemeh; Vaziri, Behrooz; Rouholamini Najafabadi, Abdolhossein; Rafati, Sima

2011-07-01

We have demonstrated that vaccination with pDNA encoding cysteine proteinase Type II (CPA) and Type I (CPB) with its unusual C-terminal extension (CTE) can partially protect BALB/c mice against cutaneous leishmanial infection. Unfortunately, this protection is insufficient to completely control infection without booster injection. Furthermore, in developing vaccines for leishmaniasis, it is necessary to consider a proper adjuvant and/or delivery system to promote an antigen specific immune response. Solid lipid nanoparticles have found their way in drug delivery system development against intracellular infections and cancer, but not Leishmania DNA vaccination. Therefore, undefined effect of cationic solid lipid nanoparticles (cSLN) as an adjuvant in enhancing the immune response toward leishmanial antigens led us to refocus our vaccine development projects. Three pDNAs encoding L. major cysteine proteinase type I and II (with or without CTE) were formulated by cSLN. BALB/c mice were immunized twice by 3-week interval, with cSLN-pcDNA-cpa/b, pcDNA-cpa/b, cSLN-pcDNA-cpa/b(-CTE), pcDNA-cpa/b(-CTE), cSLN, cSLN-pcDNA and PBS. Mice vaccinated with cSLN-pcDNA-cpa/b(-CTE) showed significantly higher levels of parasite inhibition related to protection with specific Th1 immune response development, compared to other groups. Parasite inhibition was determined by different techniques currently available in exploration vacciation efficacy, i.e., flowcytometry on footpad and lymph node, footpad caliper based measurements and imaging as well as lymph node microtitration assay. Among these techniques, lymph node flowcytometry was found to be the most rapid, sensitive and easily reproducible method for discrimination between the efficacy of vaccination strategies. This report demonstrates cSLN's ability to boost immune response magnitude of cpa/cpb(-CTE) cocktail vaccination against leishmaniasis so that the average parasite inhibition percent could be increased significantly. Hence, cSLNs can be considered as suitable adjuvant and/or delivery systems for designing third generation cocktail vaccines.

Identification of Poly(ADP-Ribose) Polymerase as a Transcriptional Coactivator of the Human T-Cell Leukemia Virus Type 1 Tax Protein

PubMed Central

Anderson, Mark G.; Scoggin, Kirsten E. S.; Simbulan-Rosenthal, Cynthia M.; Steadman, Jennifer A.

2000-01-01

Human T-cell leukemia virus type 1 (HTLV-1) encodes a transcriptional activator, Tax, whose activity is believed to contribute significantly to cellular transformation. Tax stimulates transcription from the proviral promoter as well as from promoters for a variety of cellular genes. The mechanism through which Tax communicates to the general transcription factors and RNA polymerase II has not been completely determined. We investigated whether Tax could function directly through the general transcription factors and RNA polymerase II or if other intermediary factors or coactivators were required. Our results show that a system consisting of purified recombinant TFIIA, TFIIB, TFIIE, TFIIF, CREB, and Tax, along with highly purified RNA polymerase II, affinity-purified epitope-tagged TFIID, and semipurified TFIIH, supports basal transcription of the HTLV-1 promoter but is not responsive to Tax. Two additional activities were required for Tax to stimulate transcription. We demonstrate that one of these activities is poly(ADP-ribose) polymerase (PARP), a molecule that has been previously identified to be the transcriptional coactivator PC1. PARP functions as a coactivator in our assays at molar concentrations approximately equal to those of the DNA and equal to or less than those of the transcription factors in the assay. We further demonstrate that PARP stimulates Tax-activated transcription in vivo, demonstrating that this biochemical approach has functionally identified a novel target for the retroviral transcriptional activator Tax. PMID:10666246

Dopamine 5 receptor mediates Ang II type 1 receptor degradation via a ubiquitin-proteasome pathway in mice and human cells

PubMed Central

Li, Hewang; Armando, Ines; Yu, Peiying; Escano, Crisanto; Mueller, Susette C.; Asico, Laureano; Pascua, Annabelle; Lu, Quansheng; Wang, Xiaoyan; Villar, Van Anthony M.; Jones, John E.; Wang, Zheng; Periasamy, Ammasi; Lau, Yuen-Sum; Soares-da-Silva, Patricio; Creswell, Karen; Guillemette, Gaétan; Sibley, David R.; Eisner, Gilbert; Felder, Robin A.; Jose, Pedro A.

2008-01-01

Hypertension is a multigenic disorder in which abnormal counterregulation between dopamine and Ang II plays a role. Recent studies suggest that this counterregulation results, at least in part, from regulation of the expression of both the antihypertensive dopamine 5 receptor (D5R) and the prohypertensive Ang II type 1 receptor (AT1R). In this report, we investigated the in vivo and in vitro interaction between these GPCRs. Disruption of the gene encoding D5R in mice increased both blood pressure and AT1R protein expression, and the increase in blood pressure was reversed by AT1R blockade. Activation of D5R increased the degradation of glycosylated AT1R in proteasomes in HEK cells and human renal proximal tubule cells heterologously and endogenously expressing human AT1R and D5R. Confocal microscopy, Förster/fluorescence resonance energy transfer microscopy, and fluorescence lifetime imaging microscopy revealed that activation of D5R initiated ubiquitination of the glycosylated AT1R at the plasma membrane. The regulated degradation of AT1R via a ubiquitin/proteasome pathway by activation of D5R provides what we believe to be a novel mechanism whereby blood pressure can be regulated by the interaction of 2 counterregulatory GPCRs. Our results therefore suggest that treatments for hypertension might be optimized by designing compounds that can target the AT1R and the D5R. PMID:18464932

A novel immunization method to induce cytotoxic T-lymphocyte responses (CTL) against plasmid-encoded herpes simplex virus type-1 glycoprotein D.

PubMed

Cruz, P E; Khalil, P L; Dryden, T D; Chiou, H C; Fink, P S; Berberich, S J; Bigley, N J

1999-03-05

DNA molecules complexed with an asialoglycoprotein-polycation conjugate, consisting of asialoorosomucoid (ASOR) coupled to poly-L-lysine, can enter hepatocytes which bear receptors for ASOR. We used this receptor-mediated DNA delivery system to deliver plasmid DNA encoding glycoprotein D (gD) of herpes simplex virus type 1 to ASOR-positive cells. Maximum expression of gD protein was seen at 3 days after injection of this preparation in approximately 13% of cells from BALB/c mice [hepatocytes from mice injected intravenously (i.v.) or peritoneal exudate cells from mice injected intraperitoneally (i.p.)]. In comparison with mice injected with either the plasmid vector alone or the gD-containing plasmid uncomplexed to ASOR, mice immunized with gD-containing plasmid complexed with ASOR-poly-L-lysine induced marked antigen-specific CTL responses. BALB/c mice immunized with gD-DNA developed a T-cell-mediated CTL response against target cells expressing gD and MHC class II glycoproteins, but not against cells expressing only gD and MHC class I molecules. In C3H mice, gD-DNA induced a T-cell-mediated CTL response against target cells expressing gD and class I MHC molecules. Serum anti-gD antibody in low titers were produced in both strains of mice. DNA complexed with ASOR-poly-L-lysine induced CTL responses in mice.

The detection and phylogenetic analysis of the alkane 1-monooxygenase gene of members of the genus Rhodococcus.

PubMed

Táncsics, András; Benedek, Tibor; Szoboszlay, Sándor; Veres, Péter G; Farkas, Milán; Máthé, István; Márialigeti, Károly; Kukolya, József; Lányi, Szabolcs; Kriszt, Balázs

2015-02-01

Naturally occurring and anthropogenic petroleum hydrocarbons are potential carbon sources for many bacteria. The AlkB-related alkane hydroxylases, which are integral membrane non-heme iron enzymes, play a key role in the microbial degradation of many of these hydrocarbons. Several members of the genus Rhodococcus are well-known alkane degraders and are known to harbor multiple alkB genes encoding for different alkane 1-monooxygenases. In the present study, 48 Rhodococcus strains, representing 35 species of the genus, were investigated to find out whether there was a dominant type of alkB gene widespread among species of the genus that could be used as a phylogenetic marker. Phylogenetic analysis of rhodococcal alkB gene sequences indicated that a certain type of alkB gene was present in almost every member of the genus Rhodococcus. These alkB genes were common in a unique nucleotide sequence stretch absent from other types of rhodococcal alkB genes that encoded a conserved amino acid motif: WLG(I/V/L)D(G/D)GL. The sequence identity of the targeted alkB gene in Rhodococcus ranged from 78.5 to 99.2% and showed higher nucleotide sequence variation at the inter-species level compared to the 16S rRNA gene (93.9-99.8%). The results indicated that the alkB gene type investigated might be applicable for: (i) differentiating closely related Rhodococcus species, (ii) properly assigning environmental isolates to existing Rhodococcus species, and finally (iii) assessing whether a new Rhodococcus isolate represents a novel species of the genus. Copyright © 2014 Elsevier GmbH. All rights reserved.

Stat5-mediated regulation of the human type II 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase gene: activation by prolactin.

PubMed

Feltus, F A; Groner, B; Melner, M H

1999-07-01

Altered PRL levels are associated with infertility in women. Molecular targets at which PRL elicits these effects have yet to be determined. These studies demonstrate transcriptional regulation by PRL of the gene encoding the final enzymatic step in progesterone biosynthesis: 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase (3beta-HSD). A 9/9 match with the consensus Stat5 response element was identified at -110 to -118 in the human Type II 3beta-HSD promoter. 3beta-HSD chloramphenicol acetyltransferase (CAT) reporter constructs containing either an intact or mutated Stat5 element were tested for PRL activation. Expression vectors for Stat5 and the PRL receptor were cotransfected with a -300 --> +45 3beta-HSD CAT reporter construct into HeLa cells, which resulted in a 21-fold increase in reporter activity in the presence of PRL. Promoter activity showed an increased response with a stepwise elevation of transfected Stat5 expression or by treatment with increasing concentrations of PRL (max, 250 ng/ml). This effect was dramatically reduced when the putative Stat5 response element was removed by 5'-deletion of the promoter or by the introduction of a 3-bp mutation into critical nucleotides in the element. Furthermore, 32P-labeled promoter fragments containing the Stat5 element were shifted in electrophoretic mobility shift assay experiments using nuclear extracts from cells treated with PRL, and this complex was supershifted with antibodies to Stat5. These results demonstrate that PRL has the ability to regulate expression of a key human enzyme gene (type II 3beta-HSD) in the progesterone biosynthetic pathway, which is essential for maintaining pregnancy.

Recombinant human type II collagen hydrogel provides a xeno-free 3D micro-environment for chondrogenesis of human bone marrow-derived mesenchymal stromal cells.

PubMed

Muhonen, Virpi; Narcisi, Roberto; Nystedt, Johanna; Korhonen, Matti; van Osch, Gerjo J V M; Kiviranta, Ilkka

2017-03-01

Recombinant human type II collagen (rhCII) hydrogel was tested as a xeno-free micro-environment for the chondrogenesis of human bone marrow-derived mesenchymal stromal cells (BM-MSCs). The rhCII hydrogels were seeded with BM-MSCs and cultured in a xeno-free chondro-inductive medium for 14, 28 and 84 days. High-density pellet cultures served as controls. The samples were subjected to biochemical, histological and gene expression analyses. Although the cells deposited glycosaminoglycans into the extracellular space significantly more slowly in the rhCII hydrogels compared to the high-density pellets, a similar potential of matrix deposition was reached by the end of the 84-day culture. At day 28 of culture, the gene expression level for cartilage marker genes (i.e. genes encoding for Sox9 transcription factor, Collagen type II and Aggrecan) were considerably lower in the rhCII hydrogels than in the high-density pellets, but at the end of the 84-day culture period, all the cartilage marker genes analysed were expressed at a similar level. Interestingly, the expression of the matrix metallopeptidases (MMP)-13, MMP-14 and MMP-8, i.e. extracellular collagen network-degrading enzymes, were transiently upregulated in the rhCII hydrogel, indicating active matrix reorganization. This study demonstrated that the rhCII hydrogel functions as a xeno-free platform for BM-MSC chondrogenesis, although the process is delayed. The reversible catabolic reaction evoked by the rhCII hydrogel might be beneficial in graft integration in vivo and pinpoints the need to further explore the use of hydrogels containing recombinant extracellular matrix (ECM) proteins to induce the chondrogenesis of MSCs. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

A DUF-246 family glycosyltransferase-like gene affects male fertility and the biosynthesis of pectic arabinogalactans

DOE PAGES

Stonebloom, Solomon; Ebert, Berit; Xiong, Guangyan; ...

2016-04-18

We report pectins are a group of structurally complex plant cell wall polysaccharides whose biosynthesis and function remain poorly understood. The pectic polysaccharide rhamnogalacturonan-I (RG-I) has two types of arabinogalactan side chains, type-I and type-II arabinogalactans. To date few enzymes involved in the biosynthesis of pectin have been described. Here we report the identification of a highly conserved putative glycosyltransferase encoding gene, Pectic ArabinoGalactan synthesis-Related (PAGR), affecting the biosynthesis of RG-I arabinogalactans and critical for pollen tube growth. T-DNA insertions in PAGR were identified in Arabidopsis thaliana and were found to segregate at a 1:1 ratio of heterozygotes to wildmore » type. We were unable to isolate homozygous pagr mutants as pagr mutant alleles were not transmitted via pollen. In vitro pollen germination assays revealed reduced rates of pollen tube formation in pollen from pagr heterozygotes. To characterize a loss-of-function phenotype for PAGR, the Nicotiana benthamiana orthologs, NbPAGR-A and B, were transiently silenced using Virus Induced Gene Silencing. NbPAGR-silenced plants exhibited reduced internode and petiole expansion. Cell wall materials from NbPAGR-silenced plants had reduced galactose content compared to the control. Immunological and linkage analyses support that RG-I has reduced type-I arabinogalactan content and reduced branching of the RG-I backbone in NbPAGR-silenced plants. Arabidopsis lines overexpressing PAGR exhibit pleiotropic developmental phenotypes and the loss of apical dominance as well as an increase in RG-I type-II arabinogalactan content. Together, results support a function for PAGR in the biosynthesis of RG-I arabinogalactans and illustrate the essential roles of these polysaccharides in vegetative and reproductive plant growth.« less

A DUF-246 family glycosyltransferase-like gene affects male fertility and the biosynthesis of pectic arabinogalactans

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

Stonebloom, Solomon; Ebert, Berit; Xiong, Guangyan

We report pectins are a group of structurally complex plant cell wall polysaccharides whose biosynthesis and function remain poorly understood. The pectic polysaccharide rhamnogalacturonan-I (RG-I) has two types of arabinogalactan side chains, type-I and type-II arabinogalactans. To date few enzymes involved in the biosynthesis of pectin have been described. Here we report the identification of a highly conserved putative glycosyltransferase encoding gene, Pectic ArabinoGalactan synthesis-Related (PAGR), affecting the biosynthesis of RG-I arabinogalactans and critical for pollen tube growth. T-DNA insertions in PAGR were identified in Arabidopsis thaliana and were found to segregate at a 1:1 ratio of heterozygotes to wildmore » type. We were unable to isolate homozygous pagr mutants as pagr mutant alleles were not transmitted via pollen. In vitro pollen germination assays revealed reduced rates of pollen tube formation in pollen from pagr heterozygotes. To characterize a loss-of-function phenotype for PAGR, the Nicotiana benthamiana orthologs, NbPAGR-A and B, were transiently silenced using Virus Induced Gene Silencing. NbPAGR-silenced plants exhibited reduced internode and petiole expansion. Cell wall materials from NbPAGR-silenced plants had reduced galactose content compared to the control. Immunological and linkage analyses support that RG-I has reduced type-I arabinogalactan content and reduced branching of the RG-I backbone in NbPAGR-silenced plants. Arabidopsis lines overexpressing PAGR exhibit pleiotropic developmental phenotypes and the loss of apical dominance as well as an increase in RG-I type-II arabinogalactan content. Together, results support a function for PAGR in the biosynthesis of RG-I arabinogalactans and illustrate the essential roles of these polysaccharides in vegetative and reproductive plant growth.« less

Re-engaging with the past: recapitulation of encoding operations during episodic retrieval

PubMed Central

Morcom, Alexa M.

2014-01-01

Recollection of events is accompanied by selective reactivation of cortical regions which responded to specific sensory and cognitive dimensions of the original events. This reactivation is thought to reflect the reinstatement of stored memory representations and therefore to reflect memory content, but it may also reveal processes which support both encoding and retrieval. The present study used event-related functional magnetic resonance imaging to investigate whether regions selectively engaged in encoding face and scene context with studied words are also re-engaged when the context is later retrieved. As predicted, encoding face and scene context with visually presented words elicited activity in distinct, context-selective regions. Retrieval of face and scene context also re-engaged some of the regions which had shown successful encoding effects. However, this recapitulation of encoding activity did not show the same context selectivity observed at encoding. Successful retrieval of both face and scene context re-engaged regions which had been associated with encoding of the other type of context, as well as those associated with encoding the same type of context. This recapitulation may reflect retrieval attempts which are not context-selective, but use shared retrieval cues to re-engage encoding operations in service of recollection. PMID:24904386

Characterization of HKE2: an ancient antigen encoded in the major histocompatibility complex.

PubMed

Ostrov, D A; Barnes, C L; Smith, L E; Binns, S; Brusko, T M; Brown, A C; Quint, P S; Litherland, S A; Roopenian, D C; Iczkowski, K A

2007-02-01

Genes at the centromeric end of the human leukocyte antigen region influence adaptive autoimmune diseases and cancer. In this study, we characterized protein expression of HKE2, a gene located in the centromeric portion of the class II region of the major histocompatibility complex encoding subunit 6 of prefoldin. Immunohistochemical analysis using an anti-HKE2 antibody indicated that HKE2 protein expression is dramatically upregulated as a consequence of activation. In a tissue microarray and in several tumors, HKE2 was overexpressed in certain cancers compared with normal counterparts. The localization of the HKE2 gene to the class II region, its cytoplasmic expression and putative protein-binding domain suggest that HKE2 may function in adaptive immunity and cancer.

Antisense and sense expression of cDNA coding for CYP73A15, a class II cinnamate 4-hydroxylase, leads to a delayed and reduced production of lignin in tobacco

NASA Technical Reports Server (NTRS)

Blee, K.; Choi, J. W.; O'Connell, A. P.; Jupe, S. C.; Schuch, W.; Lewis, N. G.; Bolwell, G. P.

2001-01-01

A number of plant species contain the class II of genes encoding the cytochrome P450, CYP73, the cognate protein of which cinnamic acid 4-hydroxylase, is the second enzyme of the phenylpropanoid pathway. In order to begin to determine possible functionality, tobacco has been transformed with a truncated French bean class II cinnamate hydroxylase (CYP73A15) in the sense and antisense orientations. Signals for C4H protein could be detected in vascular tissue from wild-type plants using heterologous probes. The transformed plants showed a normal phenotype, even though detectable C4H protein was much reduced in tissue prints. Young propagated transformants displayed a range of reduced C4H activities, as well as either reduced or no phloroglucinol-stainable lignin. However, all mature tobacco plants showed the accumulation of lignin, even though its deposition was apparently delayed. This was not due to induction of tyrosine ammonia-lyase activity, which was not detected, but instead it is presumed due to sufficient C4H residual activity. Analysis of the lignin content of the plants showed reductions of up to 30% with a slightly reduced syringyl to guaiacyl ratio as compared to wild type. This reduction level was favourable in comparison with some other targets in the lignification pathway that have been manipulated including that of class I cinnamate 4-hydroxylase. It is proposed that the class II cinnamate 4-hydroxylase might also function in lignification in a number of species including French bean and tobacco, based on these data.

Transcriptomic analysis reveals Toxoplasma gondii strain-specific differences in host cell response to dense granule protein GRA15.

PubMed

Liu, Qing; Gao, Wen-Wei; Elsheikha, Hany M; He, Jun-Jun; Li, Fa-Cai; Yang, Wen-Bin; Zhu, Xing-Quan

2018-06-19

Growth and replication of the protozoan parasite Toxoplasma gondii within host cell entail the production of several effector proteins, which the parasite exploits for counteracting the host's immune response. Despite considerable research to define the host signaling pathways manipulated by T. gondii and their effectors, there has been limited progress into understanding how individual members of the dense granule proteins (GRAs) modulate gene expression within host cells. The aim of this study was to evaluate whether T. gondii GRA15 protein plays any role in regulating host gene expression. Baby hamster kidney cells (BHK-21) were transfected with plasmids encoding GRA15 genes of either type I GT1 strain (GRA15 I ) or type II PRU strain (GRA15 II ). Gene expression patterns of transfected and nontransfected BHK-21 cells were investigated using RNA-sequencing analysis. GRA15 I and GRA15 II induced both known and novel transcriptional changes in the transfected BHK-21 cells compared with nontransfected cells. Pathway analysis revealed that GRA15 II was mainly involved in the regulation of tumor necrosis factor (TNF), NF-κB, HTLV-I infection, and NOD-like receptor signaling pathways. GRA15 I preferentially influenced the synthesis of unsaturated fatty acids in host cells. Our findings support the hypothesis that certain functions of GRA15 protein are strain dependent and that GRA15 modulates the expression of signaling pathways and genes with important roles in T. gondii pathophysiology. A greater understanding of host signaling pathways influenced by T. gondii effectors would allow the development of more efficient anti-T. gondii therapeutic schemes, capitalizing on disrupting parasite virulence factors to advance the treatment of toxoplasmosis.

Self-reactive VH4-34–expressing IgG B cells recognize commensal bacteria

PubMed Central

Glauzy, Salomé; Ng, Yen-Shing; Chamberlain, Nicolas; Massad, Christopher; Isnardi, Isabelle; Uzel, Gulbu; Holland, Steven M.; Picard, Capucine

2017-01-01

The germline immunoglobulin (Ig) variable heavy chain 4–34 (VH4-34) gene segment encodes in humans intrinsically self-reactive antibodies that recognize I/i carbohydrates expressed by erythrocytes with a specific motif in their framework region 1 (FWR1). VH4-34–expressing clones are common in the naive B cell repertoire but are rarely found in IgG memory B cells from healthy individuals. In contrast, CD27+IgG+ B cells from patients genetically deficient for IRAK4 or MYD88, which mediate the function of Toll-like receptors (TLRs) except TLR3, contained VH4-34–expressing clones and showed decreased somatic hypermutation frequencies. In addition, VH4-34–encoded IgGs from IRAK4- and MYD88-deficient patients often displayed an unmutated FWR1 motif, revealing that these antibodies still recognize I/i antigens, whereas their healthy donor counterparts harbored FWR1 mutations abolishing self-reactivity. However, this paradoxical self-reactivity correlated with these VH4-34–encoded IgG clones binding commensal bacteria antigens. Hence, B cells expressing germline-encoded self-reactive VH4-34 antibodies may represent an innate-like B cell population specialized in the containment of commensal bacteria when gut barriers are breached. PMID:28500047

Method and apparatus for two-dimensional absolute optical encoding

NASA Technical Reports Server (NTRS)

Leviton, Douglas B. (Inventor)

2004-01-01

This invention presents a two-dimensional absolute optical encoder and a method for determining position of an object in accordance with information from the encoder. The encoder of the present invention comprises a scale having a pattern being predetermined to indicate an absolute location on the scale, means for illuminating the scale, means for forming an image of the pattern; and detector means for outputting signals derived from the portion of the image of the pattern which lies within a field of view of the detector means, the field of view defining an image reference coordinate system, and analyzing means, receiving the signals from the detector means, for determining the absolute location of the object. There are two types of scale patterns presented in this invention: grid type and starfield type.

Noise level and MPEG-2 encoder statistics

NASA Astrophysics Data System (ADS)

Lee, Jungwoo

1997-01-01

Most software in the movie and broadcasting industries are still in analog film or tape format, which typically contains random noise that originated from film, CCD camera, and tape recording. The performance of the MPEG-2 encoder may be significantly degraded by the noise. It is also affected by the scene type that includes spatial and temporal activity. The statistical property of noise originating from camera and tape player is analyzed and the models for the two types of noise are developed. The relationship between the noise, the scene type, and encoder statistics of a number of MPEG-2 parameters such as motion vector magnitude, prediction error, and quant scale are discussed. This analysis is intended to be a tool for designing robust MPEG encoding algorithms such as preprocessing and rate control.

Host regulation of lysogenic decision in bacteriophage lambda: transmembrane modulation of FtsH (HflB), the cII degrading protease, by HflKC (HflA).

PubMed

Kihara, A; Akiyama, Y; Ito, K

1997-05-27

The cII gene product of bacteriophage lambda is unstable and required for the establishment of lysogenization. Its intracellular amount is important for the decision between lytic growth and lysogenization. Two genetic loci of Escherichia coli are crucial for these commitments of infecting lambda genome. One of them, hflA encodes the HflKC membrane protein complex, which has been believed to be a protease degrading the cII protein. However, both its absence and overproduction stabilized cII in vivo and the proposed serine protease-like sequence motif in HflC was dispensable for the lysogenization control. Moreover, the HflKC protein was found to reside on the periplasmic side of the plasma membrane. In contrast, the other host gene, ftsH (hflB) encoding an integral membrane ATPase/protease, is positively required for degradation of cII, since loss of its function stabilized cII and its overexpression accelerated the cII degradation. In vitro, purified FtsH catalyzed ATP-dependent proteolysis of cII and HflKC antagonized the FtsH action. These results, together with our previous finding that FtsH and HflKC form a complex, suggest that FtsH is the cII degrading protease and HflKC is a modulator of the FtsH function. We propose that this transmembrane modulation differentiates the FtsH actions to different substrate proteins such as the membrane-bound SecY protein and the cytosolic cII protein. This study necessitates a revision of the prevailing view about the host control over lambda lysogenic decision.

The Brown Algae Pl.LSU/2 Group II Intron-Encoded Protein Has Functional Reverse Transcriptase and Maturase Activities

PubMed Central

Zerbato, Madeleine; Holic, Nathalie; Moniot-Frin, Sophie; Ingrao, Dina; Galy, Anne; Perea, Javier

2013-01-01

Group II introns are self-splicing mobile elements found in prokaryotes and eukaryotic organelles. These introns propagate by homing into precise genomic locations, following assembly of a ribonucleoprotein complex containing the intron-encoded protein (IEP) and the spliced intron RNA. Engineered group II introns are now commonly used tools for targeted genomic modifications in prokaryotes but not in eukaryotes. We speculate that the catalytic activation of currently known group II introns is limited in eukaryotic cells. The brown algae Pylaiella littoralis Pl.LSU/2 group II intron is uniquely capable of in vitro ribozyme activity at physiological level of magnesium but this intron remains poorly characterized. We purified and characterized recombinant Pl.LSU/2 IEP. Unlike most IEPs, Pl.LSU/2 IEP displayed a reverse transcriptase activity without intronic RNA. The Pl.LSU/2 intron could be engineered to splice accurately in Saccharomyces cerevisiae and splicing efficiency was increased by the maturase activity of the IEP. However, spliced transcripts were not expressed. Furthermore, intron splicing was not detected in human cells. While further tool development is needed, these data provide the first functional characterization of the PI.LSU/2 IEP and the first evidence that the Pl.LSU/2 group II intron splicing occurs in vivo in eukaryotes in an IEP-dependent manner. PMID:23505475

Mouse Y-linked Zfy1 and Zfy2 are expressed during the male-specific interphase between meiosis I and meiosis II and promote the 2nd meiotic division.

PubMed

Vernet, Nadège; Mahadevaiah, Shantha K; Yamauchi, Yasuhiro; Decarpentrie, Fanny; Mitchell, Michael J; Ward, Monika A; Burgoyne, Paul S

2014-06-01

Mouse Zfy1 and Zfy2 encode zinc finger transcription factors that map to the short arm of the Y chromosome (Yp). They have previously been shown to promote meiotic quality control during pachytene (Zfy1 and Zfy2) and at the first meiotic metaphase (Zfy2). However, from these previous studies additional roles for genes encoded on Yp during meiotic progression were inferred. In order to identify these genes and investigate their function in later stages of meiosis, we created three models with diminishing Yp and Zfy gene complements (but lacking the Y-long-arm). Since the Y-long-arm mediates pairing and exchange with the X via their pseudoautosomal regions (PARs) we added a minute PAR-bearing X chromosome derivative to enable formation of a sex bivalent, thus avoiding Zfy2-mediated meiotic metaphase I (MI) checkpoint responses to the unpaired (univalent) X chromosome. Using these models we obtained definitive evidence that genetic information on Yp promotes meiosis II, and by transgene addition identified Zfy1 and Zfy2 as the genes responsible. Zfy2 was substantially more effective and proved to have a much more potent transactivation domain than Zfy1. We previously established that only Zfy2 is required for the robust apoptotic elimination of MI spermatocytes in response to a univalent X; the finding that both genes potentiate meiosis II led us to ask whether there was de novo Zfy1 and Zfy2 transcription in the interphase between meiosis I and meiosis II, and this proved to be the case. X-encoded Zfx was also expressed at this stage and Zfx over-expression also potentiated meiosis II. An interphase between the meiotic divisions is male-specific and we previously hypothesised that this allows meiosis II critical X and Y gene reactivation following sex chromosome silencing in meiotic prophase. The interphase transcription and meiosis II function of Zfx, Zfy1 and Zfy2 validate this hypothesis.

Mouse Y-Linked Zfy1 and Zfy2 Are Expressed during the Male-Specific Interphase between Meiosis I and Meiosis II and Promote the 2nd Meiotic Division

PubMed Central

Vernet, Nadège; Mahadevaiah, Shantha K.; Yamauchi, Yasuhiro; Decarpentrie, Fanny; Mitchell, Michael J.; Ward, Monika A.; Burgoyne, Paul S.

2014-01-01

Mouse Zfy1 and Zfy2 encode zinc finger transcription factors that map to the short arm of the Y chromosome (Yp). They have previously been shown to promote meiotic quality control during pachytene (Zfy1 and Zfy2) and at the first meiotic metaphase (Zfy2). However, from these previous studies additional roles for genes encoded on Yp during meiotic progression were inferred. In order to identify these genes and investigate their function in later stages of meiosis, we created three models with diminishing Yp and Zfy gene complements (but lacking the Y-long-arm). Since the Y-long-arm mediates pairing and exchange with the X via their pseudoautosomal regions (PARs) we added a minute PAR-bearing X chromosome derivative to enable formation of a sex bivalent, thus avoiding Zfy2-mediated meiotic metaphase I (MI) checkpoint responses to the unpaired (univalent) X chromosome. Using these models we obtained definitive evidence that genetic information on Yp promotes meiosis II, and by transgene addition identified Zfy1 and Zfy2 as the genes responsible. Zfy2 was substantially more effective and proved to have a much more potent transactivation domain than Zfy1. We previously established that only Zfy2 is required for the robust apoptotic elimination of MI spermatocytes in response to a univalent X; the finding that both genes potentiate meiosis II led us to ask whether there was de novo Zfy1 and Zfy2 transcription in the interphase between meiosis I and meiosis II, and this proved to be the case. X-encoded Zfx was also expressed at this stage and Zfx over-expression also potentiated meiosis II. An interphase between the meiotic divisions is male-specific and we previously hypothesised that this allows meiosis II critical X and Y gene reactivation following sex chromosome silencing in meiotic prophase. The interphase transcription and meiosis II function of Zfx, Zfy1 and Zfy2 validate this hypothesis. PMID:24967676

'Laminopathies': A wide spectrum of human diseases

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

Worman, Howard J.; Bonne, Gisele; Universite Pierre et Marie Curie-Paris 6, Faculte de medecine, Paris F-75013

2007-06-10

Mutations in genes encoding the intermediate filament nuclear lamins and associated proteins cause a wide spectrum of diseases sometimes called 'laminopathies.' Diseases caused by mutations in LMNA encoding A-type lamins include autosomal dominant Emery-Dreifuss muscular dystrophy and related myopathies, Dunnigan-type familial partial lipodystrophy, Charcot-Marie-Tooth disease type 2B1 and developmental and accelerated aging disorders. Duplication in LMNB1 encoding lamin B1 causes autosomal dominant leukodystrophy and mutations in LMNB2 encoding lamin B2 are associated with acquired partial lipodystrophy. Disorders caused by mutations in genes encoding lamin-associated integral inner nuclear membrane proteins include X-linked Emery-Dreifuss muscular dystrophy, sclerosing bone dysplasias, HEM/Greenberg skeletal dysplasiamore » and Pelger-Huet anomaly. While mutations and clinical phenotypes of 'laminopathies' have been carefully described, data explaining pathogenic mechanisms are only emerging. Future investigations will likely identify new 'laminopathies' and a combination of basic and clinical research will lead to a better understanding of pathophysiology and the development of therapies.« less

Characterization of the molecular defect in a feline model for type II GM2-gangliosidosis (Sandhoff disease).

PubMed Central

Muldoon, L. L.; Neuwelt, E. A.; Pagel, M. A.; Weiss, D. L.

1994-01-01

The Korat cat provides an animal model for type II GM2-gangliosidosis (Sandhoff disease) that may be suitable for tests of gene replacement therapy with the HEXB gene encoding the beta subunit of the beta-hexosaminidases. In the present report, we examined the brain and liver pathology of a typical Sandhoff-affected cat. We characterized the feline HEXB complementary DNA (cDNA) and determined the molecular defect in this feline model. cDNA libraries were produced from one normal and one affected animal, and cDNA clones homologous to human HEXB were sequenced. In the affected cDNA clone, the deletion of a cytosine residue at position +39 of the putative coding region results in a frame shift and a stop codon at base +191. This disease-related deletion was consistently detected by sequencing of cloned polymerase chain reaction amplified reverse transcribed messenger RNA from one more normal Korat and two additional affected animals. The defect was further demonstrated using single-strand conformational polymorphism analysis of the polymerase chain reaction products. In addition, alternative splicing of both normal and affected messenger RNAs was demonstrated. These results should facilitate the use of this animal model to assess gene therapy. Images Figure 1 Figure 3 Figure 4 Figure 5 PMID:8178934

Characterization of the molecular defect in a feline model for type II GM2-gangliosidosis (Sandhoff disease).

PubMed

Muldoon, L L; Neuwelt, E A; Pagel, M A; Weiss, D L

1994-05-01

The Korat cat provides an animal model for type II GM2-gangliosidosis (Sandhoff disease) that may be suitable for tests of gene replacement therapy with the HEXB gene encoding the beta subunit of the beta-hexosaminidases. In the present report, we examined the brain and liver pathology of a typical Sandhoff-affected cat. We characterized the feline HEXB complementary DNA (cDNA) and determined the molecular defect in this feline model. cDNA libraries were produced from one normal and one affected animal, and cDNA clones homologous to human HEXB were sequenced. In the affected cDNA clone, the deletion of a cytosine residue at position +39 of the putative coding region results in a frame shift and a stop codon at base +191. This disease-related deletion was consistently detected by sequencing of cloned polymerase chain reaction amplified reverse transcribed messenger RNA from one more normal Korat and two additional affected animals. The defect was further demonstrated using single-strand conformational polymorphism analysis of the polymerase chain reaction products. In addition, alternative splicing of both normal and affected messenger RNAs was demonstrated. These results should facilitate the use of this animal model to assess gene therapy.

BMP type II receptor as a therapeutic target in pulmonary arterial hypertension.

PubMed

Orriols, Mar; Gomez-Puerto, Maria Catalina; Ten Dijke, Peter

2017-08-01

Pulmonary arterial hypertension (PAH) is a chronic disease characterized by a progressive elevation in mean pulmonary arterial pressure. This occurs due to abnormal remodeling of small peripheral lung vasculature resulting in progressive occlusion of the artery lumen that eventually causes right heart failure and death. The most common cause of PAH is inactivating mutations in the gene encoding a bone morphogenetic protein type II receptor (BMPRII). Current therapeutic options for PAH are limited and focused mainly on reversal of pulmonary vasoconstriction and proliferation of vascular cells. Although these treatments can relieve disease symptoms, PAH remains a progressive lethal disease. Emerging data suggest that restoration of BMPRII signaling in PAH is a promising alternative that could prevent and reverse pulmonary vascular remodeling. Here we will focus on recent advances in rescuing BMPRII expression, function or signaling to prevent and reverse pulmonary vascular remodeling in PAH and its feasibility for clinical translation. Furthermore, we summarize the role of described miRNAs that directly target the BMPR2 gene in blood vessels. We discuss the therapeutic potential and the limitations of promising new approaches to restore BMPRII signaling in PAH patients. Different mutations in BMPR2 and environmental/genetic factors make PAH a heterogeneous disease and it is thus likely that the best approach will be patient-tailored therapies.

47 CFR 11.12 - Two-tone Attention Signal encoder and decoder.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 47 Telecommunication 1 2011-10-01 2011-10-01 false Two-tone Attention Signal encoder and decoder... SYSTEM (EAS) General § 11.12 Two-tone Attention Signal encoder and decoder. Existing two-tone Attention Signal encoder and decoder equipment type accepted for use as Emergency Broadcast System equipment under...

47 CFR 11.12 - Two-tone Attention Signal encoder and decoder.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 47 Telecommunication 1 2010-10-01 2010-10-01 false Two-tone Attention Signal encoder and decoder... SYSTEM (EAS) General § 11.12 Two-tone Attention Signal encoder and decoder. Existing two-tone Attention Signal encoder and decoder equipment type accepted for use as Emergency Broadcast System equipment under...

A missense mutation encoding Cys73Phe in neurophysin II is associated with autosomal dominant neurohypophyseal diabetes insipidus.

PubMed

Santiprabhob, Jeerunda; Browning, James; Repaske, David

2002-01-01

Autosomal dominant neurohypophyseal diabetes insipidus (ADNDI) is an inherited disease caused by progressive deficiency of the hormone arginine vasopressin (AVP) that typically becomes clinically apparent in the first decade of life. The genetic locus of ADNDI is the arginine vasopressin-neurophysin II (AVP-NPII) gene and mutations that cause ADNDI have been found in the nucleotides encoding the signal peptide, vasopressin, and neurophysin II peptides. In this study we have analyzed the AVP-NPII gene in a 20-year-old female who was diagnosed with ADNDI at 2 years of age. A heterozygous missense mutation (1684G>T) was found in exon 2 that predicts replacement of cysteine with phenylalanine at position 73 of neurophysin II. The mutation was confirmed by subcloning exon 2 PCR products to sequence each allele independently. Two out of four clones were found to have the missense mutation and two have the normal sequence, confirming the presence of the mutation and heterozygosity. Neurophysin II is an intracellular carrier protein for AVP during axonal transport from the hypothalamus to the posterior pituitary and contains 14 cysteine residues forming 7 disulfide bonds. This mutation is predicted to disrupt the disulfide bridge between Cys73 and Cys61 within the neurophysin II moiety. This finding of a novel mutation substituting cysteine with phenylalanine in one AVP-NPII gene allele supports the hypothesis that inability to form normal disulfide bonds in neurophysin II leads to ADNDI.

Entorhinal Cortical Ocean Cells Encode Specific Contexts and Drive Context-Specific Fear Memory

PubMed Central

Kitamura, Takashi; Sun, Chen; Martin, Jared; Kitch, Lacey J; Schnitzer, Mark J; Tonegawa, Susumu

2016-01-01

Summary Forming distinct representations and memories of multiple contexts and episodes is thought to be a crucial function of the hippocampal-entorhinal cortical network. The hippocampal dentate gyrus (DG) and CA3 are known to contribute to these functions but the role of the entorhinal cortex (EC) is poorly understood. Here, we show that Ocean cells, excitatory stellate neurons in the medial EC layer II projecting into DG and CA3, rapidly form a distinct representation of a novel context and drive context-specific activation of downstream CA3 cells as well as context-specific fear memory. In contrast, Island cells, excitatory pyramidal neurons in the medial EC layer II projecting into CA1, are indifferent to context-specific encoding or memory. On the other hand, Ocean cells are dispensable for temporal association learning, for which Island cells are crucial. Together, the two excitatory medial EC layer II inputs to the hippocampus have complementary roles in episodic memory. PMID:26402611

Cytochrome P460 Genes from the Methanotroph Methylococcus capsulatus Bath†

PubMed Central

Bergmann, David J.; Zahn, James A.; Hooper, Alan B.; DiSpirito, Alan A.

1998-01-01

P460 cytochromes catalyze the oxidation of hydroxylamine to nitrite. They have been isolated from the ammonia-oxidizing bacterium Nitrosomonas europaea (R. H. Erickson and A. B. Hooper, Biochim. Biophys. Acta 275:231–244, 1972) and the methane-oxidizing bacterium Methylococcus capsulatus Bath (J. A. Zahn et al., J. Bacteriol. 176:5879–5887, 1994). A degenerate oligonucleotide probe was synthesized based on the N-terminal amino acid sequence of cytochrome P460 and used to identify a DNA fragment from M. capsulatus Bath that contains cyp, the gene encoding cytochrome P460. cyp is part of a gene cluster that contains three open reading frames (ORFs), the first predicted to encode a 59,000-Da membrane-bound polypeptide, the second predicted to encode a 12,000-Da periplasmic protein, and the third (cyp) encoding cytochrome P460. The products of the first two ORFs have no apparent similarity to any proteins in the GenBank database. The overall sequence similarity of the P460 cytochromes from M. capsulatus Bath and N. europaea was low (24.3% of residues identical), although short regions of conserved residues are present in the two proteins. Both cytochromes have a C-terminal, c-heme binding motif (CXXCH) and a conserved lysine residue (K61) that may provide an additional covalent cross-link to the heme (D. M. Arciero and A. B. Hooper, FEBS Lett. 410:457–460, 1997). Gene probing using cyp indicated that a cytochrome P460 similar to that from M. capsulatus Bath may be present in the type II methanotrophs Methylosinus trichosporium OB3b and Methylocystis parvus OBBP but not in the type I methanotrophs Methylobacter marinus A45, Methylomicrobium albus BG8, and Methylomonas sp. strains MN and MM2. Immunoblot analysis with antibodies against cytochrome P460 from M. capsulatus Bath indicated that the expression level of cytochrome P460 was not affected either by expression of the two different methane monooxygenases or by addition of ammonia to the culture medium. PMID:9851984

Identification and Characterization of Non-Cellulose-Producing Mutants of Gluconacetobacter hansenii Generated by Tn5 Transposon Mutagenesis

PubMed Central

Deng, Ying; Nagachar, Nivedita; Xiao, Chaowen; Tien, Ming

2013-01-01

The acs operon of Gluconacetobacter is thought to encode AcsA, AcsB, AcsC, and AcsD proteins that constitute the cellulose synthase complex, required for the synthesis and secretion of crystalline cellulose microfibrils. A few other genes have been shown to be involved in this process, but their precise role is unclear. We report here the use of Tn5 transposon insertion mutagenesis to identify and characterize six non-cellulose-producing (Cel−) mutants of Gluconacetobacter hansenii ATCC 23769. The genes disrupted were acsA, acsC, ccpAx (encoding cellulose-complementing protein [the subscript “Ax” indicates genes from organisms formerly classified as Acetobacter xylinum]), dgc1 (encoding guanylate dicyclase), and crp-fnr (encoding a cyclic AMP receptor protein/fumarate nitrate reductase transcriptional regulator). Protein blot analysis revealed that (i) AcsB and AcsC were absent in the acsA mutant, (ii) the levels of AcsB and AcsC were significantly reduced in the ccpAx mutant, and (iii) the level of AcsD was not affected in any of the Cel− mutants. Promoter analysis showed that the acs operon does not include acsD, unlike the organization of the acs operon of several strains of closely related Gluconacetobacter xylinus. Complementation experiments confirmed that the gene disrupted in each Cel− mutant was responsible for the phenotype. Quantitative real-time PCR and protein blotting results suggest that the transcription of bglAx (encoding β-glucosidase and located immediately downstream from acsD) was strongly dependent on Crp/Fnr. A bglAx knockout mutant, generated via homologous recombination, produced only ∼16% of the wild-type cellulose level. Since the crp-fnr mutant did not produce any cellulose, Crp/Fnr may regulate the expression of other gene(s) involved in cellulose biosynthesis. PMID:24013627

Profiling of EBV-Encoded microRNAs in EBV-Associated Hemophagocytic Lymphohistiocytosis.

PubMed

Zhou, Chen; Xie, Zhengde; Gao, Liwei; Liu, Chunyan; Ai, Junhong; Zhang, Li; Shen, Kunling

2015-10-01

Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) is a life-threatening complication of EBV infection. MicroRNAs (miRNAs) were small non-coding RNA, and EBV could encode miRNAs that are involved in the progression of infection. However, the profiles of EBV-miRNAs in EBV-HLH were unknown. Here, we aimed to profile the expression of EBV-miRNAs in children with EBV-HLH by analyzing 44 known EBV-miRNAs, encoded within the BamHI fragment H rightward open reading frame 1 (BHRF1) and the BamHI-A region rightward transcript (BART), in plasma and cellular targets by real-time quantitative PCR. The study included 15 children with EBV-HLH, 15 children with infectious mononucleosis (IM), and 15 healthy controls. CD8(+) T cells were found to be the cellular target of EBV infection in EBV-HLH, while CD19(+) B cells were infected with EBV in IM. We also found the greater levels of several miRNAs encoded by BART in EBV-HLH, compared to those in IM and healthy controls, whereas the levels of BHRF1 miRNAs were lower than those in IM. The profile and pattern of EBV-miRNAs in EBV-HLH indicated that EBV could display type II latency in EBV-HLH. Importantly, the level of plasma miR-BART16-1 continued decreasing during the whole chemotherapy, suggesting that plasma miR-BART16-1 could be a potential biomarker for monitoring EBV-HLH progression. The pathogenesis of EBV-HLH might be attributed to the abundance of EBV-miRNAs in EBV-HLH. These findings help elucidate the roles of EBV miRNAs in EBV-HLH, enabling the understanding of the basis of this disease and providing clues for its treatment.

Hierarchical regulation of photosynthesis gene expression by the oxygen-responsive PrrBA and AppA-PpsR systems of Rhodobacter sphaeroides.

PubMed

Gomelsky, Larissa; Moskvin, Oleg V; Stenzel, Rachel A; Jones, Denise F; Donohue, Timothy J; Gomelsky, Mark

2008-12-01

In the facultatively phototrophic proteobacterium Rhodobacter sphaeroides, formation of the photosynthetic apparatus is oxygen dependent. When oxygen tension decreases, the response regulator PrrA of the global two-component PrrBA system is believed to directly activate transcription of the puf, puh, and puc operons, encoding structural proteins of the photosynthetic complexes, and to indirectly upregulate the photopigment biosynthesis genes bch and crt. Decreased oxygen also results in inactivation of the photosynthesis-specific repressor PpsR, bringing about derepression of the puc, bch, and crt operons. We uncovered a hierarchical relationship between these two regulatory systems, earlier thought to function independently. We also more accurately assessed the spectrum of gene targets of the PrrBA system. First, expression of the appA gene, encoding the PpsR antirepressor, is PrrA dependent, which establishes one level of hierarchical dominance of the PrrBA system over AppA-PpsR. Second, restoration of the appA transcript to the wild-type level is insufficient for rescuing phototrophic growth impairment of the prrA mutant, whereas inactivation of ppsR is sufficient. This suggests that in addition to controlling appA transcription, PrrA affects the activity of the AppA-PpsR system via an as yet unidentified mechanism(s). Third, PrrA directly activates several bch and crt genes, traditionally considered to be the PpsR targets. Therefore, in R. sphaeroides, the global PrrBA system regulates photosynthesis gene expression (i) by rigorous control over the photosynthesis-specific AppA-PpsR regulatory system and (ii) by extensive direct transcription activation of genes encoding structural proteins of photosynthetic complexes as well as genes encoding photopigment biosynthesis enzymes.

Genetic diversity and virulence profiles of Listeria monocytogenes recovered from bulk tank milk, milk filters, and milking equipment from dairies in the United States (2002 to 2014).

PubMed

Kim, Seon Woo; Haendiges, Julie; Keller, Eric N; Myers, Robert; Kim, Alexander; Lombard, Jason E; Karns, Jeffrey S; Van Kessel, Jo Ann S; Haley, Bradd J

2018-01-01

Unpasteurized dairy products are known to occasionally harbor Listeria monocytogenes and have been implicated in recent listeriosis outbreaks and numerous sporadic cases of listeriosis. However, the diversity and virulence profiles of L. monocytogenes isolates recovered from these products have not been fully described. Here we report a genomic analysis of 121 L. monocytogenes isolates recovered from milk, milk filters, and milking equipment collected from bovine dairy farms in 19 states over a 12-year period. In a multi-virulence-locus sequence typing (MVLST) analysis, 59 Virulence Types (VT) were identified, of which 25% were Epidemic Clones I, II, V, VI, VII, VIII, IX, or X, and 31 were novel VT. In a multi-locus sequence typing (MLST) analysis, 60 Sequence Types (ST) of 56 Clonal Complexes (CC) were identified. Within lineage I, CC5 and CC1 were among the most abundant, and within lineage II, CC7 and CC37 were the most abundant. Multiple CCs previously associated with central nervous system and maternal-neonatal infections were identified. A genomic analysis identified variable distribution of virulence markers, Listeria pathogenicity islands (LIPI) -1, -3, and -4, and stress survival island-1 (SSI-1). Of these, 14 virulence markers, including LIPI-3 and -4 were more frequently detected in one lineage (I or II) than the other. LIPI-3 and LIPI-4 were identified in 68% and 28% of lineage I CCs, respectively. Results of this analysis indicate that there is a high level of genetic diversity among the L. monocytogenes present in bulk tank milk in the United States with some strains being more frequently detected than others, and some being similar to those that have been isolated from previous non-dairy related outbreaks. Results of this study also demonstrate significant number of strains isolated from dairy farms encode virulence markers associated with severe human disease.

The Human Respiratory Syncytial Virus Nonstructural Protein 1 Regulates Type I and Type II Interferon Pathways*

PubMed Central

Hastie, Marcus L.; Headlam, Madeleine J.; Patel, Nirav B.; Bukreyev, Alexander A.; Buchholz, Ursula J.; Dave, Keyur A.; Norris, Emma L.; Wright, Cassandra L.; Spann, Kirsten M.; Collins, Peter L.; Gorman, Jeffrey J.

2012-01-01

Respiratory syncytial viruses encode a nonstructural protein (NS1) that interferes with type I and III interferon and other antiviral responses. Proteomic studies were conducted on human A549 type II alveolar epithelial cells and type I interferon-deficient Vero cells (African green monkey kidney cells) infected with wild-type and NS1-deficient clones of human respiratory syncytial virus to identify other potential pathway and molecular targets of NS1 interference. These analyses included two-dimensional differential gel electrophoresis and quantitative Western blotting. Surprisingly, NS1 was found to suppress the induction of manganese superoxide dismutase (SOD2) expression in A549 cells and to a much lesser degree Vero cells in response to infection. Because SOD2 is not directly inducible by type I interferons, it served as a marker to probe the impact of NS1 on signaling of other cytokines known to induce SOD2 expression and/or indirect effects of type I interferon signaling. Deductive analysis of results obtained from cell infection and cytokine stimulation studies indicated that interferon-γ signaling was a potential target of NS1, possibly as a result of modulation of STAT1 levels. However, this was not sufficient to explain the magnitude of the impact of NS1 on SOD2 induction in A549 cells. Vero cell infection experiments indicated that NS1 targeted a component of the type I interferon response that does not directly induce SOD2 expression but is required to induce another initiator of SOD2 expression. STAT2 was ruled out as a target of NS1 interference using quantitative Western blot analysis of infected A549 cells, but data were obtained to indicate that STAT1 was one of a number of potential targets of NS1. A label-free mass spectrometry-based quantitative approach is proposed as a means of more definitive identification of NS1 targets. PMID:22322095

Neomycin resistance as a selectable marker in Methanococcus maripaludis

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

Argyle, J.L.; Leigh, J.A.; Tumbula, D.L.

1996-11-01

The authors cloned the aminoglycoside phosphotransferase genes APH3{prime}I and APH3{prime}II between the Methanococcus voltae methyl reductase promoter and terminator in a plasmid containing a fragment of Methanococcus maripaludis chromosomal DNA. The resulting plasmids encoding neomycin resistance transformed M. maripaludis at frequencies similar to those observed for pKAS102 encoding puromycin resistance. The antibiotic geneticin was not inhibitory to M. maripaludis. 22 refs., 3 figs., 3 tabs.

Arabidopsis thaliana type I and II chaperonins.

PubMed

Hill, J E; Hemmingsen, S M

2001-07-01

An examination of the Arabidopsis thaliana genome sequence led to the identification of 29 predicted genes with the potential to encode members of the chaperonin family of chaperones (CPN60 and CCT), their associated cochaperonins, and the cytoplasmic chaperonin cofactor prefoldin. These comprise the first complete set of plant chaperonin protein sequences and indicate that the CPN family is more diverse than previously described. In addition to surprising sequence diversity within CPN subclasses, the genomic data also suggest the existence of previously undescribed family members, including a 10-kDa chloroplast cochaperonin. Consideration of the sequence data described in this review prompts questions about the complexities of plant CPN systems and the evolutionary relationships and functions of the component proteins, most of which have not been studied experimentally.

Overexpression, purification, crystallization and preliminary X-ray studies of Vibrio cholerae EpsG

PubMed Central

Jens, Jason; Raghunathan, Kannan; Vago, Frank; Arvidson, Dennis

2009-01-01

EpsG is the major pseudopilin protein of the Vibrio cholerae type II secretion system. An expression plasmid that encodes an N-terminally truncated form of EpsG with a C-terminal noncleavable His tag was constructed. Recombinant EpsG was expressed in Escherichia coli; the truncated protein was purified and crystallized by hanging-drop vapor diffusion against a reservoir containing 6 mM zinc sulfate, 60 mM MES pH 6.5, 15% PEG MME 550. The crystals diffracted X-rays to a resolution of 2.26 Å and belonged to space group P21, with unit-cell parameters a = 88.61, b = 70.02, c = 131.54 Å. PMID:19478449

Isolation and characterization of the chicken trypsinogen gene family.

PubMed Central

Wang, K; Gan, L; Lee, I; Hood, L

1995-01-01

Based on genomic Southern hybridizations and cDNA sequence analyses, the chicken trypsinogen gene family can be divided into two multi-member subfamilies, a six-member trypsinogen I subfamily which encodes the cationic trypsin isoenzymes and a three-member trypsinogen II subfamily which encodes the anionic trypsin isoenzymes. The chicken cDNA and genomic clones containing these two subfamilies were isolated and characterized by DNA sequence analysis. The results indicated that the chicken trypsinogen genes encoded a signal peptide of 15 to 16 amino acid residues, an activation peptide of 9 to 10 residues and a trypsin of 223 amino acid residues. The chicken trypsinogens contain all the common catalytic and structural features for trypsins, including the catalytic triad His, Asp and Ser and the six disulphide bonds. The trypsinogen I and II subfamilies share approximately 70% sequence identity at the nucleotide and amino acid level. The sequence comparison among chicken trypsinogen subfamily members and trypsin sequences from other species suggested that the chicken trypsinogen genes may have evolved in coincidental or concerted fashion. Images Figure 6 Figure 7 PMID:7733885

Production of haemolysins by strains of the Actinobacillus minor/"porcitonsillarum" complex.

PubMed

Arya, Gitanjali; Niven, Donald F

2010-03-24

Actinobacillus minor and "Actinobacillus porcitonsillarum" are distinguished by their haemolytic activities, the latter organism being haemolytic and the former, non-haemolytic. Analysis of a whole genome shotgun sequence, however, revealed that A. minor strain 202, like "A. porcitonsillarum", possesses a haemolysin-encoding apxII operon. The purpose of this study was therefore to investigate haemolysin production by this organism and also by three additional members of the A. minor/"porcitonsillarum" complex, strains 33PN and 7ATS and A. minor strain NM305(T). Primers based on sequences within the apxII genes of strain 202 allowed the amplification of appropriately sized fragments from DNA from strain 33PN suggesting that this organism also possesses an apxII operon. Analysis of a whole genome shotgun sequence failed to reveal any trace of an apxII operon in strain NM305(T) and attempts to amplify apxII genes from DNA from strain 7ATS also failed. Strains 202 and 33PN, and surprisingly, the type strain of A. minor and strain 7ATS, were all found to be haemolysin-positive as growth media from cultures of these organisms could promote the lysis of erythrocytes in suspension. The erythrocyte specificities of the haemolysins produced by strains 202 and 33PN indicated that the haemolytic activities exhibited by these organisms were due to ApxII. In keeping with the apparent lack of apxII genes in strains NM305(T) and 7ATS, the haemolysins produced by these organisms were not erythrocyte-specific and with both organisms, haemolytic activity appeared to be due to a combination of heat-stable and heat-labile components. The identities of these components, however, remain unknown. Copyright 2009 Elsevier B.V. All rights reserved.

Word form Encoding in Chinese Word Naming and Word Typing

ERIC Educational Resources Information Center

Chen, Jenn-Yeu; Li, Cheng-Yi

2011-01-01

The process of word form encoding was investigated in primed word naming and word typing with Chinese monosyllabic words. The target words shared or did not share the onset consonants with the prime words. The stimulus onset asynchrony (SOA) was 100 ms or 300 ms. Typing required the participants to enter the phonetic letters of the target word,…

Typing of Panton-Valentine Leukocidin-Encoding Phages and lukSF-PV Gene Sequence Variation in Staphylococcus aureus from China.

PubMed

Zhao, Huanqiang; Hu, Fupin; Jin, Shu; Xu, Xiaogang; Zou, Yuhan; Ding, Baixing; He, Chunyan; Gong, Fang; Liu, Qingzhong

2016-01-01

Panton-Valentine leukocidin (PVL, encoded by lukSF-PV genes), a bi-component and pore-forming toxin, is carried by different staphylococcal bacteriophages. The prevalence of PVL in Staphylococcus aureus has been reported around the globe. However, the data on PVL-encoding phage types, lukSF-PV gene variation and chromosomal phage insertion sites for PVL-positive S. aureus are limited, especially in China. In order to obtain a more complete understanding of the molecular epidemiology of PVL-positive S. aureus, an integrated and modified PCR-based scheme was applied to detect the PVL-encoding phage types. Phage insertion locus and the lukSF-PV variant were determined by PCR and sequencing. Meanwhile, the genetic background was characterized by staphylococcal cassette chromosome mec (SCCmec) typing, staphylococcal protein A (spa) gene polymorphisms typing, pulsed-field gel electrophoresis (PFGE) typing, accessory gene regulator (agr) locus typing and multilocus sequence typing (MLST). Seventy eight (78/1175, 6.6%) isolates possessed the lukSF-PV genes and 59.0% (46/78) of PVL-positive strains belonged to CC59 lineage. Eight known different PVL-encoding phage types were detected, and Φ7247PVL/ΦST5967PVL (n = 13) and ΦPVL (n = 12) were the most prevalent among them. While 25 (25/78, 32.1%) isolates, belonging to ST30, and ST59 clones, were unable to be typed by the modified PCR-based scheme. Single nucleotide polymorphisms (SNPs) were identified at five locations in the lukSF-PV genes, two of which were non-synonymous. Maximum-likelihood tree analysis of attachment sites sequences detected six SNP profiles for attR and eight for attL, respectively. In conclusion, the PVL-positive S. aureus mainly harbored Φ7247PVL/ΦST5967PVL and ΦPVL in the regions studied. lukSF-PV gene sequences, PVL-encoding phages, and phage insertion locus generally varied with lineages. Moreover, PVL-positive clones that have emerged worldwide likely carry distinct phages.

Does long-term object priming depend on the explicit detection of object identity at encoding?

PubMed Central

Gomes, Carlos A.; Mayes, Andrew

2015-01-01

It is currently unclear whether objects have to be explicitly identified at encoding for reliable behavioral long-term object priming to occur. We conducted two experiments that investigated long-term object and non-object priming using a selective-attention encoding manipulation that reduces explicit object identification. In Experiment 1, participants either counted dots flashed within an object picture (shallow encoding) or engaged in an animacy task (deep encoding) at study, whereas, at test, they performed an object-decision task. Priming, as measured by reaction times (RTs), was observed for both types of encoding, and was of equivalent magnitude. In Experiment 2, non-object priming (faster RTs for studied relative to unstudied non-objects) was also obtained under the same selective-attention encoding manipulation as in Experiment 1, and the magnitude of the priming effect was equivalent between experiments. In contrast, we observed a linear decrement in recognition memory accuracy across conditions (deep encoding of Experiment 1 > shallow encoding Experiment 1 > shallow encoding of Experiment 2), suggesting that priming was not contaminated by explicit memory strategies. We argue that our results are more consistent with the identification/production framework than the perceptual/conceptual distinction, and we conclude that priming of pictures largely ignored at encoding can be subserved by the automatic retrieval of two types of instances: one at the motor level and another at an object-decision level. PMID:25852594

P450 oxidoreductase deficiency: a disorder of steroidogenesis with multiple clinical manifestations.

PubMed

Miller, Walter L

2012-10-23

Cytochrome P450 enzymes catalyze the biosynthesis of steroid hormones and metabolize drugs. There are seven human type I P450 enzymes in mitochondria and 50 type II enzymes in endoplasmic reticulum. Type II enzymes, including both drug-metabolizing and some steroidogenic enzymes, require electron donation from a two-flavin protein, P450 oxidoreductase (POR). Although knockout of the POR gene causes embryonic lethality in mice, we discovered human POR deficiency as a disorder of steroidogenesis associated with the Antley-Bixler skeletal malformation syndrome and found mild POR mutations in phenotypically normal adults with infertility. Assay results of mutant forms of POR using the traditional but nonphysiologic assay (reduction of cytochrome c) did not correlate with patient phenotypes; assays based on the 17,20 lyase activity of P450c17 (CYP17) correlated with clinical phenotypes. The POR sequence in 842 normal individuals revealed many polymorphisms; amino acid sequence variant A503V is encoded by ~28% of human alleles. POR A503V has about 60% of wild-type activity in assays with CYP17, CYP2D6, and CYP3A4, but nearly wild-type activity with P450c21, CYP1A2, and CYP2C19. Activity of a particular POR variant with one P450 enzyme will not predict its activity with another P450 enzyme: Each POR-P450 combination must be studied individually. Human POR transcription, initiated from an untranslated exon, is regulated by Smad3/4, thyroid receptors, and the transcription factor AP-2. A promoter polymorphism reduces transcription to 60% in liver cells and to 35% in adrenal cells. POR deficiency is a newly described disorder of steroidogenesis, and POR variants may account for some genetic variation in drug metabolism.

High bit rate mass data storage device

NASA Technical Reports Server (NTRS)

1973-01-01

The HDDR-II mass data storage system consists of a Leach MTR 7114 recorder reproducer, a wire wrapped, integrated circuit flat plane and necessary power supplies for the flat plane. These units, with interconnecting cables and control panel are enclosed in a common housing mounted on casters. The electronics used in the HDDR-II double density decoding and encoding techniques are described.

Alternative splicing by participation of the group II intron ORF in extremely halotolerant and alkaliphilic Oceanobacillus iheyensis.

PubMed

Chee, Gab-Joo; Takami, Hideto

2011-01-01

Group II introns inserted into genes often undergo splicing at unexpected sites, and participate in the transcription of host genes. We identified five copies of a group II intron, designated Oi.Int, in the genome of an extremely halotolerant and alkaliphilic bacillus, Oceanobacillus iheyensis. The Oi.Int4 differs from the Oi.Int3 at four bases. The ligated exons of the Oi.Int4 could not be detected by RT-PCR assays in vivo or in vitro although group II introns can generally self-splice in vitro without the involvement of an intron-encoded open reading frame (ORF). In the Oi.Int4 mutants with base substitutions within the ORF, ligated exons were detected by in vitro self-splicing. It was clear that the ligation of exons during splicing is affected by the sequence of the intron-encoded ORF since the splice sites corresponded to the joining sites of the intron. In addition, the mutant introns showed unexpected multiple products with alternative 5' splice sites. These findings imply that alternative 5' splicing which causes a functional change of ligated exons presumably has influenced past adaptations of O. iheyensis to various environmental changes.

The effect of difference frequency on electrocommunication: chirp production and encoding in a species of weakly electric fish, Apteronotus leptorhynchus.

PubMed

Hupé, Ginette J; Lewis, John E; Benda, Jan

2008-01-01

The brown ghost knifefish, Apteronotus leptorhynchus, is a model wave-type gymnotiform used extensively in neuroethological studies. As all weakly electric fish, they produce an electric field (electric organ discharge, EOD) and can detect electric signals in their environments using electroreceptors. During social interactions, A. leptorhynchus produce communication signals by modulating the frequency and amplitude of their EOD. The Type 2 chirp, a transient increase in EOD frequency, is the most common modulation type. We will first present a description of A. leptorhynchus chirp production from a behavioural perspective, followed by a discussion of the mechanisms by which chirps are encoded by electroreceptor afferents (P-units). Both the production and encoding of chirps are influenced by the difference in EOD frequency between interacting fish, the so-called beat or difference frequency (Df). Chirps are produced most often when the Df is small, whereas attacks are more common when Dfs are large. Correlation analysis has shown that chirp production induces an echo response in interacting conspecifics and that chirps are produced when attack rates are low. Here we show that both of these relationships are strongest when Dfs are large. Electrophysiological recordings from electroreceptor afferents (P-units) have suggested that small, Type 2 chirps are encoded by increases in electroreceptor synchrony at low Dfs only. How Type 2 chirps are encoded at higher Dfs, where the signals seem to exert the greatest behavioural influence, was unknown. Here, we provide evidence that at higher Dfs, chirps could be encoded by a desynchronization of the P-unit population activity.

Genetic modification of chondrocytes with insulin-like growth factor-1 enhances cartilage healing in an equine model.

PubMed

Goodrich, L R; Hidaka, C; Robbins, P D; Evans, C H; Nixon, A J

2007-05-01

Gene therapy with insulin-like growth factor-1 (IGF-1) increases matrix production and enhances chondrocyte proliferation and survival in vitro. The purpose of this study was to determine whether arthroscopically-grafted chondrocytes genetically modified by an adenovirus vector encoding equine IGF-1 (AdIGF-1) would have a beneficial effect on cartilage healing in an equine femoropatellar joint model. A total of 16 horses underwent arthroscopic repair of a single 15 mm cartilage defect in each femoropatellar joint. One joint received 2 x 10(7) AdIGF-1 modified chondrocytes and the contralateral joint received 2 x 10(7) naive (unmodified) chondrocytes. Repairs were analysed at four weeks, nine weeks and eight months after surgery. Morphological and histological appearance, IGF-1 and collagen type II gene expression (polymerase chain reaction, in situ hybridisation and immunohistochemistry), collagen type II content (cyanogen bromide and sodium dodecyl sulphate-polyacrylamide gel electrophoresis), proteoglycan content (dimethylmethylene blue assay), and gene expression for collagen type I, matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, aggrecanase-1, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and TIMP-3 were evaluated. Genetic modification of chondrocytes significantly increased IGF-1 mRNA and ligand production in repair tissue for up to nine weeks following transplantation. The gross and histological appearance of IGF-1 modified repair tissue was improved over control defects. Gross filling of defects was significantly improved at four weeks, and a more hyaline-like tissue covered the lesions at eight months. Histological outcome at four and nine weeks post-transplantation revealed greater tissue filling of defects transplanted with genetically modified chondrocytes, whereas repair tissue in control defects was thin and irregular and more fibrous. Collagen type II expression in IGF-1 gene-transduced defects was increased 100-fold at four weeks and correlated with increased collagen type II immunoreaction up to eight months. Genetic modification of chondrocytes with AdIGF-1 prior to transplantation improved early (four to nine weeks), and to a lesser degree long-term, cartilage healing in the equine model. The equine model of cartilage healing closely resembles human clinical cartilage repair. The results of this study suggest that cartilage healing can be enhanced through genetic modification of chondrocytes prior to transplantation.

The effect of encoding strategy on the neural correlates of memory for faces.

PubMed

Bernstein, Lori J; Beig, Sania; Siegenthaler, Amy L; Grady, Cheryl L

2002-01-01

Encoding and recognition of unfamiliar faces in young adults were examined using positron emission tomography to determine whether different encoding strategies would lead to encoding/retrieval differences in brain activity. Three types of encoding were compared: a 'deep' task (judging pleasantness/unpleasantness), a 'shallow' task (judging right/left orientation), and an intentional learning task in which subjects were instructed to learn the faces for a subsequent memory test but were not provided with a specific strategy. Memory for all faces was tested with an old/new recognition test. A modest behavioral effect was obtained, with deeply-encoded faces being recognized more accurately than shallowly-encoded or intentionally-learned faces. Regardless of encoding strategy, encoding activated a primarily ventral system including bilateral temporal and fusiform regions and left prefrontal cortices, whereas recognition activated a primarily dorsal set of regions including right prefrontal and parietal areas. Within encoding, the type of strategy produced different brain activity patterns, with deep encoding being characterized by left amygdala and left anterior cingulate activation. There was no effect of encoding strategy on brain activity during the recognition conditions. Posterior fusiform gyrus activation was related to better recognition accuracy in those conditions encouraging perceptual strategies, whereas activity in left frontal and temporal areas correlated with better performance during the 'deep' condition. Results highlight three important aspects of face memory: (1) the effect of encoding strategy was seen only at encoding and not at recognition; (2) left inferior prefrontal cortex was engaged during encoding of faces regardless of strategy; and (3) differential activity in fusiform gyrus was found, suggesting that activity in this area is not only a result of automatic face processing but is modulated by controlled processes.

Risk of developing pneumonia is enhanced by the combined traits of fluoroquinolone resistance and type III secretion virulence in respiratory isolates of Pseudomonas aeruginosa.

PubMed

Sullivan, Eva; Bensman, Joyce; Lou, Mimi; Agnello, Melissa; Shriner, Kimberly; Wong-Beringer, Annie

2014-01-01

To determine the differential association of host characteristics, antimicrobial resistance, and type III secretion system virulence of Pseudomonas aeruginosa isolates with respiratory syndromes in hospitalized adult patients. Retrospective, cohort study. Community teaching hospital. Two hundred eighteen consecutive adult patients with respiratory culture positive for P. aeruginosa between January 2005 to January 2010. Medical charts were reviewed to obtain demographic, laboratory, radiographic, and clinical information. Isolates were assayed by polymerase chain reaction for genes encoding the type III secretion system effectors (ExoU, ExoS, and PcrV) and for strain relatedness using randomly amplified polymorphic DNA analysis. Levofloxacin susceptibility was determined by broth microdilution. Patients were grouped by colonization, bronchitis, or pneumonia and were compared for differential risk of developing the clinical syndrome with respect to host and microbial characteristics. Half of the study cohort (54%, 117 of 218) had pneumonia, 32% (70 of 218) had bronchitis, and 14% (31 of 218) had colonization; in-hospital mortality was 35%, 11%, and 0%, respectively. Host factors strongly associated with pneumonia development were residence in long-term care facility, healthcare-associated acquisition of P. aeruginosa, higher Acute Physiology and Chronic Health Evaluation II score, presence of enteral feeding tube, mechanical ventilation, and recent history of pneumonia. Fluoroquinolone-resistant (57% vs 34%, 16%; p < 0.0001) and multidrug-resistant (36% vs 26%, 7%; p = 0.0045) strains were more likely to cause pneumonia than bronchitis or colonization, respectively. Analysis of host and microbial factors in a multivariate regression model yielded the combined traits of fluoroquinolone resistance and gene encoding the type III secretion system ExoU effector in P. aeruginosa as the single most significant predictor of pneumonia development. These results suggest that fluoroquinolone-resistant phenotype in a type III secretion system exoU strain background contributes toward the pathogenesis of P. aeruginosa in pneumonia.

Therapeutic implications of Epstein–Barr virus infection for the treatment of nasopharyngeal carcinoma

PubMed Central

Hutajulu, Susanna Hilda; Kurnianda, Johan; Tan, I Bing; Middeldorp, Jaap M

2014-01-01

Nasopharyngeal carcinoma (NPC) is highly endemic in certain regions including the People’s Republic of China and Southeast Asia. Its etiology is unique and multifactorial, involving genetic background, epigenetic, and environment factors, including Epstein–Barr virus (EBV) infection. The presence of EBV in all tumor cells, aberrant pattern of antibodies against EBV antigens in patient sera, and elevated viral DNA in patient circulation as well as nasopharyngeal site underline the role of EBV during NPC development. In NPC tumors, EBV expresses latency type II, where three EBV-encoded proteins, Epstein–Barr nuclear antigen 1, latent membrane protein 1 and 2 (LMP1, 2), are expressed along with BamH1-A rightward reading frame 1, Epstein–Barr virus-encoded small nuclear RNAs, and BamH1-A rightward transcripts. Among all encoded proteins, LMP1 plays a central role in the propagation of NPC. Standard treatment of NPC consists of radiotherapy with or without chemotherapy for early stage, concurrent chemoradiotherapy in locally advanced tumors, and palliative systemic chemotherapy in metastatic disease. However, this standard care has limitations, allowing recurrences and disease progression in a certain proportion of cases. Although the pathophysiological link and molecular process of EBV-induced oncogenesis are not fully understood, therapeutic approaches targeting the virus may increase the cure rate and add clinical benefit. The promising results of early phase clinical trials on EBV-specific immunotherapy, epigenetic therapy, and treatment with viral lytic induction offer new options for treating NPC. PMID:25228810

Role of fimV in type II secretion system-dependent protein secretion of Pseudomonas aeruginosa on solid medium.

PubMed

Michel, Gérard P F; Aguzzi, Anthony; Ball, Geneviève; Soscia, Chantal; Bleves, Sophie; Voulhoux, Romé

2011-07-01

Although classical type II secretion systems (T2SSs) are widely present in Gram-negative bacteria, atypical T2SSs can be found in some species. In Pseudomonas aeruginosa, in addition to the classical T2SS Xcp, it was reported that two genes, xphA and xqhA, located outside the xcp locus were organized in an operon (PaQa) which encodes the orphan PaQa subunit. This subunit is able to associate with other components of the classical Xcp machinery to form a functional hybrid T2SS. In the present study, using a transcriptional lacZ fusion, we found that the PaQa operon was more efficiently expressed (i) on solid LB agar than in liquid LB medium, (ii) at 25 °C than at 37 °C and (iii) at an early stage of growth. These results suggested an adaptation of the hybrid system to particular environmental conditions. Transposon mutagenesis led to the finding that vfr and fimV genes are required for optimal expression of the orphan PaQa operon in the defined growth conditions used. Using an original culturing device designed to monitor secretion on solid medium, the ring-plate system, we found that T2SS-dependent secretion of exoproteins, namely the elastase LasB, was affected in a fimV deletion mutant. Our findings led to the discovery of an interplay between FimV and the global regulator Vfr triggering the modulation of the level of Vfr and consequently the modulation of T2SS-dependent secretion on solid medium.

Integration-deficient lentivectors: an effective strategy to purify and differentiate human embryonic stem cell-derived hepatic progenitors.

PubMed

Yang, Guanghua; Si-Tayeb, Karim; Corbineau, Sébastien; Vernet, Rémi; Gayon, Régis; Dianat, Noushin; Martinet, Clémence; Clay, Denis; Goulinet-Mainot, Sylvie; Tachdjian, Gérard; Tachdjian, Gérard; Burks, Deborah; Vallier, Ludovic; Bouillé, Pascale; Dubart-Kupperschmitt, Anne; Weber, Anne

2013-07-19

Human pluripotent stem cells (hPSCs) hold great promise for applications in regenerative medicine. However, the safety of cell therapy using differentiated hPSC derivatives must be improved through methods that will permit the transplantation of homogenous populations of a specific cell type. To date, purification of progenitors and mature cells generated from either embryonic or induced pluripotent stem cells remains challenging with use of conventional methods. We used lentivectors encoding green fluorescent protein (GFP) driven by the liver-specific apoliprotein A-II (APOA-II) promoter to purify human hepatic progenitors. We evaluated both integrating and integration-defective lentivectors in combination with an HIV integrase inhibitor. A human embryonic stem cell line was differentiated into hepatic progenitors using a chemically defined protocol. Subsequently, cells were transduced and sorted at day 16 of differentiation to obtain a cell population enriched in hepatic progenitor cells. After sorting, more than 99% of these APOA-II-GFP-positive cells expressed hepatoblast markers such as α-fetoprotein and cytokeratin 19. When further cultured for 16 days, these cells underwent differentiation into more mature cells and exhibited hepatocyte properties such as albumin secretion. Moreover, they were devoid of vector DNA integration. We have developed an effective strategy to purify human hepatic cells from cultures of differentiating hPSCs, producing a novel tool that could be used not only for cell therapy but also for in vitro applications such as drug screening. The present strategy should also be suitable for the purification of a broad range of cell types derived from either pluripotent or adult stem cells.

Differential gene expression in Staphylococcus aureus exposed to Orange II and Sudan III azo dyes

PubMed Central

Pan, Hongmiao; Xu, Joshua; Kweon, Oh-Gew; Zou, Wen; Feng, Jinhui; He, Gui-Xin; Cerniglia, Carl E.

2018-01-01

We previously demonstrated the effects of azo dyes and their reduction metabolites on bacterial cell growth and cell viability. In this report, the effects of Orange II and Sudan III on gene expression profiling in Staphylococcus aureus ATCC BAA 1556 were analyzed using microarray and quantitative RT-PCR technology. Upon exposure to 6 μg/ml Orange II for 18 h, 21 genes were found to be differently expressed. Among them, 8 and 13 genes were up- and down-regulated, respectively. Most proteins encoded by these differentially expressed genes involve stress response caused by drug metabolism, oxidation, and alkaline shock indicating that S. aureus could adapt to Orange II exposure through a balance between up and down regulated gene expression. Whereas, after exposure to 6 μg/ml Sudan III for 18 h, 57 genes were differentially expressed. In which, 51 genes were up-regulated and 6 were down-regulated. Most proteins encoded by these differentially expressed genes involve in cell wall/membrane biogenesis and biosynthesis, nutrient uptake, transport and metabolite, and stress response, suggesting that Sudan III damages the bacterial cell wall or/and membrane due to binding of the dye. Further analysis indicated that all differentially expressed genes encoded membrane proteins were up-regulated and most of them serve as transporters. The result suggested that these genes might contribute to survival, persistence and growth in the presence of Sudan III. Only one gene msrA, which plays an important role in oxidative stress resistance, was found to be down-regulated after exposure to both Orange II and Sudan III. The present results suggested that both these two azo dyes can cause stress in S. aureus and the response of the bacterium to the stress is mainly related to characteristics of the azo dyes. PMID:25720844

Transplantation of autoimmune regulator-encoding bone marrow cells delays the onset of experimental autoimmune encephalomyelitis.

PubMed

Ko, Hyun-Ja; Kinkel, Sarah A; Hubert, François-Xavier; Nasa, Zeyad; Chan, James; Siatskas, Christopher; Hirubalan, Premila; Toh, Ban-Hock; Scott, Hamish S; Alderuccio, Frank

2010-12-01

The autoimmune regulator (AIRE) promotes "promiscuous" expression of tissue-restricted antigens (TRA) in thymic medullary epithelial cells to facilitate thymic deletion of autoreactive T-cells. Here, we show that AIRE-deficient mice showed an earlier development of myelin oligonucleotide glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). To determine the outcome of ectopic Aire expression, we used a retroviral transduction system to over-express Aire in vitro, in cell lines and in bone marrow (BM). In the cell lines that included those of thymic medullary and dendritic cell origin, ectopically expressed Aire variably promoted expression of TRA including Mog and Ins2 (proII) autoantigens associated, respectively, with the autoimmune diseases multiple sclerosis and type 1 diabetes. BM chimeras generated from BM transduced with a retrovirus encoding Aire displayed elevated levels of Mog and Ins2 expression in thymus and spleen. Following induction of EAE with MOG(35-55), transplanted mice displayed significant delay in the onset of EAE compared with control mice. To our knowledge, this is the first example showing that in vivo ectopic expression of AIRE can modulate TRA expression and alter autoimmune disease development. Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Engineered Biosynthesis of a Novel Amidated Polyketide, Using the Malonamyl-Specific Initiation Module from the Oxytetracycline Polyketide Synthase

PubMed Central

Zhang, Wenjun; Ames, Brian D.; Tsai, Shiou-Chuan; Tang, Yi

2006-01-01

Tetracyclines are aromatic polyketides biosynthesized by bacterial type II polyketide synthases (PKSs). Understanding the biochemistry of tetracycline PKSs is an important step toward the rational and combinatorial manipulation of tetracycline biosynthesis. To this end, we have sequenced the gene cluster of oxytetracycline (oxy and otc genes) PKS genes from Streptomyces rimosus. Sequence analysis revealed a total of 21 genes between the otrA and otrB resistance genes. We hypothesized that an amidotransferase, OxyD, synthesizes the malonamate starter unit that is a universal building block for tetracycline compounds. In vivo reconstitution using strain CH999 revealed that the minimal PKS and OxyD are necessary and sufficient for the biosynthesis of amidated polyketides. A novel alkaloid (WJ35, or compound 2) was synthesized as the major product when the oxy-encoded minimal PKS, the C-9 ketoreductase (OxyJ), and OxyD were coexpressed in CH999. WJ35 is an isoquinolone compound derived from an amidated decaketide backbone and cyclized with novel regioselectivity. The expression of OxyD with a heterologous minimal PKS did not afford similarly amidated polyketides, suggesting that the oxy-encoded minimal PKS possesses novel starter unit specificity. PMID:16597959

Activation of Exogenous Fatty Acids to Acyl-Acyl Carrier Protein Cannot Bypass FabI Inhibition in Neisseria*

PubMed Central

Yao, Jiangwei; Bruhn, David F.; Frank, Matthew W.; Lee, Richard E.; Rock, Charles O.

2016-01-01

Neisseria is a Gram-negative pathogen with phospholipids composed of straight chain saturated and monounsaturated fatty acids, the ability to incorporate exogenous fatty acids, and lipopolysaccharides that are not essential. The FabI inhibitor, AFN-1252, was deployed as a chemical biology tool to determine whether Neisseria can bypass the inhibition of fatty acid synthesis by incorporating exogenous fatty acids. Neisseria encodes a functional FabI that was potently inhibited by AFN-1252. AFN-1252 caused a dose-dependent inhibition of fatty acid synthesis in growing Neisseria, a delayed inhibition of growth phenotype, and minimal inhibition of DNA, RNA, and protein synthesis, showing that its mode of action is through inhibiting fatty acid synthesis. Isotopic fatty acid labeling experiments showed that Neisseria encodes the ability to incorporate exogenous fatty acids into its phospholipids by an acyl-acyl carrier protein-dependent pathway. However, AFN-1252 remained an effective antibacterial when Neisseria were supplemented with exogenous fatty acids. These results demonstrate that extracellular fatty acids are activated by an acyl-acyl carrier protein synthetase (AasN) and validate type II fatty acid synthesis (FabI) as a therapeutic target against Neisseria. PMID:26567338

Cache directory lookup reader set encoding for partial cache line speculation support

DOEpatents

Gara, Alan; Ohmacht, Martin

2014-10-21

In a multiprocessor system, with conflict checking implemented in a directory lookup of a shared cache memory, a reader set encoding permits dynamic recordation of read accesses. The reader set encoding includes an indication of a portion of a line read, for instance by indicating boundaries of read accesses. Different encodings may apply to different types of speculative execution.

Laboratory and Genetic Investigation of Mutations Accounting for Congenital Fibrinogen Disorders.

PubMed

Neerman-Arbez, Marguerite; de Moerloose, Philippe; Casini, Alessandro

2016-06-01

Congenital fibrinogen disorders are classified into two types of plasma fibrinogen defects: type I (quantitative fibrinogen deficiencies), that is, hypofibrinogenemia or afibrinogenemia, in which there are low or absent plasma fibrinogen antigen levels, respectively, and type II (qualitative fibrinogen deficiencies), that is, dysfibrinogenemia or hypodysfibrinogenemia, in which there are normal or reduced antigen levels associated with disproportionately low functional activity. These disorders are caused by mutations in the three fibrinogen-encoding genes FGA, FGB, and FGG. Afibrinogenemia is associated with mild to severe bleeding, whereas hypofibrinogenemia is often asymptomatic. For these quantitative disorders, the majority of mutations prevent protein production. However, in some cases, missense or late-truncating nonsense mutations allow synthesis of the mutant fibrinogen chain, but intracellular fibrinogen assembly and/or secretion are impaired. Qualitative fibrinogen disorders are associated with bleeding, thrombosis, or both thrombosis and bleeding, but many dysfibrinogenemias are asymptomatic. The majority of cases are caused by heterozygous missense mutations. Here, we review the laboratory and genetic diagnosis of fibrinogen gene anomalies with an updated discussion of causative mutations identified. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

SAXS fingerprints of aldehyde dehydrogenase oligomers.

PubMed

Tanner, John J

2015-12-01

Enzymes of the aldehyde dehydrogenase (ALDH) superfamily catalyze the nicotinamide adenine dinucleotide-dependent oxidation of aldehydes to carboxylic acids. ALDHs are important in detoxification of aldehydes, amino acid metabolism, embryogenesis and development, neurotransmission, oxidative stress, and cancer. Mutations in genes encoding ALDHs cause metabolic disorders, including alcohol flush reaction (ALDH2), Sjögren-Larsson syndrome (ALDH3A2), hyperprolinemia type II (ALDH4A1), γ-hydroxybutyric aciduria (ALDH5A1), methylmalonic aciduria (ALDH6A1), pyridoxine dependent epilepsy (ALDH7A1), and hyperammonemia (ALDH18A1). We previously reported crystal structures and small-angle X-ray scattering (SAXS) analyses of ALDHs exhibiting dimeric, tetrameric, and hexameric oligomeric states (Luo et al., Biochemistry 54 (2015) 5513-5522; Luo et al., J. Mol. Biol. 425 (2013) 3106-3120). Herein I provide the SAXS curves, radii of gyration, and distance distribution functions for the three types of ALDH oligomer. The SAXS curves and associated analysis provide diagnostic fingerprints that allow rapid identification of the type of ALDH oligomer that is present in solution. The data sets provided here serve as a benchmark for characterizing oligomerization of ALDHs.

Novel encoding methods for DNA-templated chemical libraries.

PubMed

Li, Gang; Zheng, Wenlu; Liu, Ying; Li, Xiaoyu

2015-06-01

Among various types of DNA-encoded chemical libraries, DNA-templated library takes advantage of the sequence-specificity of DNA hybridization, enabling not only highly effective DNA-templated chemical reactions, but also high fidelity in library encoding. This brief review summarizes recent advances that have been made on the encoding strategies for DNA-templated libraries, and it also highlights their respective advantages and limitations for the preparation of DNA-encoded libraries. Copyright © 2015 Elsevier Ltd. All rights reserved.

Ly49 Receptors: Innate and Adaptive Immune Paradigms

PubMed Central

Rahim, Mir Munir A.; Tu, Megan M.; Mahmoud, Ahmad Bakur; Wight, Andrew; Abou-Samra, Elias; Lima, Patricia D. A.; Makrigiannis, Andrew P.

2014-01-01

The Ly49 receptors are type II C-type lectin-like membrane glycoproteins encoded by a family of highly polymorphic and polygenic genes within the mouse natural killer (NK) gene complex. This gene family is designated Klra, and includes genes that encode both inhibitory and activating Ly49 receptors in mice. Ly49 receptors recognize class I major histocompatibility complex-I (MHC-I) and MHC-I-like proteins on normal as well as altered cells. Their functional homologs in humans are the killer cell immunoglobulin-like receptors, which recognize HLA class I molecules as ligands. Classically, Ly49 receptors are described as being expressed on both the developing and mature NK cells. The inhibitory Ly49 receptors are involved in NK cell education, a process in which NK cells acquire function and tolerance toward cells that express “self-MHC-I.” On the other hand, the activating Ly49 receptors recognize altered cells expressing activating ligands. New evidence shows a broader Ly49 expression pattern on both innate and adaptive immune cells. Ly49 receptors have been described on multiple NK cell subsets, such as uterine NK and memory NK cells, as well as NKT cells, dendritic cells, plasmacytoid dendritic cells, macrophages, neutrophils, and cells of the adaptive immune system, such as activated T cells and regulatory CD8+ T cells. In this review, we discuss the expression pattern and proposed functions of Ly49 receptors on various immune cells and their contribution to immunity. PMID:24765094

Interrelations of secondary structure stability and DNA-binding affinity in the bacteriophage SPO1-encoded type II DNA-binding protein TF1.

PubMed

Andera, L; Spangler, C J; Galeone, A; Mayol, L; Geiduschek, E P

1994-02-11

TF1, a homodimeric DNA-binding and -bending protein with a preference for hydroxymethyluracil-containing DNA is the Bacillus subtilis-encoded homolog of the bacterial HU proteins and of the E. coli integration host factor. A temperature-sensitive mutation at amino acid 25 of TF1 (L25-->A) and two intragenic second site revertants at amino acids 15 (E15-->G) and 32 (L32-->I) were previously identified and their effects on virus development were examined. The DNA-binding properties of these proteins and the thermal stability of their secondary structures have now been analyzed. Amino acids 15 and 32 are far removed from the putative DNA-binding domains of TF1 but changes there exert striking effects on DNA affinity that correlate with effects on structure. The double mutant protein TF1-G15I32 binds to a preferred site in hydroxymethyluracil-containing DNA 40 times more tightly, denatures at higher temperature (delta tm = 21 degrees C), and also exchanges subunits much more slowly than does the wild-type protein. The L25-->A mutation makes TF1 secondary structure and DNA-binding highly salt concentration-dependent. The E15-->G mutation partly suppresses this effect: secondary structure of TF1-A25G15 is restored at 21 degrees C by 1 M NaCl or, at low NaCl concentration, by binding to DNA.

The effects of divided attention on encoding processes under incidental and intentional learning instructions: underlying mechanisms?

PubMed

Naveh-Benjamin, Moshe; Guez, Jonathan; Hara, Yoko; Brubaker, Matthew S; Lowenschuss-Erlich, Iris

2014-01-01

Divided attention (DA) at encoding has been shown to significantly disrupt later memory for the studied information. However, what type of processing gets disrupted during DA remains unresolved. In this study, we assessed the degree to which strategic effortful processes are affected under DA by comparing the effects of DA at encoding under intentional and pure incidental learning instructions. In three experiments, participants studied list of words or word pairs under either full or divided attention. Results of three experiments, which used different methodologies, converged to show that the effects of DA at encoding reduce memory performance to the same degree under incidental and intentional learning. Secondary task performance indicated that encoding under intentional learning instructions was more effortful than under incidental learning instructions. In addition, the results indicated enhanced attention to the initial appearance of the words under both types of learning instructions. Results are interpreted to imply that other processes, rather than only strategic effortful ones, might be affected by DA at encoding.

The path to memory is guided by strategy: distinct networks are engaged in associative encoding under visual and verbal strategy and influence memory performance in healthy and impaired individuals

PubMed Central

Hales, J. B.; Brewer, J. B.

2018-01-01

Given the diversity of stimuli encountered in daily life, a variety of strategies must be used for learning new information. Relating and encoding visual and verbal stimuli into memory has been probed using various tasks and stimulus-types. Engagement of specific subsequent memory and cortical processing regions depends on the stimulus modality of studied material; however, it remains unclear whether different encoding strategies similarly influence regional activity when stimulus-type is held constant. In this study, subjects encoded object pairs using a visual or verbal associative strategy during functional magnetic resonance imaging (fMRI), and subsequent memory was assessed for pairs encoded under each strategy. Each strategy elicited distinct regional processing and subsequent memory effects: middle / superior frontal, lateral parietal, and lateral occipital for visually-associated pairs and inferior frontal, medial frontal, and medial occipital for verbally-associated pairs. This regional selectivity mimics the effects of stimulus modality, suggesting that cortical involvement in associative encoding is driven by strategy, and not simply by stimulus-type. The clinical relevance of these findings, probed in two patients with recent aphasic strokes, suggest that training with strategies utilizing unaffected cortical regions might improve memory ability in patients with brain damage. PMID:22390467

Plasmids encoding therapeutic agents

DOEpatents

Keener, William K [Idaho Falls, ID

2007-08-07

Plasmids encoding anti-HIV and anti-anthrax therapeutic agents are disclosed. Plasmid pWKK-500 encodes a fusion protein containing DP178 as a targeting moiety, the ricin A chain, an HIV protease cleavable linker, and a truncated ricin B chain. N-terminal extensions of the fusion protein include the maltose binding protein and a Factor Xa protease site. C-terminal extensions include a hydrophobic linker, an L domain motif peptide, a KDEL ER retention signal, another Factor Xa protease site, an out-of-frame buforin II coding sequence, the lacZ.alpha. peptide, and a polyhistidine tag. More than twenty derivatives of plasmid pWKK-500 are described. Plasmids pWKK-700 and pWKK-800 are similar to pWKK-500 wherein the DP178-encoding sequence is substituted by RANTES- and SDF-1-encoding sequences, respectively. Plasmid pWKK-900 is similar to pWKK-500 wherein the HIV protease cleavable linker is substituted by a lethal factor (LF) peptide-cleavable linker.

Identification of a Xylogalacturonan Xylosyltransferase Involved in Pectin Biosynthesis in Arabidopsis

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

Pauly, Markus; Sorensen, Susanne Oxenboll; Harholt, Jesper

2009-08-19

Xylogalacturonan (XGA) is a class of pectic polysaccharide found in plant cell walls. The Arabidopsis thaliana locus At5g33290 encodes a predicted Type II membrane protein, and insertion mutants of the At5g33290 locus had decreased cell wall xylose. Immunological studies, enzymatic extraction of polysaccharides, monosaccharide linkage analysis, and oligosaccharide mass profiling were employed to identify the affected cell wall polymer. Pectic XGA was reduced to much lower levels in mutant than in wild-type leaves, indicating a role of At5g33290 in XGA biosynthesis. The mutated gene was designated xylogalacturonan deficient1 (xgd1). Transformation of the xgd1-1 mutant with the wild-type gene restored XGAmore » to wild-type levels. XGD1 protein heterologously expressed in Nicotiana benthamiana catalyzed the transfer of xylose from UDP-xylose onto oligogalacturonides and endogenous acceptors. The products formed could be hydrolyzed with an XGA-specific hydrolase. These results confirm that the XGD1 protein is a XGA xylosyltransferase. The protein was shown by expression of a fluorescent fusion protein in N. benthamiana to be localized in the Golgi vesicles as expected for a glycosyltransferase involved in pectin biosynthesis.« less

RNA Polymerase II Stalling Promotes Nucleosome Occlusion and pTEFb Recruitment to Drive Immortalization by Epstein-Barr Virus

PubMed Central

Palermo, Richard D.; Webb, Helen M.; West, Michelle J.

2011-01-01

Epstein-Barr virus (EBV) immortalizes resting B-cells and is a key etiologic agent in the development of numerous cancers. The essential EBV-encoded protein EBNA 2 activates the viral C promoter (Cp) producing a message of ∼120 kb that is differentially spliced to encode all EBNAs required for immortalization. We have previously shown that EBNA 2-activated transcription is dependent on the activity of the RNA polymerase II (pol II) C-terminal domain (CTD) kinase pTEFb (CDK9/cyclin T1). We now demonstrate that Cp, in contrast to two shorter EBNA 2-activated viral genes (LMP 1 and 2A), displays high levels of promoter-proximally stalled pol II despite being constitutively active. Consistent with pol II stalling, we detect considerable pausing complex (NELF/DSIF) association with Cp. Significantly, we observe substantial Cp-specific pTEFb recruitment that stimulates high-level pol II CTD serine 2 phosphorylation at distal regions (up to +75 kb), promoting elongation. We reveal that Cp-specific pol II accumulation is directed by DNA sequences unfavourable for nucleosome assembly that increase TBP access and pol II recruitment. Stalled pol II then maintains Cp nucleosome depletion. Our data indicate that pTEFb is recruited to Cp by the bromodomain protein Brd4, with polymerase stalling facilitating stable association of pTEFb. The Brd4 inhibitor JQ1 and the pTEFb inhibitors DRB and Flavopiridol significantly reduce Cp, but not LMP1 transcript production indicating that Brd4 and pTEFb are required for Cp transcription. Taken together our data indicate that pol II stalling at Cp promotes transcription of essential immortalizing genes during EBV infection by (i) preventing promoter-proximal nucleosome assembly and ii) necessitating the recruitment of pTEFb thereby maintaining serine 2 CTD phosphorylation at distal regions. PMID:22046134

Expression of a Truncated ATHB17 Protein in Maize Increases Ear Weight at Silking

PubMed Central

Creelman, Robert A.; Griffith, Cara; Ahrens, Jeffrey E.; Taylor, J. Philip; Murphy, Lesley R.; Manjunath, Siva; Thompson, Rebecca L.; Lingard, Matthew J.; Back, Stephanie L.; Larue, Huachun; Brayton, Bonnie R.; Burek, Amanda J.; Tiwari, Shiv; Adam, Luc; Morrell, James A.; Caldo, Rico A.; Huai, Qing; Kouadio, Jean-Louis K.; Kuehn, Rosemarie; Sant, Anagha M.; Wingbermuehle, William J.; Sala, Rodrigo; Foster, Matt; Kinser, Josh D.; Mohanty, Radha; Jiang, Dongming; Ziegler, Todd E.; Huang, Mingya G.; Kuriakose, Saritha V.; Skottke, Kyle; Repetti, Peter P.; Reuber, T. Lynne; Ruff, Thomas G.; Petracek, Marie E.; Loida, Paul J.

2014-01-01

ATHB17 (AT2G01430) is an Arabidopsis gene encoding a member of the α-subclass of the homeodomain leucine zipper class II (HD-Zip II) family of transcription factors. The ATHB17 monomer contains four domains common to all class II HD-Zip proteins: a putative repression domain adjacent to a homeodomain, leucine zipper, and carboxy terminal domain. However, it also possesses a unique N-terminus not present in other members of the family. In this study we demonstrate that the unique 73 amino acid N-terminus is involved in regulation of cellular localization of ATHB17. The ATHB17 protein is shown to function as a transcriptional repressor and an EAR-like motif is identified within the putative repression domain of ATHB17. Transformation of maize with an ATHB17 expression construct leads to the expression of ATHB17Δ113, a truncated protein lacking the first 113 amino acids which encodes a significant portion of the repression domain. Because ATHB17Δ113 lacks the repression domain, the protein cannot directly affect the transcription of its target genes. ATHB17Δ113 can homodimerize, form heterodimers with maize endogenous HD-Zip II proteins, and bind to target DNA sequences; thus, ATHB17Δ113 may interfere with HD-Zip II mediated transcriptional activity via a dominant negative mechanism. We provide evidence that maize HD-Zip II proteins function as transcriptional repressors and that ATHB17Δ113 relieves this HD-Zip II mediated transcriptional repression activity. Expression of ATHB17Δ113 in maize leads to increased ear size at silking and, therefore, may enhance sink potential. We hypothesize that this phenotype could be a result of modulation of endogenous HD-Zip II pathways in maize. PMID:24736658

Expression of a truncated ATHB17 protein in maize increases ear weight at silking.

PubMed

Rice, Elena A; Khandelwal, Abha; Creelman, Robert A; Griffith, Cara; Ahrens, Jeffrey E; Taylor, J Philip; Murphy, Lesley R; Manjunath, Siva; Thompson, Rebecca L; Lingard, Matthew J; Back, Stephanie L; Larue, Huachun; Brayton, Bonnie R; Burek, Amanda J; Tiwari, Shiv; Adam, Luc; Morrell, James A; Caldo, Rico A; Huai, Qing; Kouadio, Jean-Louis K; Kuehn, Rosemarie; Sant, Anagha M; Wingbermuehle, William J; Sala, Rodrigo; Foster, Matt; Kinser, Josh D; Mohanty, Radha; Jiang, Dongming; Ziegler, Todd E; Huang, Mingya G; Kuriakose, Saritha V; Skottke, Kyle; Repetti, Peter P; Reuber, T Lynne; Ruff, Thomas G; Petracek, Marie E; Loida, Paul J

2014-01-01

ATHB17 (AT2G01430) is an Arabidopsis gene encoding a member of the α-subclass of the homeodomain leucine zipper class II (HD-Zip II) family of transcription factors. The ATHB17 monomer contains four domains common to all class II HD-Zip proteins: a putative repression domain adjacent to a homeodomain, leucine zipper, and carboxy terminal domain. However, it also possesses a unique N-terminus not present in other members of the family. In this study we demonstrate that the unique 73 amino acid N-terminus is involved in regulation of cellular localization of ATHB17. The ATHB17 protein is shown to function as a transcriptional repressor and an EAR-like motif is identified within the putative repression domain of ATHB17. Transformation of maize with an ATHB17 expression construct leads to the expression of ATHB17Δ113, a truncated protein lacking the first 113 amino acids which encodes a significant portion of the repression domain. Because ATHB17Δ113 lacks the repression domain, the protein cannot directly affect the transcription of its target genes. ATHB17Δ113 can homodimerize, form heterodimers with maize endogenous HD-Zip II proteins, and bind to target DNA sequences; thus, ATHB17Δ113 may interfere with HD-Zip II mediated transcriptional activity via a dominant negative mechanism. We provide evidence that maize HD-Zip II proteins function as transcriptional repressors and that ATHB17Δ113 relieves this HD-Zip II mediated transcriptional repression activity. Expression of ATHB17Δ113 in maize leads to increased ear size at silking and, therefore, may enhance sink potential. We hypothesize that this phenotype could be a result of modulation of endogenous HD-Zip II pathways in maize.

Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing.

PubMed

Mohr, Sabine; Ghanem, Eman; Smith, Whitney; Sheeter, Dennis; Qin, Yidan; King, Olga; Polioudakis, Damon; Iyer, Vishwanath R; Hunicke-Smith, Scott; Swamy, Sajani; Kuersten, Scott; Lambowitz, Alan M

2013-07-01

Mobile group II introns encode reverse transcriptases (RTs) that function in intron mobility ("retrohoming") by a process that requires reverse transcription of a highly structured, 2-2.5-kb intron RNA with high processivity and fidelity. Although the latter properties are potentially useful for applications in cDNA synthesis and next-generation RNA sequencing (RNA-seq), group II intron RTs have been difficult to purify free of the intron RNA, and their utility as research tools has not been investigated systematically. Here, we developed general methods for the high-level expression and purification of group II intron-encoded RTs as fusion proteins with a rigidly linked, noncleavable solubility tag, and we applied them to group II intron RTs from bacterial thermophiles. We thus obtained thermostable group II intron RT fusion proteins that have higher processivity, fidelity, and thermostability than retroviral RTs, synthesize cDNAs at temperatures up to 81°C, and have significant advantages for qRT-PCR, capillary electrophoresis for RNA-structure mapping, and next-generation RNA sequencing. Further, we find that group II intron RTs differ from the retroviral enzymes in template switching with minimal base-pairing to the 3' ends of new RNA templates, making it possible to efficiently and seamlessly link adaptors containing PCR-primer binding sites to cDNA ends without an RNA ligase step. This novel template-switching activity enables facile and less biased cloning of nonpolyadenylated RNAs, such as miRNAs or protein-bound RNA fragments. Our findings demonstrate novel biochemical activities and inherent advantages of group II intron RTs for research, biotechnological, and diagnostic methods, with potentially wide applications.

Ensemble codes involving hippocampal neurons are at risk during delayed performance tests.

PubMed

Hampson, R E; Deadwyler, S A

1996-11-26

Multielectrode recording techniques were used to record ensemble activity from 10 to 16 simultaneously active CA1 and CA3 neurons in the rat hippocampus during performance of a spatial delayed-nonmatch-to-sample task. Extracted sources of variance were used to assess the nature of two different types of errors that accounted for 30% of total trials. The two types of errors included ensemble "miscodes" of sample phase information and errors associated with delay-dependent corruption or disappearance of sample information at the time of the nonmatch response. Statistical assessment of trial sequences and associated "strength" of hippocampal ensemble codes revealed that miscoded error trials always followed delay-dependent error trials in which encoding was "weak," indicating that the two types of errors were "linked." It was determined that the occurrence of weakly encoded, delay-dependent error trials initiated an ensemble encoding "strategy" that increased the chances of being correct on the next trial and avoided the occurrence of further delay-dependent errors. Unexpectedly, the strategy involved "strongly" encoding response position information from the prior (delay-dependent) error trial and carrying it forward to the sample phase of the next trial. This produced a miscode type error on trials in which the "carried over" information obliterated encoding of the sample phase response on the next trial. Application of this strategy, irrespective of outcome, was sufficient to reorient the animal to the proper between trial sequence of response contingencies (nonmatch-to-sample) and boost performance to 73% correct on subsequent trials. The capacity for ensemble analyses of strength of information encoding combined with statistical assessment of trial sequences therefore provided unique insight into the "dynamic" nature of the role hippocampus plays in delay type memory tasks.

Effects of exogenous pyoverdines on Fe availability and their impacts on Mn(II) oxidation by Pseudomonas putida GB-1

PubMed Central

Lee, Sung-Woo; Parker, Dorothy L.; Geszvain, Kati; Tebo, Bradley M.

2014-01-01

Pseudomonas putida GB-1 is a Mn(II)-oxidizing bacterium that produces pyoverdine-type siderophores (PVDs), which facilitate the uptake of Fe(III) but also influence MnO2 formation. Recently, a non-ribosomal peptide synthetase mutant that does not synthesize PVD was described. Here we identified a gene encoding the PVDGB-1 (PVD produced by strain GB-1) uptake receptor (PputGB1_4082) of strain GB-1 and confirmed its function by in-frame mutagenesis. Growth and other physiological responses of these two mutants and of wild type were compared during cultivation in the presence of three chemically distinct sets of PVDs (siderotypes n°1, n°2, and n°4) derived from various pseudomonads. Under iron-limiting conditions, Fe(III) complexes of various siderotype n°1 PVDs (including PVDGB-1) allowed growth of wild type and the synthetase mutant, but not the receptor mutant, confirming that iron uptake with any tested siderotype n°1 PVD depended on PputGB1_4082. Fe(III) complexes of a siderotype n°2 PVD were not utilized by any strain and strongly induced PVD synthesis. In contrast, Fe(III) complexes of siderotype n°4 PVDs promoted the growth of all three strains and did not induce PVD synthesis by the wild type, implying these complexes were utilized for iron uptake independent of PputGB1_4082. These differing properties of the three PVD types provided a way to differentiate between effects on MnO2 formation that resulted from iron limitation and others that required participation of the PVDGB-1 receptor. Specifically, MnO2 production was inhibited by siderotype n°1 but not n°4 PVDs indicating PVD synthesis or PputGB1_4082 involvement rather than iron-limitation caused the inhibition. In contrast, iron limitation was sufficient to explain the inhibition of Mn(II) oxidation by siderotype n°2 PVDs. Collectively, our results provide insight into how competition for iron via siderophores influences growth, iron nutrition and MnO2 formation in more complex environmental systems. PMID:25009534

The euryhaline yeast Debaryomyces hansenii has two catalase genes encoding enzymes with differential activity profile.

PubMed

Segal-Kischinevzky, Claudia; Rodarte-Murguía, Beatriz; Valdés-López, Victor; Mendoza-Hernández, Guillermo; González, Alicia; Alba-Lois, Luisa

2011-03-01

Debaryomyces hansenii is a spoilage yeast able to grow in a variety of ecological niches, from seawater to dairy products. Results presented in this article show that (i) D. hansenii has an inherent resistance to H2O2 which could be attributed to the fact that this yeast has a basal catalase activity which is several-fold higher than that observed in Saccharomyces cerevisiae under the same culture conditions, (ii) D. hansenii has two genes (DhCTA1 and DhCTT1) encoding two catalase isozymes with a differential enzymatic activity profile which is not strictly correlated with a differential expression profile of the encoding genes.

Mutations in FKBP10, which result in Bruck syndrome and recessive forms of osteogenesis imperfecta, inhibit the hydroxylation of telopeptide lysines in bone collagen

PubMed Central

Schwarze, Ulrike; Cundy, Tim; Pyott, Shawna M.; Christiansen, Helena E.; Hegde, Madhuri R.; Bank, Ruud A.; Pals, Gerard; Ankala, Arunkanth; Conneely, Karen; Seaver, Laurie; Yandow, Suzanne M.; Raney, Ellen; Babovic-Vuksanovic, Dusica; Stoler, Joan; Ben-Neriah, Ziva; Segel, Reeval; Lieberman, Sari; Siderius, Liesbeth; Al-Aqeel, Aida; Hannibal, Mark; Hudgins, Louanne; McPherson, Elizabeth; Clemens, Michele; Sussman, Michael D.; Steiner, Robert D.; Mahan, John; Smith, Rosemarie; Anyane-Yeboa, Kwame; Wynn, Julia; Chong, Karen; Uster, Tami; Aftimos, Salim; Sutton, V. Reid; Davis, Elaine C.; Kim, Lammy S.; Weis, Mary Ann; Eyre, David; Byers, Peter H.

2013-01-01

Although biallelic mutations in non-collagen genes account for <10% of individuals with osteogenesis imperfecta, the characterization of these genes has identified new pathways and potential interventions that could benefit even those with mutations in type I collagen genes. We identified mutations in FKBP10, which encodes the 65 kDa prolyl cis–trans isomerase, FKBP65, in 38 members of 21 families with OI. These include 10 families from the Samoan Islands who share a founder mutation. Of the mutations, three are missense; the remainder either introduce premature termination codons or create frameshifts both of which result in mRNA instability. In four families missense mutations result in loss of most of the protein. The clinical effects of these mutations are short stature, a high incidence of joint contractures at birth and progressive scoliosis and fractures, but there is remarkable variability in phenotype even within families. The loss of the activity of FKBP65 has several effects: type I procollagen secretion is slightly delayed, the stabilization of the intact trimer is incomplete and there is diminished hydroxylation of the telopeptide lysyl residues involved in intermolecular cross-link formation in bone. The phenotype overlaps with that seen with mutations in PLOD2 (Bruck syndrome II), which encodes LH2, the enzyme that hydroxylates the telopeptide lysyl residues. These findings define a set of genes, FKBP10, PLOD2 and SERPINH1, that act during procollagen maturation to contribute to molecular stability and post-translational modification of type I procollagen, without which bone mass and quality are abnormal and fractures and contractures result. PMID:22949511

Bioinformatic Analysis of Pathogenic Missense Mutations of Activin Receptor Like Kinase 1 Ectodomain

PubMed Central

Scotti, Claudia; Olivieri, Carla; Boeri, Laura; Canzonieri, Cecilia; Ornati, Federica; Buscarini, Elisabetta; Pagella, Fabio; Danesino, Cesare

2011-01-01

Activin A receptor, type II-like kinase 1 (also called ALK1), is a serine-threonine kinase predominantly expressed on endothelial cells surface. Mutations in its ACVRL1 encoding gene (12q11-14) cause type 2 Hereditary Haemorrhagic Telangiectasia (HHT2), an autosomal dominant multisystem vascular dysplasia. The study of the structural effects of mutations is crucial to understand their pathogenic mechanism. However, while an X-ray structure of ALK1 intracellular domain has recently become available (PDB ID: 3MY0), structure determination of ALK1 ectodomain (ALK1EC) has been elusive so far. We here describe the building of a homology model for ALK1EC, followed by an extensive bioinformatic analysis, based on a set of 38 methods, of the effect of missense mutations at the sequence and structural level. ALK1EC potential interaction mode with its ligand BMP9 was then predicted combining modelling and docking data. The calculated model of the ALK1EC allowed mapping and a preliminary characterization of HHT2 associated mutations. Major structural changes and loss of stability of the protein were predicted for several mutations, while others were found to interfere mainly with binding to BMP9 or other interactors, like Endoglin (CD105), whose encoding ENG gene (9q34) mutations are known to cause type 1 HHT. This study gives a preliminary insight into the potential structure of ALK1EC and into the structural effects of HHT2 associated mutations, which can be useful to predict the potential effect of each single mutation, to devise new biological experiments and to interpret the biological significance of new mutations, private mutations, or non-synonymous polymorphisms. PMID:22028876

Input-output relation and energy efficiency in the neuron with different spike threshold dynamics.

PubMed

Yi, Guo-Sheng; Wang, Jiang; Tsang, Kai-Ming; Wei, Xi-Le; Deng, Bin

2015-01-01

Neuron encodes and transmits information through generating sequences of output spikes, which is a high energy-consuming process. The spike is initiated when membrane depolarization reaches a threshold voltage. In many neurons, threshold is dynamic and depends on the rate of membrane depolarization (dV/dt) preceding a spike. Identifying the metabolic energy involved in neural coding and their relationship to threshold dynamic is critical to understanding neuronal function and evolution. Here, we use a modified Morris-Lecar model to investigate neuronal input-output property and energy efficiency associated with different spike threshold dynamics. We find that the neurons with dynamic threshold sensitive to dV/dt generate discontinuous frequency-current curve and type II phase response curve (PRC) through Hopf bifurcation, and weak noise could prohibit spiking when bifurcation just occurs. The threshold that is insensitive to dV/dt, instead, results in a continuous frequency-current curve, a type I PRC and a saddle-node on invariant circle bifurcation, and simultaneously weak noise cannot inhibit spiking. It is also shown that the bifurcation, frequency-current curve and PRC type associated with different threshold dynamics arise from the distinct subthreshold interactions of membrane currents. Further, we observe that the energy consumption of the neuron is related to its firing characteristics. The depolarization of spike threshold improves neuronal energy efficiency by reducing the overlap of Na(+) and K(+) currents during an action potential. The high energy efficiency is achieved at more depolarized spike threshold and high stimulus current. These results provide a fundamental biophysical connection that links spike threshold dynamics, input-output relation, energetics and spike initiation, which could contribute to uncover neural encoding mechanism.

Antennal transcriptome analysis of the piercing moth Oraesia emarginata (Lepidoptera: Noctuidae)

PubMed Central

Feng, Bo; Guo, Qianshuang; Zheng, Kaidi; Qin, Yuanxia; Du, Yongjun

2017-01-01

The piercing fruit moth Oraesia emarginata is an economically significant pest; however, our understanding of its olfactory mechanisms in infestation is limited. The present study conducted antennal transcriptome analysis of olfactory genes using real-time quantitative reverse transcription PCR analysis (RT-qPCR). We identified a total of 104 candidate chemosensory genes from several gene families, including 35 olfactory receptors (ORs), 41 odorant-binding proteins, 20 chemosensory proteins, 6 ionotropic receptors, and 2 sensory neuron membrane proteins. Seven candidate pheromone receptors (PRs) and 3 candidate pheromone-binding proteins (PBPs) for sex pheromone recognition were found. OemaOR29 and OemaPBP1 had the highest fragments per kb per million fragments (FPKM) values in all ORs and OBPs, respectively. Eighteen olfactory genes were upregulated in females, including 5 candidate PRs, and 20 olfactory genes were upregulated in males, including 2 candidate PRs (OemaOR29 and 4) and 2 PBPs (OemaPBP1 and 3). These genes may have roles in mediating sex-specific behaviors. Most candidate olfactory genes of sex pheromone recognition (except OemaOR29 and OemaPBP3) in O. emarginata were not clustered with those of studied noctuid species (type I pheromone). In addition, OemaOR29 was belonged to cluster PRIII, which comprise proteins that recognize type II pheromones instead of type I pheromones. The structure and function of olfactory genes that encode sex pheromones in O. emarginata might thus differ from those of other studied noctuids. The findings of the present study may help explain the molecular mechanism underlying olfaction and the evolution of olfactory genes encoding sex pheromones in O. emarginata. PMID:28614384

Input-output relation and energy efficiency in the neuron with different spike threshold dynamics

PubMed Central

Yi, Guo-Sheng; Wang, Jiang; Tsang, Kai-Ming; Wei, Xi-Le; Deng, Bin

2015-01-01

Neuron encodes and transmits information through generating sequences of output spikes, which is a high energy-consuming process. The spike is initiated when membrane depolarization reaches a threshold voltage. In many neurons, threshold is dynamic and depends on the rate of membrane depolarization (dV/dt) preceding a spike. Identifying the metabolic energy involved in neural coding and their relationship to threshold dynamic is critical to understanding neuronal function and evolution. Here, we use a modified Morris-Lecar model to investigate neuronal input-output property and energy efficiency associated with different spike threshold dynamics. We find that the neurons with dynamic threshold sensitive to dV/dt generate discontinuous frequency-current curve and type II phase response curve (PRC) through Hopf bifurcation, and weak noise could prohibit spiking when bifurcation just occurs. The threshold that is insensitive to dV/dt, instead, results in a continuous frequency-current curve, a type I PRC and a saddle-node on invariant circle bifurcation, and simultaneously weak noise cannot inhibit spiking. It is also shown that the bifurcation, frequency-current curve and PRC type associated with different threshold dynamics arise from the distinct subthreshold interactions of membrane currents. Further, we observe that the energy consumption of the neuron is related to its firing characteristics. The depolarization of spike threshold improves neuronal energy efficiency by reducing the overlap of Na+ and K+ currents during an action potential. The high energy efficiency is achieved at more depolarized spike threshold and high stimulus current. These results provide a fundamental biophysical connection that links spike threshold dynamics, input-output relation, energetics and spike initiation, which could contribute to uncover neural encoding mechanism. PMID:26074810

Genome-wide identification and expression analysis of the ClTCP transcription factors in Citrullus lanatus.

PubMed

Shi, Pibiao; Guy, Kateta Malangisha; Wu, Weifang; Fang, Bingsheng; Yang, Jinghua; Zhang, Mingfang; Hu, Zhongyuan

2016-04-12

The plant-specific TCP transcription factor family, which is involved in the regulation of cell growth and proliferation, performs diverse functions in multiple aspects of plant growth and development. However, no comprehensive analysis of the TCP family in watermelon (Citrullus lanatus) has been undertaken previously. A total of 27 watermelon TCP encoding genes distributed on nine chromosomes were identified. Phylogenetic analysis clustered the genes into 11 distinct subgroups. Furthermore, phylogenetic and structural analyses distinguished two homology classes within the ClTCP family, designated Class I and Class II. The Class II genes were differentiated into two subclasses, the CIN subclass and the CYC/TB1 subclass. The expression patterns of all members were determined by semi-quantitative PCR. The functions of two ClTCP genes, ClTCP14a and ClTCP15, in regulating plant height were confirmed by ectopic expression in Arabidopsis wild-type and ortholog mutants. This study represents the first genome-wide analysis of the watermelon TCP gene family, which provides valuable information for understanding the classification and functions of the TCP genes in watermelon.

A Local DCT-II Feature Extraction Approach for Personal Identification Based on Palmprint

NASA Astrophysics Data System (ADS)

Choge, H. Kipsang; Oyama, Tadahiro; Karungaru, Stephen; Tsuge, Satoru; Fukumi, Minoru

Biometric applications based on the palmprint have recently attracted increased attention from various researchers. In this paper, a method is presented that differs from the commonly used global statistical and structural techniques by extracting and using local features instead. The middle palm area is extracted after preprocessing for rotation, position and illumination normalization. The segmented region of interest is then divided into blocks of either 8×8 or 16×16 pixels in size. The type-II Discrete Cosine Transform (DCT) is applied to transform the blocks into DCT space. A subset of coefficients that encode the low to medium frequency components is selected using the JPEG-style zigzag scanning method. Features from each block are subsequently concatenated into a compact feature vector and used in palmprint verification experiments with palmprints from the PolyU Palmprint Database. Results indicate that this approach achieves better results than many conventional transform-based methods, with an excellent recognition accuracy above 99% and an Equal Error Rate (EER) of less than 1.2% in palmprint verification.

Targeted Modification of Homogalacturonan by Transgenic Expression of a Fungal Polygalacturonase Alters Plant Growth1

PubMed Central

Capodicasa, Cristina; Vairo, Donatella; Zabotina, Olga; McCartney, Lesley; Caprari, Claudio; Mattei, Benedetta; Manfredini, Cinzia; Aracri, Benedetto; Benen, Jacques; Knox, J. Paul; De Lorenzo, Giulia; Cervone, Felice

2004-01-01

Pectins are a highly complex family of cell wall polysaccharides comprised of homogalacturonan (HGA), rhamnogalacturonan I and rhamnogalacturonan II. We have specifically modified HGA in both tobacco (Nicotiana tabacum) and Arabidopsis by expressing the endopolygalacturonase II of Aspergillus niger (AnPGII). Cell walls of transgenic tobacco plants showed a 25% reduction in GalUA content as compared with the wild type and a reduced content of deesterified HGA as detected by antibody labeling. Neutral sugars remained unchanged apart from a slight increase of Rha, Ara, and Gal. Both transgenic tobacco and Arabidopsis were dwarfed, indicating that unesterified HGA is a critical factor for plant cell growth. The dwarf phenotypes were associated with AnPGII activity as demonstrated by the observation that the mutant phenotype of tobacco was completely reverted by crossing the dwarfed plants with plants expressing PGIP2, a strong inhibitor of AnPGII. The mutant phenotype in Arabidopsis did not appear when transformation was performed with a gene encoding AnPGII inactivated by site directed mutagenesis. PMID:15247378

SOLO: a meiotic protein required for centromere cohesion, coorientation, and SMC1 localization in Drosophila melanogaster.

PubMed

Yan, Rihui; Thomas, Sharon E; Tsai, Jui-He; Yamada, Yukihiro; McKee, Bruce D

2010-02-08

Sister chromatid cohesion is essential to maintain stable connections between homologues and sister chromatids during meiosis and to establish correct centromere orientation patterns on the meiosis I and II spindles. However, the meiotic cohesion apparatus in Drosophila melanogaster remains largely uncharacterized. We describe a novel protein, sisters on the loose (SOLO), which is essential for meiotic cohesion in Drosophila. In solo mutants, sister centromeres separate before prometaphase I, disrupting meiosis I centromere orientation and causing nondisjunction of both homologous and sister chromatids. Centromeric foci of the cohesin protein SMC1 are absent in solo mutants at all meiotic stages. SOLO and SMC1 colocalize to meiotic centromeres from early prophase I until anaphase II in wild-type males, but both proteins disappear prematurely at anaphase I in mutants for mei-S332, which encodes the Drosophila homologue of the cohesin protector protein shugoshin. The solo mutant phenotypes and the localization patterns of SOLO and SMC1 indicate that they function together to maintain sister chromatid cohesion in Drosophila meiosis.

[Differentiation of spa types and staphylococcal cassette chromosome mec (SCCmec) in clinical methicillin-resistant Staphylococcus aureus isolated in medical sites of Gdańsk region].

PubMed

Kasprzyk, Joanna; Piechowicz, Lidia; Wiśniewska, Katarzyna; Dziewit, Łukasz; Bronk, Marek; Świeć, Krystyna

2015-01-01

Methicillin-resistant Staphylococcus aureus bacteria are one of the key etiological factors of hospital-acquired and community-acquired infections. MRSA strains have an ability of causing a broad spectrum infections: from a relatively mild skin infections to severe life-threatening systemic infections. They are characterized by multi-drug resistance, virulence of a number of factors, may clonally spread within the hospitals and between hospitals. The study embraced a number of 75 isolates of MRSA isolated from patients of 7 medical sites of the Gdansk region within the period of six months (June to December 2013). Strains have derived from various clinical materials, both of hospitalized patients (n=59) and outpatient (n=16). The isolates were tested for the susceptibility to antimicrobial agents accordance with the guidelines EUCAST. To estimate of the variability of occurrence of S. aureus clones used were standard spa gene, consisting in the amplified polymorphic region of the X gene encoding the protein A gene (spa). After receiving the results, a spa types were identified using international database Ridom Spa Server (www.spaserver.ridom.de). To determine the polymorphism cassette carrying the inecA gene from MRSA strains, used typing five major chromosomal cassette SCCmec (I-V) by multiplex PCR. MRSA population genetic analysis carried out on the basis of typing SCCmec cassettes and spa gene has showed a predominance of strains with SCCmec type II casette (46.7%) and SCCmec IV casette (38.7%). Less frequently detected were strains containing SCCmec I cassette (12.0%) and SCCmec III cassette (2.6%). Spa typing revealed the presence of 13 gene types in MRSA. The most frequently observed spa types were: t151 (24.0%), t003 (16.0%) in strains of the SCCmec II cassette and t437 (16.0%) and t008 (14.8%) in the isolates with SCCmec cassette IV, whereas staphylococcus with the type of spa t011 (12.0%) had SCCmec cassette I. In our population most frequent strains cassette SCCmec II (46.7%), in most representing types of spa t151 (51.4%) and t003 (34.3%), generally resistant not only to β-lactam antibiotics, but as erythromycin, clindamycin and norfloxacin (82.8%), the more frequently they were isolated from patients than a hospital outpatient centers. The strains SCCmec IV that represent the majority of outpatient centers (68.8%), the most represented type t437 (41.4%) and often occurred in hospital centers.

Novel Type V Staphylococcal Cassette Chromosome mec Driven by a Novel Cassette Chromosome Recombinase, ccrC

PubMed Central

Ito, Teruyo; Ma, Xiao Xue; Takeuchi, Fumihiko; Okuma, Keiko; Yuzawa, Harumi; Hiramatsu, Keiichi

2004-01-01

Staphylococcal cassette chromosome mec (SCCmec) is a mobile genetic element composed of the mec gene complex, which encodes methicillin resistance, and the ccr gene complex, which encodes the recombinases responsible for its mobility. The mec gene complex has been classified into four classes, and the ccr gene complex has been classified into three allotypes. Different combinations of mec gene complex classes and ccr gene complex types have so far defined four types of SCCmec elements. Now we introduce the fifth allotype of SCCmec, which was found on the chromosome of a community-acquired methicillin-resistant Staphylococcus aureus strain (strain WIS [WBG8318]) isolated in Australia. The element shared the same chromosomal integration site with the four extant types of SCCmec and the characteristic nucleotide sequences at the chromosome-SCCmec junction regions. The novel SCCmec carried mecA bracketed by IS431 (IS431-mecA-ΔmecR1-IS431), which is designated the class C2 mec gene complex; and instead of ccrA and ccrB genes, it carried a single copy of a gene homologue that encoded cassette chromosome recombinase. Since the open reading frame (ORF) was found to encode an enzyme which catalyzes the precise excision as well as site- and orientation-specific integration of the element, we designated the ORF cassette chromosome recombinase C (ccrC), and we designated the element type V SCCmec. Type V SCCmec is a small SCCmec element (28 kb) and does not carry any antibiotic resistance genes besides mecA. Unlike the extant SCCmec types, it carries a set of foreign genes encoding a restriction-modification system that might play a role in the stabilization of the element on the chromosome. PMID:15215121

Encoders for block-circulant LDPC codes

NASA Technical Reports Server (NTRS)

Andrews, Kenneth; Dolinar, Sam; Thorpe, Jeremy

2005-01-01

In this paper, we present two encoding methods for block-circulant LDPC codes. The first is an iterative encoding method based on the erasure decoding algorithm, and the computations required are well organized due to the block-circulant structure of the parity check matrix. The second method uses block-circulant generator matrices, and the encoders are very similar to those for recursive convolutional codes. Some encoders of the second type have been implemented in a small Field Programmable Gate Array (FPGA) and operate at 100 Msymbols/second.

Structural basis of human β-cell killing by CD8+ T cells in Type 1 diabetes

PubMed Central

Bulek, Anna M.; Cole, David K.; Skowera, Ania; Dolton, Garry; Gras, Stephanie; Madura, Florian; Fuller, Anna; Miles, John J.; Gostick, Emma; Price, David A.; Drijfhout, Jan W.; Knight, Robin R.; Huang, Guo C.; Lissin, Nikolai; Molloy, Peter E.; Wooldridge, Linda; Jakobsen, Bent K.; Rossjohn, Jamie; Peakman, Mark; Rizkallah, Pierre J.; Sewell, Andrew K.

2011-01-01

The structural characteristics of autoreactive-T cell receptor (TCR) engagement of major histocompatability (MHC) class II-restricted self-antigens is established, but how autoimmune-TCRs interact with self-MHC class I has been unclear. We examined how CD8+ T cells kill human islet β-cells, in Type-1 diabetes, via autoreactive-TCR (1E6) recognition of an HLA-A*0201-restricted glucose-sensitive preproinsulin peptide. Rigid ‘lock-and-key’ binding underpinned the 1E6-HLA-A*0201-peptide interaction, whereby 1E6 docked similarly to most MHCI-restricted TCRs. However, this interaction was extraordinarily weak, due to limited contacts with MHCI. TCR binding was highly peptide-centric, dominated by two CDR3-loop-encoded residues, acting as an ‘aromatic-cap’, over the peptide MHCI (pMHCI). Thus, highly focused peptide-centric interactions associated with suboptimal TCR-pMHCI binding affinities might lead to thymic escape and potential CD8+ T cell-mediated autoreactivity. PMID:22245737

A Time Diversity Coding Experiment for a UHF/VHF Satellite Channel with Scintillation: Equipment Description

DTIC Science & Technology

1977-09-01

to state as successive input bits are brought into the encoder. We can more easily follow our progress on the equivalent lattice diagram where...Pg.Pj.. STATE DIAGRAM INPUT PATH i ,i.,i ,L.. = 1001 1’ 2’𔃽’ V Fig. 12. Convolutional Encoder, State Diagram and Lattice . 39 represented...and can in fact be traced. The Viterbi algorithm can be simply described with the aid of this lattice . Note that the nodes of the lattice represent

Roles of BOR2, a Boron Exporter, in Cross Linking of Rhamnogalacturonan II and Root Elongation under Boron Limitation in Arabidopsis1[W

PubMed Central

Miwa, Kyoko; Wakuta, Shinji; Takada, Shigeki; Ide, Koji; Takano, Junpei; Naito, Satoshi; Omori, Hiroyuki; Matsunaga, Toshiro; Fujiwara, Toru

2013-01-01

Boron (B) is required for cross linking of the pectic polysaccharide rhamnogalacturonan II (RG-II) and is consequently essential for the maintenance of cell wall structure. Arabidopsis (Arabidopsis thaliana) BOR1 is an efflux B transporter for xylem loading of B. Here, we describe the roles of BOR2, the most similar paralog of BOR1. BOR2 encodes an efflux B transporter localized in plasma membrane and is strongly expressed in lateral root caps and epidermis of elongation zones of roots. Transfer DNA insertion of BOR2 reduced root elongation by 68%, whereas the mutation in BOR1 reduced it by 32% under low B availability (0.1 µm), but the reduction in shoot growth was not as obvious as that in the BOR1 mutant. A double mutant of BOR1 and BOR2 exhibited much more severe growth defects in both roots and shoots under B-limited conditions than the corresponding single mutants. All single and double mutants grew normally under B-sufficient conditions. These results suggest that both BOR1 and BOR2 are required under B limitation and that their roles are, at least in part, different. The total B concentrations in roots of BOR2 mutants were not significantly different from those in wild-type plants, but the proportion of cross-linked RG-II was reduced under low B availability. Such a reduction in RG-II cross linking was not evident in roots of the BOR1 mutant. Thus, we propose that under B-limited conditions, transport of boric acid/borate by BOR2 from symplast to apoplast is required for effective cross linking of RG-II in cell wall and root cell elongation. PMID:24114060

Comparative Genomics of Two ST 195 Carbapenem-Resistant Acinetobacter baumannii with Different Susceptibility to Polymyxin Revealed Underlying Resistance Mechanism.

PubMed

Lean, Soo-Sum; Yeo, Chew Chieng; Suhaili, Zarizal; Thong, Kwai-Lin

2015-01-01

Acinetobacter baumannii is a Gram-negative nosocomial pathogen of importance due to its uncanny ability to acquire resistance to most antimicrobials. These include carbapenems, which are the drugs of choice for treating A. baumannii infections, and polymyxins, the drugs of last resort. Whole genome sequencing was performed on two clinical carbapenem-resistant A. baumannii AC29 and AC30 strains which had an indistinguishable ApaI pulsotype but different susceptibilities to polymyxin. Both genomes consisted of an approximately 3.8 Mbp circular chromosome each and several plasmids. AC29 (susceptible to polymyxin) and AC30 (resistant to polymyxin) belonged to the ST195 lineage and are phylogenetically clustered under the International Clone II (IC-II) group. An AbaR4-type resistance island (RI) interrupted the comM gene in the chromosomes of both strains and contained the bla OXA-23 carbapenemase gene and determinants for tetracycline and streptomycin resistance. AC29 harbored another copy of bla OXA-23 in a large (~74 kb) conjugative plasmid, pAC29b, but this gene was absent in a similar plasmid (pAC30c) found in AC30. A 7 kb Tn1548::armA RI which encodes determinants for aminoglycoside and macrolide resistance, is chromosomally-located in AC29 but found in a 16 kb plasmid in AC30, pAC30b. Analysis of known determinants for polymyxin resistance in AC30 showed mutations in the pmrA gene encoding the response regulator of the two-component pmrAB signal transduction system as well as in the lpxD, lpxC, and lpsB genes that encode enzymes involved in the biosynthesis of lipopolysaccharide (LPS). Experimental evidence indicated that impairment of LPS along with overexpression of pmrAB may have contributed to the development of polymyxin resistance in AC30. Cloning of a novel variant of the bla AmpC gene from AC29 and AC30, and its subsequent expression in E. coli also indicated its likely function as an extended-spectrum cephalosporinase.

Arabidopsis thaliana type I and II chaperonins

PubMed Central

Hill, Janet E.; Hemmingsen, Sean M.

2001-01-01

An examination of the Arabidopsis thaliana genome sequence led to the identification of 29 predicted genes with the potential to encode members of the chaperonin family of chaperones (CPN60 and CCT), their associated cochaperonins, and the cytoplasmic chaperonin cofactor prefoldin. These comprise the first complete set of plant chaperonin protein sequences and indicate that the CPN family is more diverse than previously described. In addition to surprising sequence diversity within CPN subclasses, the genomic data also suggest the existence of previously undescribed family members, including a 10-kDa chloroplast cochaperonin. Consideration of the sequence data described in this review prompts questions about the complexities of plant CPN systems and the evolutionary relationships and functions of the component proteins, most of which have not been studied experimentally. PMID:11599560

Nuclear magnetic resonance-based model of a TF1/HmU-DNA complex.

PubMed

Silva, M V; Pasternack, L B; Kearns, D R

1997-12-15

Transcription factor 1 (TF1), a type II DNA-binding protein encoded by the Bacillus subtilis bacteriophage SPO1, has the capacity for sequence-selective DNA binding and a preference for 5-hydroxymethyl-2'-deoxyuridine (HmU)-containing DNA. In NMR studies of the TF1/HmU-DNA complex, intermolecular NOEs indicate that the flexible beta-ribbon and C-terminal alpha-helix are involved in the DNA-binding site of TF1, placing it in the beta-sheet category of DNA-binding proteins proposed to bind by wrapping two beta-ribbon "arms" around the DNA. Intermolecular and intramolecular NOEs were used to generate an energy-minimized model of the protein-DNA complex in which both DNA bending and protein structure changes are evident.

Chronic shin splints. Classification and management of medial tibial stress syndrome.

PubMed

Detmer, D E

1986-01-01

A clinical classification and treatment programme has been developed for chronic medial tibial stress syndrome. Medial tibial stress syndrome has been reported to be either tibial stress fracture or microfracture, tibial periostitis, or distal deep posterior chronic compartment syndrome. Three chronic types exist and may coexist: Type I (tibial microfracture, bone stress reaction or cortical fracture); type II (periostalgia from chronic avulsion of the periosteum at the periosteal-fascial junction); and type III (chronic compartment syndrome syndrome). Type I disease is treated nonoperatively. Operations for resistant types II and III medial tibial stress syndrome were performed in 41 patients. Bilaterality was common (type II, 50% type III, 88%). Seven had coexistent type II/III; one had type I/II. Preoperative symptoms averaged 24 months in type II, 6 months in type III, and 33 months in types II/III. Mean age was 22 years (15 to 51). Resting compartment pressures were normal in type II (mean 12 mm Hg) and elevated in type III and type II/III (mean 23 mm Hg). Type II and type II/III patients received fasciotomy plus periosteal cauterisation. Type III patients had fasciotomy only. All procedures were performed on an outpatient basis using local anaesthesia. Follow up was complete and averaged 6 months (2 to 14 months). Improved performance was as follows: type II, 93%, type III, 100%; type II/III, 86%. Complete cures were as follows: type II, 78%; type III, 75%; and type II/III, 57%. This experience suggests that with precise diagnosis and treatment involving minimal risk and cost the athlete has a reasonable chance of return to full activity.

Methods of increasing secretion of polypeptides having biological activity

DOEpatents

Merino, Sandra

2014-05-27

The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.

Methods of increasing secretion of polypeptides having biological activity

DOEpatents

Merino, Sandra

2014-10-28

The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.

Methods of increasing secretion of polypeptides having biological activity

DOEpatents

Merino, Sandra

2015-04-14

The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.

Methods of increasing secretion of polypeptides having biological activity

DOEpatents

Merino, Sandra

2013-10-01

The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.

Collagen IX is required for the integrity of collagen II fibrils and the regulation of vascular plexus formation in Zebrafish caudal fins

PubMed Central

Huang, Cheng-chen; Wang, Tai-Chuan; Lin, Bo-Hung; Wang, Yi-Wen; Johnson, Stephen L.; Yu, John

2013-01-01

Capillary plexuses form during both vasculogenesis and angiogenesis and are remodeled into mature vessel types and patterns which are delicately orchestrated with the sizes and shapes of other tissues and organs. We isolated a zebrafish mutation named prp (for persistent plexus) that causes persistent formation of vascular plexuses in the caudal fins and consequent mispatterning of bony fin rays and the fin shape. Detailed analyses revealed that the prp mutation causes a significant reduction in the size and dramatic structural defects in collagen II-rich extracellular matrices called actinotrichia of both embryonic finfolds and adult fins. prp was mapped to chromosome 19 and found to encode the zebrafish collagen9α1 (col9α1) gene which is abundantly expressed in developing finfolds. A point mutation resulting in a leucine-to-histidine change was detected in the thrombospondin domain of the col9α1 gene in prp. Morpholino-mediated knockdown of col9α1 phenocopied the prp small-finfold phenotype in wild-type embryos, and an injection of plasmids containing the col9α1 cDNA into prp embryos locally restored the finfold size. Furthermore, we found that osteoblasts in prp mutants were mispatterned apparently following the abnormal vascular plexus pattern, demonstrating that blood vessels play an important role in the patterning of bony rays in zebrafish caudal fins. PMID:19501583

Collagen IX is required for the integrity of collagen II fibrils and the regulation of vascular plexus formation in zebrafish caudal fins.

PubMed

Huang, Cheng-chen; Wang, Tai-Chuan; Lin, Bo-Hung; Wang, Yi-Wen; Johnson, Stephen L; Yu, John

2009-08-15

Capillary plexuses form during both vasculogenesis and angiogenesis and are remodeled into mature vessel types and patterns which are delicately orchestrated with the sizes and shapes of other tissues and organs. We isolated a zebrafish mutation named prp (for persistent plexus) that causes persistent formation of vascular plexuses in the caudal fins and consequent mispatterning of bony fin rays and the fin shape. Detailed analyses revealed that the prp mutation causes a significant reduction in the size and dramatic structural defects in collagen II-rich extracellular matrices called actinotrichia of both embryonic finfolds and adult fins. prp was mapped to chromosome 19 and found to encode the zebrafish collagen9alpha1 (col9alpha1) gene which is abundantly expressed in developing finfolds. A point mutation resulting in a leucine-to-histidine change was detected in the thrombospondin domain of the col9alpha1 gene in prp. Morpholino-mediated knockdown of col9alpha1 phenocopied the prp small-finfold phenotype in wild-type embryos, and an injection of plasmids containing the col9alpha1 cDNA into prp embryos locally restored the finfold size. Furthermore, we found that osteoblasts in prp mutants were mispatterned apparently following the abnormal vascular plexus pattern, demonstrating that blood vessels play an important role in the patterning of bony rays in zebrafish caudal fins.

CNS-directed gene therapy for the treatment of neurologic and somatic mucopolysaccharidosis type II (Hunter syndrome).

PubMed

Motas, Sandra; Haurigot, Virginia; Garcia, Miguel; Marcó, Sara; Ribera, Albert; Roca, Carles; Sánchez, Xavier; Sánchez, Víctor; Molas, Maria; Bertolin, Joan; Maggioni, Luca; León, Xavier; Ruberte, Jesús; Bosch, Fatima

2016-06-16

Mucopolysaccharidosis type II (MPSII) is an X-linked lysosomal storage disease characterized by severe neurologic and somatic disease caused by deficiency of iduronate-2-sulfatase (IDS), an enzyme that catabolizes the glycosaminoglycans heparan and dermatan sulphate. Intravenous enzyme replacement therapy (ERT) currently constitutes the only approved therapeutic option for MPSII. However, the inability of recombinant IDS to efficiently cross the blood-brain barrier (BBB) limits ERT efficacy in treating neurological symptoms. Here, we report a gene therapy approach for MPSII through direct delivery of vectors to the CNS. Through a minimally invasive procedure, we administered adeno-associated virus vectors encoding IDS (AAV9- Ids ) to the cerebrospinal fluid of MPSII mice with already established disease. Treated mice showed a significant increase in IDS activity throughout the encephalon, with full resolution of lysosomal storage lesions, reversal of lysosomal dysfunction, normalization of brain transcriptomic signature, and disappearance of neuroinflammation. Moreover, our vector also transduced the liver, providing a peripheral source of therapeutic protein that corrected storage pathology in visceral organs, with evidence of cross-correction of nontransduced organs by circulating enzyme. Importantly, AAV9- Ids -treated MPSII mice showed normalization of behavioral deficits and considerably prolonged survival. These results provide a strong proof of concept for the clinical translation of our approach for the treatment of Hunter syndrome patients with cognitive impairment.

CNS-directed gene therapy for the treatment of neurologic and somatic mucopolysaccharidosis type II (Hunter syndrome)

PubMed Central

Motas, Sandra; Haurigot, Virginia; Garcia, Miguel; Marcó, Sara; Ribera, Albert; Roca, Carles; Sánchez, Víctor; Molas, Maria; Bertolin, Joan; Maggioni, Luca; León, Xavier; Ruberte, Jesús; Bosch, Fatima

2016-01-01

Mucopolysaccharidosis type II (MPSII) is an X-linked lysosomal storage disease characterized by severe neurologic and somatic disease caused by deficiency of iduronate-2-sulfatase (IDS), an enzyme that catabolizes the glycosaminoglycans heparan and dermatan sulphate. Intravenous enzyme replacement therapy (ERT) currently constitutes the only approved therapeutic option for MPSII. However, the inability of recombinant IDS to efficiently cross the blood-brain barrier (BBB) limits ERT efficacy in treating neurological symptoms. Here, we report a gene therapy approach for MPSII through direct delivery of vectors to the CNS. Through a minimally invasive procedure, we administered adeno-associated virus vectors encoding IDS (AAV9-Ids) to the cerebrospinal fluid of MPSII mice with already established disease. Treated mice showed a significant increase in IDS activity throughout the encephalon, with full resolution of lysosomal storage lesions, reversal of lysosomal dysfunction, normalization of brain transcriptomic signature, and disappearance of neuroinflammation. Moreover, our vector also transduced the liver, providing a peripheral source of therapeutic protein that corrected storage pathology in visceral organs, with evidence of cross-correction of nontransduced organs by circulating enzyme. Importantly, AAV9-Ids-treated MPSII mice showed normalization of behavioral deficits and considerably prolonged survival. These results provide a strong proof of concept for the clinical translation of our approach for the treatment of Hunter syndrome patients with cognitive impairment. PMID:27699273

Enoyl-Acyl Carrier Protein Reductase I (FabI) Is Essential for the Intracellular Growth of Listeria monocytogenes

PubMed Central

Ericson, Megan E.; Frank, Matthew W.

2016-01-01

Enoyl-acyl carrier protein reductase catalyzes the last step in each elongation cycle of type II bacterial fatty acid synthesis and is a key regulatory protein in bacterial fatty acid synthesis. Genes of the facultative intracellular pathogen Listeria monocytogenes encode two functional enoyl-acyl carrier protein isoforms based on their ability to complement the temperature-sensitive growth phenotype of Escherichia coli strain JP1111 [fabI(Ts)]. The FabI isoform was inactivated by the FabI selective inhibitor AFN-1252, but the FabK isoform was not affected by the drug, as expected. Inhibition of FabI by AFN-1252 decreased endogenous fatty acid synthesis by 80% and lowered the growth rate of L. monocytogenes in laboratory medium. Robust exogenous fatty acid incorporation was not detected in L. monocytogenes unless the pathway was partially inactivated by AFN-1252 treatment. However, supplementation with exogenous fatty acids did not restore normal growth in the presence of AFN-1252. FabI inactivation prevented the intracellular growth of L. monocytogenes, showing that neither FabK nor the incorporation of host cellular fatty acids was sufficient to support the intracellular growth of L. monocytogenes. Our results show that FabI is the primary enoyl-acyl carrier protein reductase of type II bacterial fatty acid synthesis and is essential for the intracellular growth of L. monocytogenes. PMID:27736774

A Dedicated Type II NADPH Dehydrogenase Performs the Penultimate Step in the Biosynthesis of Vitamin K1 in Synechocystis and Arabidopsis

PubMed Central

Fatihi, Abdelhak; Latimer, Scott; Schmollinger, Stefan; Block, Anna; Dussault, Patrick H.; Vermaas, Wim F.J.; Merchant, Sabeeha S.; Basset, Gilles J.

2015-01-01

Mutation of Arabidopsis thaliana NAD(P)H DEHYDROGENASE C1 (NDC1; At5g08740) results in the accumulation of demethylphylloquinone, a late biosynthetic intermediate of vitamin K1. Gene coexpression and phylogenomics analyses showed that conserved functional associations occur between vitamin K biosynthesis and NDC1 homologs throughout the prokaryotic and eukaryotic lineages. Deletion of Synechocystis ndbB, which encodes for one such homolog, resulted in the same defects as those observed in the cyanobacterial demethylnaphthoquinone methyltransferase knockout. Chemical modeling and assay of purified demethylnaphthoquinone methyltransferase demonstrated that, by virtue of the strong electrophilic nature of S-adenosyl-l-methionine, the transmethylation of the demethylated precursor of vitamin K is strictly dependent on the reduced form of its naphthoquinone ring. NDC1 was shown to catalyze such a prerequisite reduction by using NADPH and demethylphylloquinone as substrates and flavine adenine dinucleotide as a cofactor. NDC1 displayed Michaelis-Menten kinetics and was markedly inhibited by dicumarol, a competitive inhibitor of naphthoquinone oxidoreductases. These data demonstrate that the reduction of the demethylnaphthoquinone ring represents an authentic step in the biosynthetic pathway of vitamin K, that this reaction is enzymatically driven, and that a selection pressure is operating to retain type II NAD(P)H dehydrogenases in this process. PMID:26023160

Characterization and Exploitation of CRISPR Loci in Bifidobacterium longum

PubMed Central

Hidalgo-Cantabrana, Claudio; Crawley, Alexandra B.; Sanchez, Borja; Barrangou, Rodolphe

2017-01-01

Diverse CRISPR-Cas systems provide adaptive immunity in many bacteria and most archaea, via a DNA-encoded, RNA-mediated, nucleic-acid targeting mechanism. Over time, CRISPR loci expand via iterative uptake of invasive DNA sequences into the CRISPR array during the adaptation process. These genetic vaccination cards thus provide insights into the exposure of strains to phages and plasmids in space and time, revealing the historical predatory exposure of a strain. These genetic loci thus constitute a unique basis for genotyping of strains, with potential of resolution at the strain-level. Here, we investigate the occurrence and diversity of CRISPR-Cas systems in the genomes of various Bifidobacterium longum strains across three sub-species. Specifically, we analyzed the genomic content of 66 genomes belonging to B. longum subsp. longum, B. longum subsp. infantis and B. longum subsp. suis, and identified 25 strains that carry 29 total CRISPR-Cas systems. We identify various Type I and Type II CRISPR-Cas systems that are widespread in this species, notably I-C, I-E, and II-C. Noteworthy, Type I-C systems showed extended CRISPR arrays, with extensive spacer diversity. We show how these hypervariable loci can be used to gain insights into strain origin, evolution and phylogeny, and can provide discriminatory sequences to distinguish even clonal isolates. By investigating CRISPR spacer sequences, we reveal their origin and implicate phages and prophages as drivers of CRISPR immunity expansion in this species, with redundant targeting of select prophages. Analysis of CRISPR spacer origin also revealed novel PAM sequences. Our results suggest that CRISPR-Cas immune systems are instrumental in mounting diversified viral resistance in B. longum, and show that these sequences are useful for typing across three subspecies. PMID:29033911

Characterization and Exploitation of CRISPR Loci in Bifidobacterium longum.

PubMed

Hidalgo-Cantabrana, Claudio; Crawley, Alexandra B; Sanchez, Borja; Barrangou, Rodolphe

2017-01-01

Diverse CRISPR-Cas systems provide adaptive immunity in many bacteria and most archaea, via a DNA-encoded, RNA-mediated, nucleic-acid targeting mechanism. Over time, CRISPR loci expand via iterative uptake of invasive DNA sequences into the CRISPR array during the adaptation process. These genetic vaccination cards thus provide insights into the exposure of strains to phages and plasmids in space and time, revealing the historical predatory exposure of a strain. These genetic loci thus constitute a unique basis for genotyping of strains, with potential of resolution at the strain-level. Here, we investigate the occurrence and diversity of CRISPR-Cas systems in the genomes of various Bifidobacterium longum strains across three sub-species. Specifically, we analyzed the genomic content of 66 genomes belonging to B. longum subsp. longum, B. longum subsp. infantis and B. longum subsp. suis , and identified 25 strains that carry 29 total CRISPR-Cas systems. We identify various Type I and Type II CRISPR-Cas systems that are widespread in this species, notably I-C, I-E, and II-C. Noteworthy, Type I-C systems showed extended CRISPR arrays, with extensive spacer diversity. We show how these hypervariable loci can be used to gain insights into strain origin, evolution and phylogeny, and can provide discriminatory sequences to distinguish even clonal isolates. By investigating CRISPR spacer sequences, we reveal their origin and implicate phages and prophages as drivers of CRISPR immunity expansion in this species, with redundant targeting of select prophages. Analysis of CRISPR spacer origin also revealed novel PAM sequences. Our results suggest that CRISPR-Cas immune systems are instrumental in mounting diversified viral resistance in B. longum , and show that these sequences are useful for typing across three subspecies.

KNS4/UPEX1: A Type II Arabinogalactan β-(1,3)-Galactosyltransferase Required for Pollen Exine Development1[OPEN

PubMed Central

Suzuki, Toshiya; Narciso, Joan Oñate; Zeng, Wei; van de Meene, Allison; Yasutomi, Masayuki; Takemura, Shunsuke

2017-01-01

Pollen exine is essential for protection from the environment of the male gametes of seed-producing plants, but its assembly and composition remain poorly understood. We previously characterized Arabidopsis (Arabidopsis thaliana) mutants with abnormal pollen exine structure and morphology that we named kaonashi (kns). Here we describe the identification of the causal gene of kns4 that was found to be a member of the CAZy glycosyltransferase 31 gene family, identical to UNEVEN PATTERN OF EXINE1, and the biochemical characterization of the encoded protein. The characteristic exine phenotype in the kns4 mutant is related to an abnormality of the primexine matrix laid on the surface of developing microspores. Using light microscopy with a combination of type II arabinogalactan (AG) antibodies and staining with the arabinogalactan-protein (AGP)-specific β-Glc Yariv reagent, we show that the levels of AGPs in the kns4 microspore primexine are considerably diminished, and their location differs from that of wild type, as does the distribution of pectin labeling. Furthermore, kns4 mutants exhibit reduced fertility as indicated by shorter fruit lengths and lower seed set compared to the wild type, confirming that KNS4 is critical for pollen viability and development. KNS4 was heterologously expressed in Nicotiana benthamiana, and was shown to possess β-(1,3)-galactosyltransferase activity responsible for the synthesis of AG glycans that are present on both AGPs and/or the pectic polysaccharide rhamnogalacturonan I. These data demonstrate that defects in AGP/pectic glycans, caused by disruption of KNS4 function, impact pollen development and viability in Arabidopsis. PMID:27837085

Genome-wide analysis of the Zn(II)2Cys6 zinc cluster-encoding gene family in Aspergillus flavus

USDA-ARS?s Scientific Manuscript database

Proteins with a Zn(II)2Cys6 domain, Cys-X2-Cys-X6-Cys-X5-12-Cys-X2-Cys-X6-9-Cys (hereafter, referred to as the C6 domain), form a subclass of zinc finger proteins found exclusively in fungi and yeast. Genome sequence databases of Saccharomyces cerevisiae and Candida albicans have provided an overvie...

Constitutive signaling by the phototaxis receptor sensory rhodopsin II from disruption of its protonated Schiff base–Asp-73 interhelical salt bridge

PubMed Central

Spudich, Elena N.; Zhang, Weisheng; Alam, Maqsudul; Spudich, John L.

1997-01-01

Sensory rhodopsin II (SRII) is a repellent phototaxis receptor in the archaeon Halobacterium salinarum, similar to visual pigments in its seven-helix structure and linkage of retinal to the protein by a protonated Schiff base in helix G. Asp-73 in helix C is shown by spectroscopic analysis to be a counterion to the protonated Schiff base in the unphotolyzed SRII and to be the proton acceptor from the Schiff base during photoconversion to the receptor signaling state. Coexpression of the genes encoding mutated SRII with Asn substituted for Asp-73 (D73N) and the SRII transducer HtrII in H. salinarum cells results in a 3-fold higher swimming reversal frequency accompanied by demethylation of HtrII in the dark, showing that D73N SRII produces repellent signals in its unphotostimulated state. Analogous constitutive signaling has been shown to be produced by the similar neutral residue substitution of the Schiff base counterion and proton acceptor Glu-113 in human rod rhodopsin. The interpretation for both seven-helix receptors is that light activation of the wild-type protein is caused primarily by photoisomerization-induced transfer of the Schiff base proton on helix G to its primary carboxylate counterion on helix C. Therefore receptor activation by helix C–G salt-bridge disruption in the photoactive site is a general mechanism in retinylidene proteins spanning the vast evolutionary distance between archaea and humans. PMID:9144172

The role of bile salt export pump mutations in progressive familial intrahepatic cholestasis type II

PubMed Central

Wang, Lin; Soroka, Carol J.; Boyer, James L.

2002-01-01

PFIC II is a subtype of progressive familial intrahepatic cholestasis (PFIC) that is associated with mutations in the ABCB11 gene encoding the bile salt export pump (BSEP). However it is not known how these mutations cause this disease. To evaluate these mechanisms, we introduced seven PFIC II–associated missense mutations into rat Bsep and assessed their effects on Bsep membrane localization and transport function in MDCK and Sf9 cells, respectively. Five mutations, G238V, E297G, G982R, R1153C, and R1268Q, prevented the protein from trafficking to the apical membrane, and E297G, G982R, R1153C, and R1268Q also abolished taurocholate transport activity, possibly by causing Bsep to misfold. Mutation C336S affected neither Bsep transport activity nor the apical trafficking of Bsep, suggesting that this mutation alone may not cause this disease. D482G did not affect the apical expression but partially decreased the transport activity of Bsep. Mutant G238V was rapidly degraded in both MDCK and Sf9 cells, and proteasome inhibitor resulted in intracellular accumulation of this and other mutants, suggesting proteasome-mediated degradation plays an important role in expression of these PFIC II mutants. Our studies highlight the heterogeneous nature of PFIC II mutations and illustrate the significance of these mutations in the function and expression of Bsep. PMID:12370274

ERP Subsequent Memory Effects Differ between Inter-Item and Unitization Encoding Tasks

PubMed Central

Kamp, Siri-Maria; Bader, Regine; Mecklinger, Axel

2017-01-01

The “subsequent memory paradigm” is an analysis tool to identify brain activity elicited during episodic encoding that is associated with successful subsequent retrieval. Two commonly observed event-related potential “subsequent memory effects” (SMEs) are the parietal SME in the P300 time window and the frontal slow wave SME, but to date a clear characterization of the circumstances under which each SME is observed is missing. To test the hypothesis that the parietal SME occurs when aspects of an experience are unitized into a single item representation, while inter-item associative encoding is reflected in the frontal slow wave effect, participants were assigned to one of two conditions that emphasized one of the encoding types under otherwise matched study phases of a recognition memory experiment. Word pairs were presented either in the context of a definition that allowed to combine the word pairs into a new concept (unitization or item encoding) or together with a sentence frame (inter-item encoding). Performance on the recognition test did not differ between the groups. The parietal SME was only found in the definition group, supporting the idea that this SME occurs when the components of an association are integrated in a unitized item representation. An early prefrontal negativity also exhibited an SME only in this group, suggesting that the formation of novel units occurs through interactions of multiple brain areas. The frontal slow wave SME was pronounced in both groups and may thus reflect processes generally involved in encoding of associations. Our results provide evidence for a partial dissociation of the eliciting conditions of the two types of SMEs and therefore provide a tool for future studies to characterize the different types of episodic encoding. PMID:28194105

ERP Subsequent Memory Effects Differ between Inter-Item and Unitization Encoding Tasks.

PubMed

Kamp, Siri-Maria; Bader, Regine; Mecklinger, Axel

2017-01-01

The "subsequent memory paradigm" is an analysis tool to identify brain activity elicited during episodic encoding that is associated with successful subsequent retrieval. Two commonly observed event-related potential "subsequent memory effects" (SMEs) are the parietal SME in the P300 time window and the frontal slow wave SME, but to date a clear characterization of the circumstances under which each SME is observed is missing. To test the hypothesis that the parietal SME occurs when aspects of an experience are unitized into a single item representation, while inter-item associative encoding is reflected in the frontal slow wave effect, participants were assigned to one of two conditions that emphasized one of the encoding types under otherwise matched study phases of a recognition memory experiment. Word pairs were presented either in the context of a definition that allowed to combine the word pairs into a new concept (unitization or item encoding) or together with a sentence frame (inter-item encoding). Performance on the recognition test did not differ between the groups. The parietal SME was only found in the definition group, supporting the idea that this SME occurs when the components of an association are integrated in a unitized item representation. An early prefrontal negativity also exhibited an SME only in this group, suggesting that the formation of novel units occurs through interactions of multiple brain areas. The frontal slow wave SME was pronounced in both groups and may thus reflect processes generally involved in encoding of associations. Our results provide evidence for a partial dissociation of the eliciting conditions of the two types of SMEs and therefore provide a tool for future studies to characterize the different types of episodic encoding.

Positive Feedback between Transcriptional and Kinase Suppression in Nematodes with Extraordinary Longevity and Stress Resistance

PubMed Central

Bharill, Puneet; Shmookler Reis, Robert J.

2009-01-01

Insulin/IGF-1 signaling (IIS) regulates development and metabolism, and modulates aging, of Caenorhabditis elegans. In nematodes, as in mammals, IIS is understood to operate through a kinase-phosphorylation cascade that inactivates the DAF-16/FOXO transcription factor. Situated at the center of this pathway, phosphatidylinositol 3-kinase (PI3K) phosphorylates PIP2 to form PIP3, a phospholipid required for membrane tethering and activation of many signaling molecules. Nonsense mutants of age-1, the nematode gene encoding the class-I catalytic subunit of PI3K, produce only a truncated protein lacking the kinase domain, and yet confer 10-fold greater longevity on second-generation (F2) homozygotes, and comparable gains in stress resistance. Their F1 parents, like weaker age-1 mutants, are far less robust—implying that maternally contributed trace amounts of PI3K activity or of PIP3 block the extreme age-1 phenotypes. We find that F2-mutant adults have <10% of wild-type kinase activity in vitro and <60% of normal phosphoprotein levels in vivo. Inactivation of PI3K not only disrupts PIP3-dependent kinase signaling, but surprisingly also attenuates transcripts of numerous IIS components, even upstream of PI3K, and those of signaling molecules that cross-talk with IIS. The age-1(mg44) nonsense mutation results, in F2 adults, in changes to kinase profiles and to expression levels of multiple transcripts that distinguish this mutant from F1 age-1 homozygotes, a weaker age-1 mutant, or wild-type adults. Most but not all of those changes are reversed by a second mutation to daf-16, implicating both DAF-16/ FOXO–dependent and –independent mechanisms. RNAi, silencing genes that are downregulated in long-lived worms, improves oxidative-stress resistance of wild-type adults. It is therefore plausible that attenuation of those genes in age-1(mg44)-F2 adults contributes to their exceptional survival. IIS in nematodes (and presumably in other species) thus involves transcriptional as well as kinase regulation in a positive-feedback circuit, favoring either survival or reproduction. Hyperlongevity of strong age-1(mg44) mutants may result from their inability to reset this molecular switch to the reproductive mode. PMID:19360094

Weak encoding of faces predicts socially influenced judgments of facial attractiveness.

PubMed

Schnuerch, Robert; Koppehele-Gossel, Judith; Gibbons, Henning

2015-01-01

Conforming to the majority can be seen as a heuristic type of judgment, as it allows the individual to easily choose the most accurate or most socially acceptable type of behavior. People who process the currently to-be-judged items in a superficial, heuristic way should tend to conform to group judgment more than people processing these items in a systematic and elaborate way. We investigated this hypothesis using electroencephalography (EEG), analyzing whether the strength of neural encoding of faces was related to the tendency to adopt a group's evaluative judgments regarding these faces. As expected, we found that the amplitude of the N170, a specific neural correlate of face encoding, was inversely related to conformity across participants: The weaker the faces were encoded, the more the majority response regarding the faces' attractiveness was adopted instead of relying on the actual qualities of the faces. Applying neurophysiological methodology, we thus provide support for previous claims, based on behavioral data and theorizing, that social conformity is a heuristic type of judgment. We propose that weak encoding of judgment-relevant information is a typical, possibly even necessary, precursor of socially adjusted judgments, irrespective of one's current motivational goal (i.e., to be accurate or accepted).

A Eukaryotic-Type Serine/Threonine Protein Kinase Is Required for Biofilm Formation, Genetic Competence, and Acid Resistance in Streptococcus mutans

PubMed Central

Hussain, Haitham; Branny, Pavel; Allan, Elaine

2006-01-01

We report an operon encoding a eukaryotic-type serine/threonine protein kinase (STPK) and its cognate phosphatase (STPP) in Streptococcus mutans. Mutation of the gene encoding the STPK produced defects in biofilm formation, genetic competence, and acid resistance, determinants important in caries pathogenesis. PMID:16452447

NMR structures of anti-HIV D-peptides derived from the N-terminus of viral chemokine vMIP-II

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

Mori, Mayuko; Liu Dongxiang; Kumar, Santosh

2005-09-30

The viral macrophage inflammatory protein-II (vMIP-II) encoded by Kaposi's sarcoma-associated herpesvirus has unique biological activities in that it blocks the cell entry by several different human immunodeficiency virus type 1 (HIV-1) strains via chemokine receptors including CXCR4 and CCR5. In this paper, we report the solution structure of all-D-amino acid peptides derived from the N-terminus of vMIP-II, which have been shown to have strong CXCR4 binding activity and potently inhibit HIV-1 entry via CXCR4, by using long mixing time two-dimensional nuclear Overhauser enhancement spectroscopy experiments. Both of all-D-peptides vMIP-II (1-10) and vMIP-II (1-21), which are designated as DV3 and DV1,more » respectively, have higher CXCR4 binding ability than their L-peptide counterparts. They are partially structured in aqueous solution, displaying a turn-like structure over residues 5-8. The small temperature coefficients of His-6 amide proton for both peptides also suggest the formation of a small hydrophobic pocket centered on His-6. The structural features of DV3 are very similar to the reported solution structure of all-L-peptide vMIP-II (1-10) [M.P. Crump, E. Elisseeva, J. Gong, I. Clark-Lewis, B.D. Sykes, Structure/function of human herpesvirus-8 MIP-II (1-71) and the antagonist N-terminal segment (1-10), FEBS Lett. 489 (2001) 171], which is consistent with the notion that D- and L-enantiomeric peptides can adopt mirror image conformations. The NMR structures of the D-peptides provide a structural basis to understand their mechanism of action and design new peptidomimetic analogs to further explore the structure-activity relationship of D-peptide ligand binding to CXCR4.« less

In vitro inhibition of African swine fever virus-topoisomerase II disrupts viral replication.

PubMed

Freitas, Ferdinando B; Frouco, Gonçalo; Martins, Carlos; Leitão, Alexandre; Ferreira, Fernando

2016-10-01

African swine fever virus (ASFV) is the etiological agent of a highly-contagious and fatal disease of domestic pigs, leading to serious socio-economic impact in affected countries. To date, neither a vaccine nor a selective anti-viral drug are available for prevention or treatment of African swine fever (ASF), emphasizing the need for more detailed studies at the role of ASFV proteins involved in viral DNA replication and transcription. Notably, ASFV encodes for a functional type II topoisomerase (ASFV-Topo II) and we recently showed that several fluoroquinolones (bacterial DNA topoisomerase inhibitors) fully abrogate ASFV replication in vitro. Here, we report that ASFV-Topo II gene is actively transcribed throughout infection, with transcripts being detected as early as 2 hpi and reaching a maximum peak concentration around 16 hpi, when viral DNA synthesis, transcription and translation are more active. siRNA knockdown experiments showed that ASFV-Topo II plays a critical role in viral DNA replication and gene expression, with transfected cells presenting lower viral transcripts (up to 89% decrease) and reduced cytopathic effect (-66%) when compared to the control group. Further, a significant decrease in the number of both infected cells (75.5%) and viral factories per cell and in virus yields (up to 99.7%, 2.5 log) was found only in cells transfected with siRNA targeting ASFV-Topo II. We also demonstrate that a short exposure to enrofloxacin during the late phase of infection (from 15 to 1 hpi) induces fragmentation of viral genomes, whereas no viral genomes were detected when enrofloxacin was added from the early phase of infection (from 2 to 16 hpi), suggesting that fluoroquinolones are ASFV-Topo II poisons. Altogether, our results demonstrate that ASFV-Topo II enzyme has an essential role during viral genome replication and transcription, emphasizing the idea that this enzyme can be a potential target for drug and vaccine development against ASF. Copyright © 2016 Elsevier B.V. All rights reserved.

Encoding electric signals by Gymnotus omarorum: heuristic modeling of tuberous electroreceptor organs.

PubMed

Cilleruelo, Esteban R; Caputi, Angel Ariel

2012-01-24

The role of different substructures of electroreceptor organs in signal encoding was explored using a heuristic computational model. This model consists of four modules representing the pre-receptor structures, the transducer cells, the synapses and the afferent fiber, respectively. Simulations reproduced previously obtained experimental data. We showed that different electroreceptor types described in the literature can be qualitative modeled with the same set of equations by changing only two parameters, one affecting the filtering properties of the pre-receptor, and the other affecting the transducer module. We studied the responses of different electroreceptor types to natural stimuli using simulations derived from an experimentally-obtained database in which the fish were exposed to resistive or capacitive objects. Our results indicate that phase and frequency spectra are differentially encoded by different subpopulations of tuberous electroreceptors. A different type of receptor responses to the same input is a necessary condition for encoding a multidimensional space of stimuli as in the waveform of the EOD. Our simulation analysis suggested that the electroreceptive mosaic may perform a waveform analysis of electrosensory signals. As in color vision or tactile texture perception, a secondary attribute, "electric color" may be encoded as a parallel activity of various electroreceptor types. This article is part of a Special Issue entitled Neural Coding. Copyright © 2011 Elsevier B.V. All rights reserved.

Human autoantibody to topoisomerase II.

PubMed

Hoffmann, A; Heck, M M; Bordwell, B J; Rothfield, N F; Earnshaw, W C

1989-02-01

The rheumatic diseases are characterized by the production of autoantibodies that are usually directed against components of the cell nucleus. In this communication, we describe autoantibodies that recognize DNA topoisomerase II (anti-topoII) present in the serum of a patient with systemic lupus erythematosus. Several lines of evidence indicate that this antibody recognizes topoisomerase II. First, it binds to the native enzyme in soluble extracts prepared from isolated chromosomes and effectively depletes such extracts of active enzyme. Second, the serum binds to topoisomerase II in immunoblots of mitotic chromosomes and chromosome scaffolds. Finally, the antiserum binds strongly to a fusion protein encoded by a cloned cDNA and expressed in Escherichia coli that (based on immunological evidence) represents the carboxy-terminal portion of chicken topoisomerase II. Autoantibodies such as the one described here may provide useful reagents for the study of human topoisomerase II.

SU43. The Effect of Implementation Intention on Different Types of Prospective Memory Performance in Patients With Schizophrenia

PubMed Central

Wang, Ya; Liu, Lu-lu; Gan, Ming-yuan; Tan, Shu-ping; Shum, David; Chan, Raymond

2017-01-01

Abstract Background: Prospective memory (PM) refers to remembering to execute a planned intention in the future, which can been divided as event-based PM (focal, nonfocal) and time-based PM according to the nature of the cue. Focal event-based PM, where the ongoing task requires processing of the characteristics of PM cues, has been found to be benefited from implementation intention (II, ie, an encoding strategy in the format of “if I see X, then I will do Y”). However, to date, it is unclear whether implementation intention can produce a positive effect on nonfocal event-based PM (where the ongoing task is irrelevant with the PM cues) and time-based PM. Moreover, patients with schizophrenia (SCZ) were found to have impairments in these types of PM, and few studies have been conducted to examine the effect of II on these types of PM. This study investigated whether (and how) implementation intention can improve nonfocal event-based PM and time-based PM performance in patients with SCZ. Methods: Forty-two patients with SCZ and 42 healthy control participants were administered both computerized nonfocal event-based PM task and time-based PM task. Patients and healthy controls were further randomly allocated to implementation intention condition (N = 21) and typical instruction condition (N = 21). Results: Patients with SCZ in the implementation intention group showed higher PM accuracy than the typical instruction group in both nonfocal event-based PM task (0.51 ± 0.32 vs 0.19 ± 0.29, t(40) = 3.39, P = .002) and time-based PM task (0.72 ± 0.31 vs 0.39 ± 0.40, t(40) = 2.98, P = .005). Similarly, healthy controls in the II group also showed better PM performance than the typical instruction group in both tasks (all P’s < 0.05). Time check frequency of time-based PM task in the II group of all the participants was significantly higher than the typical instruction group. Conclusion: Implementation intention is an effective strategy for improving different types of PM performance in patients with schizophrenia and can be applied for clinical settings.

Subtypes of the Type II Pit Pattern Reflect Distinct Molecular Subclasses in the Serrated Neoplastic Pathway.

PubMed

Aoki, Hironori; Yamamoto, Eiichiro; Yamano, Hiro-O; Sugai, Tamotsu; Kimura, Tomoaki; Tanaka, Yoshihito; Matsushita, Hiro-O; Yoshikawa, Kenjiro; Takagi, Ryo; Harada, Eiji; Nakaoka, Michiko; Yoshida, Yuko; Harada, Taku; Sudo, Gota; Eizuka, Makoto; Yorozu, Akira; Kitajima, Hiroshi; Niinuma, Takeshi; Kai, Masahiro; Nojima, Masanori; Suzuki, Hiromu; Nakase, Hiroshi

2018-03-15

Colorectal serrated lesions (SLs) are important premalignant lesions whose clinical and biological features are not fully understood. We aimed to establish accurate colonoscopic diagnosis and treatment of SLs through evaluation of associations among the morphological, pathological, and molecular characteristics of SLs. A total of 388 premalignant and 18 malignant colorectal lesions were studied. Using magnifying colonoscopy, microsurface structures were assessed based on Kudo's pit pattern classification system, and the Type II pit pattern was subcategorized into classical Type II, Type II-Open (Type II-O) and Type II-Long (Type II-L). BRAF/KRAS mutations and DNA methylation of CpG island methylator phenotype (CIMP) markers (MINT1, - 2, - 12, - 31, p16, and MLH1) were analyzed through pyrosequencing. Type II-O was tightly associated with sessile serrated adenoma/polyps (SSA/Ps) with BRAF mutation and CIMP-high. Most lesions with simple Type II or Type II-L were hyperplastic polyps, while mixtures of Type II or Type II-L plus more advanced pit patterns (III/IV) were characteristic of traditional serrated adenomas (TSAs). Type II-positive TSAs frequently exhibited BRAF mutation and CIMP-low, while Type II-L-positive TSAs were tightly associated with KRAS mutation and CIMP-low. Analysis of lesions containing both premalignant and cancerous components suggested Type II-L-positive TSAs may develop into KRAS-mutated/CIMP-low/microsatellite stable cancers, while Type II-O-positive SSA/Ps develop into BRAF-mutated/CIMP-high/microsatellite unstable cancers. These results suggest that Type II subtypes reflect distinct molecular subclasses in the serrated neoplasia pathway and that they could be useful hallmarks for identifying SLs at high risk of developing into CRC.

Mechanisms Used for Genomic Proliferation by Thermophilic Group II Introns

PubMed Central

Mohr, Georg; Ghanem, Eman; Lambowitz, Alan M.

2010-01-01

Mobile group II introns, which are found in bacterial and organellar genomes, are site-specific retroelments hypothesized to be evolutionary ancestors of spliceosomal introns and retrotransposons in higher organisms. Most bacteria, however, contain no more than one or a few group II introns, making it unclear how introns could have proliferated to higher copy numbers in eukaryotic genomes. An exception is the thermophilic cyanobacterium Thermosynechococcus elongatus, which contains 28 closely related copies of a group II intron, constituting ∼1.3% of the genome. Here, by using a combination of bioinformatics and mobility assays at different temperatures, we identified mechanisms that contribute to the proliferation of T. elongatus group II introns. These mechanisms include divergence of DNA target specificity to avoid target site saturation; adaptation of some intron-encoded reverse transcriptases to splice and mobilize multiple degenerate introns that do not encode reverse transcriptases, leading to a common splicing apparatus; and preferential insertion within other mobile introns or insertion elements, which provide new unoccupied sites in expanding non-essential DNA regions. Additionally, unlike mesophilic group II introns, the thermophilic T. elongatus introns rely on elevated temperatures to help promote DNA strand separation, enabling access to a larger number of DNA target sites by base pairing of the intron RNA, with minimal constraint from the reverse transcriptase. Our results provide insight into group II intron proliferation mechanisms and show that higher temperatures, which are thought to have prevailed on Earth during the emergence of eukaryotes, favor intron proliferation by increasing the accessibility of DNA target sites. We also identify actively mobile thermophilic introns, which may be useful for structural studies, gene targeting in thermophiles, and as a source of thermostable reverse transcriptases. PMID:20543989

The multiscale classification system and grid encoding mode of ecological land in China

NASA Astrophysics Data System (ADS)

Wang, Jing; Liu, Aixia; Lin, Yifan

2017-10-01

Ecological land provides goods and services that have direct or indirect benefic to eco-environment and human welfare. In recent years, researches on ecological land have become important in the field of land changes and ecosystem management. In the study, a multi-scale classification scheme of ecological land was developed for land management based on combination of the land-use classification and the ecological function zoning in China, including eco-zone, eco-region, eco-district, land ecosystem, and ecological land-use type. The geographical spatial unit leads toward greater homogeneity from macro to micro scale. The term "ecological land-use type" is the smallest one, being important to maintain the key ecological processes in land ecosystem. Ecological land-use type was categorized into main-functional and multi-functional ecological land-use type according to its ecological function attributes and production function attributes. Main-functional type was defined as one kind of land-use type mainly providing ecological goods and function attributes, such as river, lake, swampland, shoaly land, glacier and snow, while multi-functional type not only providing ecological goods and function attributes but also productive goods and function attributes, such as arable land, forestry land, and grassland. Furthermore, a six-level grid encoding mode was proposed for modern management of ecological land and data update under cadastral encoding. The six-level irregular grid encoding from macro to micro scale included eco-zone, eco-region, eco-district, cadastral area, land ecosystem, land ownership type, ecological land-use type, and parcel. Besides, the methodologies on ecosystem management were discussed for integrated management of natural resources in China.

Interaction Between Encoding and Retrieval Operations in Cued Recall

ERIC Educational Resources Information Center

Fisher, Ronald P.; Craik, Fergus I. M.

1977-01-01

Three experiments are described in which the qualitative nature of memorial processing was manipulated at both input (encoding) and output (retrieval). As in earlier research, it was found that retention levels were highest when the same type of information was used as a retrieval cue. Concludes that the notions of encoding specificity and depth…

The recent emergence in hospitals of multidrug-resistant community-associated sequence type 1 and spa type t127 methicillin-resistant Staphylococcus aureus investigated by whole-genome sequencing: Implications for screening

PubMed Central

Earls, Megan R.; Kinnevey, Peter M.; Brennan, Gráinne I.; Lazaris, Alexandros; Skally, Mairead; O’Connell, Brian; Humphreys, Hilary; Shore, Anna C.

2017-01-01

Community-associated spa type t127/t922 methicillin-resistant Staphylococcus aureus (MRSA) prevalence increased from 1%-7% in Ireland between 2010–2015. This study tracked the spread of 89 such isolates from June 2013-June 2016. These included 78 healthcare-associated and 11 community associated-MRSA isolates from a prolonged hospital outbreak (H1) (n = 46), 16 other hospitals (n = 28), four other healthcare facilities (n = 4) and community-associated sources (n = 11). Isolates underwent antimicrobial susceptibility testing, DNA microarray profiling and whole-genome sequencing. Minimum spanning trees were generated following core-genome multilocus sequence typing and pairwise single nucleotide variation (SNV) analysis was performed. All isolates were sequence type 1 MRSA staphylococcal cassette chromosome mec type IV (ST1-MRSA-IV) and 76/89 were multidrug-resistant. Fifty isolates, including 40/46 from H1, were high-level mupirocin-resistant, carrying a conjugative 39 kb iles2-encoding plasmid. Two closely related ST1-MRSA-IV strains (I and II) and multiple sporadic strains were identified. Strain I isolates (57/89), including 43/46 H1 and all high-level mupirocin-resistant isolates, exhibited ≤80 SNVs. Two strain I isolates from separate H1 healthcare workers differed from other H1/strain I isolates by 7–47 and 12–53 SNVs, respectively, indicating healthcare worker involvement in this outbreak. Strain II isolates (19/89), including the remaining H1 isolates, exhibited ≤127 SNVs. For each strain, the pairwise SNVs exhibited by healthcare-associated and community-associated isolates indicated recent transmission of ST1-MRSA-IV within and between multiple hospitals, healthcare facilities and communities in Ireland. Given the interchange between healthcare-associated and community-associated isolates in hospitals, the risk factors that inform screening for MRSA require revision. PMID:28399151

New Ultra-High Sensitivity, Absolute, Linear, and Rotary Encoders

NASA Technical Reports Server (NTRS)

Leviton, Douglas B.

1998-01-01

Several new types of absolute optical encoders of both rotary and linear function are discussed. The means for encoding are complete departures from conventional optical encoders and offer advantages of compact form, immunity to damage-induced dropouts of position information, and about an order of magnitude higher sensitivity over what is commercially available. Rotary versions have sensitivity from 0.02 arcseconds down to 0.003 arcsecond while linear models have sensitivity of 10 nm.

Differential expression of α-L-arabinofuranosidases during maize (Zea mays L.) root elongation.

PubMed

Kozlova, Liudmila V; Gorshkov, Oleg V; Mokshina, Natalia E; Gorshkova, Tatyana A

2015-05-01

Specific α- l -arabinofuranosidases are involved in the realisation of elongation growth process in cells with type II cell walls. Elongation growth in a plant cell is largely based on modification of the cell wall. In type II cell walls, the Ara/Xyl ratio is known to decrease during elongation due to the partial removal of Ara residues from glucuronoarabinoxylan. We searched within the maize genome for the genes of all predicted α-L-arabinofuranosidases that may be responsible for such a process and related their expression to the activity of the enzyme and the amount of free arabinose measured in six zones of a growing maize root. Eight genes of the GH51 family (ZmaABFs) and one gene of the GH3 family (ZmaARA-I) were identified. The abundance of ZmaABF1 and 3-6 transcripts was highly correlated with the measured enzymatic activity and free arabinose content that significantly increased during elongation. The transcript abundances also coincided with the pattern of changes in the Ara/Xyl ratio of the xylanase-extractable glucuronoarabinoxylan described in previous studies. The expression of ZmaABF3, 5 and 6 was especially up-regulated during elongation although corresponding proteins are devoid of the catalytic glutamate at the proper position. ZmaABF2 transcripts were specifically enriched in the root cap and meristem. A single ZmaARA-I gene was not expressed as a whole gene but instead as splice variants that encode the C-terminal end of the protein. Changes in the ZmaARA-I transcript level were rather moderate and had no significant correlation with free arabinose content. Thus, elongation growth of cells with type II cell walls is accompanied by the up-regulation of specific and predicted α-L-arabinofuranosidase genes, and the corresponding activity is indeed pronounced and is important for the modification of glucuronoarabinoxylan, which plays a key role in the modification of the cell wall supramolecular organisation.

Structure of adenovirus bound to cellular receptor car

DOEpatents

Freimuth, Paul I.

2007-01-02

Disclosed is a mutant CAR-DI-binding adenovirus which has a genome comprising one or more mutations in sequences which encode the fiber protein knob domain wherein the mutation causes the encoded viral particle to have a significantly weakened binding affinity for CAR-DI relative to wild-type adenovirus. Such mutations may be in sequences which encode either the AB loop, or the HI loop of the fiber protein knob domain. Specific residues and mutations are described. Also disclosed is a method for generating a mutant adenovirus which is characterized by a receptor binding affinity or specificity which differs substantially from wild type.

Adaptive evolution in the Arabidopsis MADS-box gene family inferred from its complete resolved phylogeny

PubMed Central

Martínez-Castilla, León Patricio; Alvarez-Buylla, Elena R.

2003-01-01

Gene duplication is a substrate of evolution. However, the relative importance of positive selection versus relaxation of constraints in the functional divergence of gene copies is still under debate. Plant MADS-box genes encode transcriptional regulators key in various aspects of development and have undergone extensive duplications to form a large family. We recovered 104 MADS sequences from the Arabidopsis genome. Bayesian phylogenetic trees recover type II lineage as a monophyletic group and resolve a branching sequence of monophyletic groups within this lineage. The type I lineage is comprised of several divergent groups. However, contrasting gene structure and patterns of chromosomal distribution between type I and II sequences suggest that they had different evolutionary histories and support the placement of the root of the gene family between these two groups. Site-specific and site-branch analyses of positive Darwinian selection (PDS) suggest that different selection regimes could have affected the evolution of these lineages. We found evidence for PDS along the branch leading to flowering time genes that have a direct impact on plant fitness. Sites with high probabilities of having been under PDS were found in the MADS and K domains, suggesting that these played important roles in the acquisition of novel functions during MADS-box diversification. Detected sites are targets for further experimental analyses. We argue that adaptive changes in MADS-domain protein sequences have been important for their functional divergence, suggesting that changes within coding regions of transcriptional regulators have influenced phenotypic evolution of plants. PMID:14597714

Differences in virulence genes and genome patterns of mastitis-associated Staphylococcus aureus among goat, cow, and human isolates in Taiwan.

PubMed

Chu, Chishih; Wei, Yajiun; Chuang, Shih-Te; Yu, Changyou; Changchien, Chih-Hsuan; Su, Yaochi

2013-03-01

A total of 117 mastitis-associated Staphylococcus aureus isolates from cow, goat, and human patients were analyzed for differences in antibiotic susceptibility, virulence genes, and genotypes using accessory gene regulator (agr) typing, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Multidrug-resistant (MDR) S. aureus were commonly found in all sources, though they were predominantly found in human and goat isolates. Almost 70% of the isolates were resistant to ampicillin and penicillin. Host-associated virulence genes were identified as follows: tst, a gene encoding toxic shock syndrome toxin, was found in goat isolates; lukED and lukM, genes encoding leukocidin, found in cow isolates; lukPV, a gene encoding leukocidin, found in human isolates; and eta, a gene encoding for exfoliative toxin, found in both human and cow isolates. All four types of hemolysin, α, β, γ, and δ, were identified in human isolates, three types (α, γ, and δ), were identified in cow isolates, and two types (α and δ) were identified in goat isolates. Agr-typing determined agr1 to be the main subtype in human and cow isolates. PFGE and MLST analysis revealed the presence of diverse genotypes among the three sources. In conclusion, mastitis-associated, genetically diverse strains of MDR S. aureus differed in virulence genes among human, cow, and goat isolates.

FZD4S, a splicing variant of frizzled-4, encodes a soluble-type positive regulator of the WNT signaling pathway.

PubMed

Sagara, N; Kirikoshi, H; Terasaki, H; Yasuhiko, Y; Toda, G; Shiokawa, K; Katoh, M

2001-04-06

Frizzled-1 (FZD1)-FZD10 are seven-transmembrane-type WNT receptors, and SFRP1-SFRP5 are soluble-type WNT antagonists. These molecules are encoded by mutually distinct genes. We have previously isolated and characterized the 7.7-kb FZD4 mRNA, encoding a seven-transmembrane receptor with the extracellular cysteine-rich domain (CRD). Here, we have cloned and characterized FZD4S, a splicing variant of the FZD4 gene. FZD4S, corresponding to the 10.0-kb FZD4 mRNA, consisted of exon 1, intron 1, and exon 2 of the FZD4 gene. FZD4S encoded a soluble-type polypeptide with the N-terminal part of CRD, and was expressed in human fetal kidney. Injection of synthetic FZD4S mRNA into the ventral marginal zone of Xenopus embryos at the 4-cell stage did not induce axis duplication by itself, but augmented the axis duplication potential of coinjected Xwnt-8 mRNA. These results indicate that the FZD4 gene gives rise to soluble-type FZD4S as well as seven-transmembrane-type FZD4 due to alternative splicing, and strongly suggest that FZD4S plays a role as a positive regulator of the WNT signaling pathway. Copyright 2001 Academic Press.

CYTOMEGALOVIRUS VECTORS VIOLATE CD8+ T CELL EPITOPE RECOGNITION PARADIGMS

PubMed Central

Hansen, Scott G.; Sacha, Jonah B.; Hughes, Colette M.; Ford, Julia C.; Burwitz, Benjamin J.; Scholz, Isabel; Gilbride, Roxanne M.; Lewis, Matthew S.; Gilliam, Awbrey N.; Ventura, Abigail B.; Malouli, Daniel; Xu, Guangwu; Richards, Rebecca; Whizin, Nathan; Reed, Jason S.; Hammond, Katherine B.; Fischer, Miranda; Turner, John M.; Legasse, Alfred W.; Axthelm, Michael K.; Edlefsen, Paul T.; Nelson, Jay A.; Lifson, Jeffrey D.; Früh, Klaus; Picker, Louis J.

2013-01-01

CD8+ T cell responses focus on a small fraction of pathogen- or vaccine-encoded peptides, and for some pathogens, these restricted recognition hierarchies limit the effectiveness of anti-pathogen immunity. We found that simian immunodeficiency virus (SIV) protein-expressing Rhesus Cytomegalovirus (RhCMV) vectors elicit SIV-specific CD8+ T cells that recognize unusual, diverse and highly promiscuous epitopes, including dominant responses to epitopes restricted by class II major histocompatibility complex (MHC) molecules. Induction of canonical SIV epitope-specific CD8+ T cell responses is suppressed by the RhCMV-encoded Rh189 (US11) gene, and the promiscuous MHC class I- and class II-restricted CD8+ T cell responses only occur in the absence of the Rh157.4-.6 (UL128-131) genes. Thus, CMV vectors can be genetically programmed to achieve distinct patterns of CD8+ T cell epitope recognition. PMID:23704576

Random codon re-encoding induces stable reduction of replicative fitness of Chikungunya virus in primate and mosquito cells.

PubMed

Nougairede, Antoine; De Fabritus, Lauriane; Aubry, Fabien; Gould, Ernest A; Holmes, Edward C; de Lamballerie, Xavier

2013-02-01

Large-scale codon re-encoding represents a powerful method of attenuating viruses to generate safe and cost-effective vaccines. In contrast to specific approaches of codon re-encoding which modify genome-scale properties, we evaluated the effects of random codon re-encoding on the re-emerging human pathogen Chikungunya virus (CHIKV), and assessed the stability of the resultant viruses during serial in cellulo passage. Using different combinations of three 1.4 kb randomly re-encoded regions located throughout the CHIKV genome six codon re-encoded viruses were obtained. Introducing a large number of slightly deleterious synonymous mutations reduced the replicative fitness of CHIKV in both primate and arthropod cells, demonstrating the impact of synonymous mutations on fitness. Decrease of replicative fitness correlated with the extent of re-encoding, an observation that may assist in the modulation of viral attenuation. The wild-type and two re-encoded viruses were passaged 50 times either in primate or insect cells, or in each cell line alternately. These viruses were analyzed using detailed fitness assays, complete genome sequences and the analysis of intra-population genetic diversity. The response to codon re-encoding and adaptation to culture conditions occurred simultaneously, resulting in significant replicative fitness increases for both re-encoded and wild type viruses. Importantly, however, the most re-encoded virus failed to recover its replicative fitness. Evolution of these viruses in response to codon re-encoding was largely characterized by the emergence of both synonymous and non-synonymous mutations, sometimes located in genomic regions other than those involving re-encoding, and multiple convergent and compensatory mutations. However, there was a striking absence of codon reversion (<0.4%). Finally, multiple mutations were rapidly fixed in primate cells, whereas mosquito cells acted as a brake on evolution. In conclusion, random codon re-encoding provides important information on the evolution and genetic stability of CHIKV viruses and could be exploited to develop a safe, live attenuated CHIKV vaccine.

Light-modulated abundance of an mRNA encoding a calmodulin-regulated, chromatin-associated NTPase in pea

NASA Technical Reports Server (NTRS)

Hsieh, H. L.; Tong, C. G.; Thomas, C.; Roux, S. J.

1996-01-01

A CDNA encoding a 47 kDa nucleoside triphosphatase (NTPase) that is associated with the chromatin of pea nuclei has been cloned and sequenced. The translated sequence of the cDNA includes several domains predicted by known biochemical properties of the enzyme, including five motifs characteristic of the ATP-binding domain of many proteins, several potential casein kinase II phosphorylation sites, a helix-turn-helix region characteristic of DNA-binding proteins, and a potential calmodulin-binding domain. The deduced primary structure also includes an N-terminal sequence that is a predicted signal peptide and an internal sequence that could serve as a bipartite-type nuclear localization signal. Both in situ immunocytochemistry of pea plumules and immunoblots of purified cell fractions indicate that most of the immunodetectable NTPase is within the nucleus, a compartment proteins typically reach through nuclear pores rather than through the endoplasmic reticulum pathway. The translated sequence has some similarity to that of human lamin C, but not high enough to account for the earlier observation that IgG against human lamin C binds to the NTPase in immunoblots. Northern blot analysis shows that the NTPase MRNA is strongly expressed in etiolated plumules, but only poorly or not at all in the leaf and stem tissues of light-grown plants. Accumulation of NTPase mRNA in etiolated seedlings is stimulated by brief treatments with both red and far-red light, as is characteristic of very low-fluence phytochrome responses. Southern blotting with pea genomic DNA indicates the NTPase is likely to be encoded by a single gene.

Cyclomodulins in Urosepsis Strains of Escherichia coli▿

PubMed Central

Dubois, Damien; Delmas, Julien; Cady, Anne; Robin, Frédéric; Sivignon, Adeline; Oswald, Eric; Bonnet, Richard

2010-01-01

Determinants of urosepsis in Escherichia coli remain incompletely defined. Cyclomodulins (CMs) are a growing functional family of toxins that hijack the eukaryotic cell cycle. Four cyclomodulin types are actually known in E. coli: cytotoxic necrotizing factors (CNFs), cycle-inhibiting factor (Cif), cytolethal distending toxins (CDTs), and the pks-encoded toxin. In the present study, the distribution of CM-encoding genes and the functionality of these toxins were investigated in 197 E. coli strains isolated from patients with community-acquired urosepsis (n = 146) and from uninfected subjects (n = 51). This distribution was analyzed in relation to the phylogenetic background, clinical origin, and antibiotic resistance of the strains. It emerged from this study that strains harboring the pks island and the cnf1 gene (i) were strongly associated with the B2 phylogroup (P, <0.001), (ii) frequently harbored both toxin-encoded genes in phylogroup B2 (33%), and (iii) were predictive of a urosepsis origin (P, <0.001 to 0.005). However, the prevalences of the pks island among phylogroup B2 strains, in contrast to those of the cnf1 gene, were not significantly different between fecal and urosepsis groups, suggesting that the pks island is more important for the colonization process and the cnf1 gene for virulence. pks- or cnf1-harboring strains were significantly associated with susceptibility to antibiotics (amoxicillin, cotrimoxazole, and quinolones [P, <0.001 to 0.043]). Otherwise, only 6% and 1% of all strains harbored the cdtB and cif genes, respectively, with no particular distribution by phylogenetic background, antimicrobial susceptibility, or clinical origin. PMID:20375237

Mimicry by asx- and ST-turns of the four main types of beta-turn in proteins.

PubMed

Duddy, William J; Nissink, J Willem M; Allen, Frank H; Milner-White, E James

2004-11-01

Hydrogen-bonded beta-turns in proteins occur in four categories: type I (the most common), type II, type II', and type I'. Asx-turns resemble beta-turns, in that both have an NH. . .OC hydrogen bond forming a ring of 10 atoms. Serine and threonine side chains also commonly form hydrogen-bonded turns, here called ST-turns. Asx-turns and ST-turns can be categorized into four classes, based on side chain rotamers and the conformation of the central turn residue, which are geometrically equivalent to the four types of beta-turns. We propose asx- and ST-turns be named using the type I, II, I', and II' beta-turn nomenclature. Using this, the frequency of occurrence of both asx- and ST-turns is: type II' > type I > type II > type I', whereas for beta-turns it is type I > type II > type I' > type II'. Almost all type II asx-turns occur as a recently described three residue feature named an asx-nest.

HSD3B2, HSD17B1, HSD17B2, ESR1, ESR2 and AR expression in infertile women with endometriosis.

PubMed

Osiński, Maciej; Wirstlein, Przemysław; Wender-Ożegowska, Ewa; Mikołajczyk, Mateusz; Jagodziński, Paweł Piotr; Szczepańska, Małgorzata

2018-01-01

The development of endometriosis is associated with changes in the expression of genes encoding the 3β-hydroxysteroid dehydrogenase type II (HSD3B2) and 17β-hydroxysteroid dehydrogenase type II (HSD17B2), estrogen receptors 1 (ESR1) and 2 (ESR2) and the androgen receptor (AR). However, little is known about the expression of HSD3B2, HSD17B1, HSD17B2, ESR1 ESR2 and AR during the endometrial phases in eutopic endometrium from infertile women with endometriosis. Using RT-qPCR analysis, we assessed the expression of the studied genes in the follicular and luteal phases in eutopic endometrium from fertile women (n = 17) and infertile women (n = 35) with endometriosis. In the mid-follicular eutopic endometrium, we observed a significant increase in HSD3B2 transcript levels in all infertile women with endometriosis (p = 0.003), in infertile women with stage I/II endometriosis (p = 0.008) and in infertile women with stage III/IV endometriosis (p = 0.009) compared to all fertile women. There was a significant increase in ESR1 tran-scripts in all infertile women with endometriosis (p = 0.008) and in infertile women with stage I/II endometriosis (p = 0.019) and in infertile women with stage III/IV endometriosis (p = 0.023) compared to all fertile women. In the mid-luteal eutopic endometrium, we did not observe significant differences in HSD3B2, HSD17B1, HSD17B2, ESR1, ESR2 and AR transcripts between infertile women with endometriosis and fertile women. Observed significant increase in HSD3B2 and ESR1 transcripts in follicular eutopic endometrium from infer-tile women with endometriosis may be related to abnormal biological effect of E2 in endometrium, further affecting the development of human embryos.

Structure of the Bacillus subtilis phage SPO1-encoded type II DNA-binding protein TF1 in solution.

PubMed

Jia, X; Grove, A; Ivancic, M; Hsu, V L; Geiduscheck, E P; Kearns, D R

1996-10-25

The solution structure of a type II DNA-binding protein, the bacteriophage SPO1-encoded transcription factor 1 (TF1), was determined using NMR spectroscopy. Selective 2H-labeling, 13C-labeling and isotopic heterodimers were used to distinguish contacts between and within monomers of the dimeric protein. A total of 1914 distance and dihedral angle constraints derived from NMR experiments were used in structure calculations using restrained molecular dynamics and simulated annealing protocols. The ensemble of 30 calculated structures has a root-mean-square deviation (r.m.s.d.) of 0.9 A, about the average structure for the backbone atoms, and 1.2 A for all heavy-atoms of the dimeric core (helices 1 and 2) and the beta-sheets. A severe helix distortion at residues 92-93 in the middle of helix 3 is associated with r.m.s.d. of approximately 1.5 A for the helix 3 backbone. Deviations of approximately 5 A or larger are noted for the very flexible beta-ribbon arms that constitute part of a proposed DNA-binding region. A structural model of TF1 has been calculated based on the previously reported crystal structure of the homologous HU protein and this model was used as the starting structure for calculations. A comparison between the calculated average solution structure of TF1 and a solution structure of HU indicates a similarity in the dimeric core (excluding the nine amino acid residue tail) with pairwise deviations of 2 to 3 A. The largest deviations between the average structure and the HU solution structure were found in the beta-ribbon arms, as expected. A 4 A deviation is found at residue 15 of TF1 which is in a loop connecting two helical segments; it has been reported that substitution of Glu15 by Gly increases the thermostability of TF1. The homology between TF1 and other proteins of this family leads us to anticipate similar tertiary structures.

Uncertainty Considerations for Ballistic Limit Equations

NASA Technical Reports Server (NTRS)

Schonberg, W. P.; Evans, H. J.; Williamsen, J. E.; Boyer, R. L.; Nakayama, G. S.

2005-01-01

The overall risk for any spacecraft system is typically determined using a Probabilistic Risk Assessment (PRA). A PRA attempts to determine the overall risk associated with a particular mission by factoring in all known risks (and their corresponding uncertainties, if known) to the spacecraft during its mission. The threat to mission and human life posed by the mircro-meteoroid & orbital debris (MMOD) environment is one of the risks. NASA uses the BUMPER II program to provide point estimate predictions of MMOD risk for the Space Shuttle and the International Space Station. However, BUMPER II does not provide uncertainty bounds or confidence intervals for its predictions. With so many uncertainties believed to be present in the models used within BUMPER II, providing uncertainty bounds with BUMPER II results would appear to be essential to properly evaluating its predictions of MMOD risk. The uncertainties in BUMPER II come primarily from three areas: damage prediction/ballistic limit equations, environment models, and failure criteria definitions. In order to quantify the overall uncertainty bounds on MMOD risk predictions, the uncertainties in these three areas must be identified. In this paper, possible approaches through which uncertainty bounds can be developed for the various damage prediction and ballistic limit equations encoded within the shuttle and station versions of BUMPER II are presented and discussed. We begin the paper with a review of the current approaches used by NASA to perform a PRA for the Space Shuttle and the International Space Station, followed by a review of the results of a recent sensitivity analysis performed by NASA using the shuttle version of the BUMPER II code. Following a discussion of the various equations that are encoded in BUMPER II, we propose several possible approaches for establishing uncertainty bounds for the equations within BUMPER II. We conclude with an evaluation of these approaches and present a recommendation regarding which of them would be the most appropriate to follow.

A transcriptomic approach to search for novel phenotypic regulators in McArdle disease.

PubMed

Nogales-Gadea, Gisela; Consuegra-García, Inés; Rubio, Juan C; Arenas, Joaquin; Cuadros, Marc; Camara, Yolanda; Torres-Torronteras, Javier; Fiuza-Luces, Carmen; Lucia, Alejandro; Martín, Miguel A; García-Arumí, Elena; Andreu, Antoni L

2012-01-01

McArdle disease is caused by lack of glycogen phosphorylase (GP) activity in skeletal muscle. Patients experience exercise intolerance, presenting as early fatigue and contractures. In this study, we investigated the effects produced by a lack of GP on several genes and proteins of skeletal muscle in McArdle patients. Muscle tissue of 35 patients and 7 healthy controls were used to identify abnormalities in the patients' transcriptomic profile using low-density arrays. Gene expression was analyzed for the influence of variables such as sex and clinical severity. Differences in protein expression were studied by immunoblotting and 2D electrophoresis analysis, and protein complexes were examined by two-dimensional, blue native gel electrophoresis (BN-PAGE). A number of genes including those encoding acetyl-coA carboxylase beta, m-cadherin, calpain III, creatine kinase, glycogen synthase (GS), and sarcoplasmic reticulum calcium ATPase 1 (SERCA1), were found to be downregulated in patients. Specifically, compared to controls, GS and SERCA1 proteins were reduced by 50% and 75% respectively; also, unphosphorylated GS and SERCA1 were highly downregulated. On BN-PAGE analysis, GP was present with GS in two muscle protein complexes. Our findings revealed some issues that could be important in understanding the physiological consequences of McArdle disease: (i) SERCA1 downregulation in patients could result in impaired calcium transport in type II (fast-twitch) muscle fibers, leading to early fatigability during exercise tasks involving type II fibers (which mostly use glycolytic metabolism), i.e. isometric exercise, lifting weights or intense dynamic exercise (stair climbing, bicycling, walking at a very brisk pace), (ii) GP and GS were found together in two protein complexes, which suggests a new regulatory mechanism in the activity of these glycogen enzymes.

Evolution of group I introns in Porifera: new evidence for intron mobility and implications for DNA barcoding.

PubMed

Schuster, Astrid; Lopez, Jose V; Becking, Leontine E; Kelly, Michelle; Pomponi, Shirley A; Wörheide, Gert; Erpenbeck, Dirk; Cárdenas, Paco

2017-03-20

Mitochondrial introns intermit coding regions of genes and feature characteristic secondary structures and splicing mechanisms. In metazoans, mitochondrial introns have only been detected in sponges, cnidarians, placozoans and one annelid species. Within demosponges, group I and group II introns are present in six families. Based on different insertion sites within the cox1 gene and secondary structures, four types of group I and two types of group II introns are known, which can harbor up to three encoding homing endonuclease genes (HEG) of the LAGLIDADG family (group I) and/or reverse transcriptase (group II). However, only little is known about sponge intron mobility, transmission, and origin due to the lack of a comprehensive dataset. We analyzed the largest dataset on sponge mitochondrial group I introns to date: 95 specimens, from 11 different sponge genera which provided novel insights into the evolution of group I introns. For the first time group I introns were detected in four genera of the sponge family Scleritodermidae (Scleritoderma, Microscleroderma, Aciculites, Setidium). We demonstrated that group I introns in sponges aggregate in the most conserved regions of cox1. We showed that co-occurrence of two introns in cox1 is unique among metazoans, but not uncommon in sponges. However, this combination always associates an active intron with a degenerating one. Earlier hypotheses of HGT were confirmed and for the first time VGT and secondary losses of introns conclusively demonstrated. This study validates the subclass Spirophorina (Tetractinellida) as an intron hotspot in sponges. Our analyses confirm that most sponge group I introns probably originated from fungi. DNA barcoding is discussed and the application of alternative primers suggested.

The 2016 revision of the WHO Classification of Central Nervous System Tumours: retrospective application to a cohort of diffuse gliomas.

PubMed

Rogers, Te Whiti; Toor, Gurvinder; Drummond, Katharine; Love, Craig; Field, Kathryn; Asher, Rebecca; Tsui, Alpha; Buckland, Michael; Gonzales, Michael

2018-03-01

The classification of central nervous system tumours has more recently been shaped by a focus on molecular pathology rather than histopathology. We re-classified 82 glial tumours according to the molecular-genetic criteria of the 2016 revision of the World Health Organization (WHO) Classification of Tumours of the Central Nervous System. Initial diagnoses and grading were based on the morphological criteria of the 2007 WHO scheme. Because of the impression of an oligodendroglial component on initial histological assessment, each tumour was tested for co-deletion of chromosomes 1p and 19q and mutations of isocitrate dehydrogenase (IDH-1 and 2) genes. Additionally, expression of proteins encoded by alpha-thalassemia X-linked mental retardation (ATRX) and TP53 genes was assessed by immunohistochemistry. We found that all but two tumours could be assigned to a specific category in the 2016 revision. The most common change in diagnosis was from oligoastrocytoma to specifically astrocytoma or oligodendroglioma. Analysis of progression free survival (PFS) for WHO grade II and III tumours showed that the objective criteria of the 2016 revision separated diffuse gliomas into three distinct molecular categories: chromosome 1p/19q co-deleted/IDH mutant, intact 1p/19q/IDH mutant and IDH wild type. No significant difference in PFS was found when comparing IDH mutant grade II and III tumours suggesting that IDH status is more informative than tumour grade. The segregation into distinct molecular sub-types that is achieved by the 2016 revision provides an objective evidence base for managing patients with grade II and III diffuse gliomas based on prognosis.

Far-red light photoacclimation (FaRLiP) in Synechococcus sp. PCC 7335. II.Characterization of phycobiliproteins produced during acclimation to far-red light.

PubMed

Ho, Ming-Yang; Gan, Fei; Shen, Gaozhong; Bryant, Donald A

2017-02-01

Phycobilisomes (PBS) are antenna complexes that harvest light for photosystem (PS) I and PS II in cyanobacteria and some algae. A process known as far-red light photoacclimation (FaRLiP) occurs when some cyanobacteria are grown in far-red light (FRL). They synthesize chlorophylls d and f and remodel PS I, PS II, and PBS using subunits paralogous to those produced in white light. The FaRLiP strain, Leptolyngbya sp. JSC-1, replaces hemidiscoidal PBS with pentacylindrical cores, which are produced when cells are grown in red or white light, with PBS with bicylindrical cores when cells are grown in FRL. This study shows that the PBS of another FaRLiP strain, Synechococcus sp. PCC 7335, are not remodeled in cells grown in FRL. Instead, cells grown in FRL produce bicylindrical cores that uniquely contain the paralogous allophycocyanin subunits encoded in the FaRLiP cluster, and these bicylindrical cores coexist with red-light-type PBS with tricylindrical cores. The bicylindrical cores have absorption maxima at 650 and 711 nm and a low-temperature fluorescence emission maximum at 730 nm. They contain ApcE2:ApcF:ApcD3:ApcD2:ApcD5:ApcB2 in the approximate ratio 2:2:4:6:12:22, and a structural model is proposed. Time course experiments showed that bicylindrical cores were detectable about 48 h after cells were transferred from RL to FRL and that synthesis of red-light-type PBS continued throughout a 21-day growth period. When considered in comparison with results for other FaRLiP cyanobacteria, the results here show that acclimation responses to FRL can differ considerably among FaRLiP cyanobacteria.

Insulin-like growth factors I and II in starry flounder (Platichthys stellatus): molecular cloning and differential expression during embryonic development.

PubMed

Xu, Yongjiang; Zang, Kun; Liu, Xuezhou; Shi, Bao; Li, Cunyu; Shi, Xueying

2015-02-01

In order to elucidate the possible roles of insulin-like growth factors I and II (IGF-I and IGF-II) in the embryonic development of Platichthys stellatus, their cDNAs were isolated and their spatial expression pattern in adult organs and temporal expression pattern throughout embryonic development were examined by quantitative real-time PCR assay. The IGF-I cDNA sequence was 1,268 bp in length and contained an open reading frame (ORF) of 558 bp, which encoded 185 amino acid residues. With respect to IGF-II, the full-length cDNA was 899 bp in length and contained a 648-bp ORF, which encoded 215 amino acid residues. The amino acid sequences of IGF-I and IGF-II exhibited high identities with their fish counterparts. The highest IGF-I mRNA level was found in the liver for both sexes, whereas the IGF-II gene was most abundantly expressed in female liver and male liver, gill, and brain. The sex-specific and spatial expression patterns of IGF-I and IGF-II mRNAs are thought to be related to the sexually dimorphic growth and development of starry flounder. Both IGF-I and IGF-II mRNAs were detected in unfertilized eggs, which indicated that IGF-I and IGF-II were parentally transmitted. Nineteen embryonic development stages were tested. IGF-I mRNA level remained high from unfertilized eggs to low blastula followed by a significant decrease at early gastrula and then maintained a lower level. In contrast, IGF-II mRNA level was low from unfertilized eggs to high blastula and peaked at low blastula followed by a gradual decrease. Moreover, higher levels of IGF-I mRNA than that of IGF-II were found from unfertilized eggs to high blastula, vice versa from low blastula to newly hatched larva, and the different expression pattern verified the differential roles of IGF-I and IGF-II in starry flounder embryonic development. These results could help in understanding the endocrine mechanism involved in the early development and growth of starry flounder.

Diagnostic value of succinate ubiquinone reductase activity in the identification of patients with mitochondrial DNA depletion.

PubMed

Hargreaves, P; Rahman, S; Guthrie, P; Taanman, J W; Leonard, J V; Land, J M; Heales, S J R

2002-02-01

Mitochondrial DNA (mtDNA) depletion syndrome (McKusick 251880) is characterized by a progressive quantitative loss of mtDNA resulting in severe mitochondrial dysfunction. A diagnosis of mtDNA depletion can only be confirmed after Southern blot analysis of affected tissue. Only a limited number of centres have the facilities to offer this service, and this is frequently on an irregular basis. There is therefore a need for a test that can refine sample selection as well as complementing the molecular analysis. In this study we compared the activities of the nuclear-encoded succinate ubiquinone reductase (complex II) to the activities of the combined mitochondrial and nuclear-encoded mitochondrial electron transport chain (ETC) complexes; NADH:ubiquinone reductase (complex I), ubiquinol-cytochrome-c reductase (complex III), and cytochrome-c oxidase (complex IV), in skeletal muscle biopsies from 7 patients with confirmed mtDNA depletion. In one patient there was no evidence of an ETC defect. However, the remaining 6 patients exhibited reduced complex I and IV activities. Five of these patients also displayed reduced complex II-III (succinate:cytochrome-c reductase) activity. Individual measurement of complex II and complex III activities demonstrated normal levels of complex II activity compared to complex III, which was reduced in the 5 biopsies assayed. These findings suggest a possible diagnostic value for the detection of normal levels of complex II activity in conjunction with reduced complex I, III and IV activity in the identification of likely candidates for mtDNA depletion syndrome

Functional and Expression Analysis of the Metal-Inducible dmeRF System from Rhizobium leguminosarum bv. viciae

PubMed Central

Rubio-Sanz, L.; Prieto, R. I.; Imperial, J.; Brito, B.

2013-01-01

A gene encoding a homolog to the cation diffusion facilitator protein DmeF from Cupriavidus metallidurans has been identified in the genome of Rhizobium leguminosarum UPM791. The R. leguminosarum dmeF gene is located downstream of an open reading frame (designated dmeR) encoding a protein homologous to the nickel- and cobalt-responsive transcriptional regulator RcnR from Escherichia coli. Analysis of gene expression showed that the R. leguminosarum dmeRF genes are organized as a transcriptional unit whose expression is strongly induced by nickel and cobalt ions, likely by alleviating the repressor activity of DmeR on dmeRF transcription. An R. leguminosarum dmeRF mutant strain displayed increased sensitivity to Co(II) and Ni(II), whereas no alterations of its resistance to Cd(II), Cu(II), or Zn(II) were observed. A decrease of symbiotic performance was observed when pea plants inoculated with an R. leguminosarum dmeRF deletion mutant strain were grown in the presence of high concentrations of nickel and cobalt. The same mutant induced significantly lower activity levels of NiFe hydrogenase in microaerobic cultures. These results indicate that the R. leguminosarum DmeRF system is a metal-responsive efflux mechanism acting as a key element for metal homeostasis in R. leguminosarum under free-living and symbiotic conditions. The presence of similar dmeRF gene clusters in other Rhizobiaceae suggests that the dmeRF system is a conserved mechanism for metal tolerance in legume endosymbiotic bacteria. PMID:23934501

Blue light photoreceptors and methods of using the same

DOEpatents

Cashmore, Anthony Robert; Ahmad, Margaret; Lin, Chentao

1998-01-01

The invention features a substantially pure preparation of a nucleic acid encoding a HY4 or a HY4-related gene. The invention further features transgenic plants encoding a HY4 gene having a shorter stem than substantially homozygous wild type nontransgenic plants; and, transgenic plants comprising complementary HY4 sequences having a longer stem than substantially homozygous wild type nontransgenic plants.

The fast milk acidifying phenotype of Streptococcus thermophilus can be acquired by natural transformation of the genomic island encoding the cell-envelope proteinase PrtS.

PubMed

Dandoy, Damien; Fremaux, Christophe; de Frahan, Marie Henry; Horvath, Philippe; Boyaval, Patrick; Hols, Pascal; Fontaine, Laetitia

2011-08-30

In industrial fermentation processes, the rate of milk acidification by Streptococcus thermophilus is of major technological importance. The cell-envelope proteinase PrtS was previously shown to be a key determinant of the milk acidification activity in this species. The PrtS enzyme is tightly anchored to the cell wall via a mechanism involving the typical sortase A (SrtA) and initiates the breakdown of milk casein into small oligopeptides. The presence or absence of PrtS divides the S. thermophilus strains into two phenotypic groups i.e. the slow and the fast acidifying strains. The aim of this study was to improve the milk acidification rate of slow S. thermophilus strains, and hence optimise the fermentation process of dairy products. In the present work, we developed for the first time a strategy based on natural transformation to confer the rapid acidification phenotype to slow acidifying starter strains of S. thermophilus. First, we established by gene disruption that (i) prtS, encoding the cell-envelope proteinase, is a key factor responsible for rapid milk acidification in fast acidifying strains, and that (ii) srtA, encoding sortase A, is not absolutely required to express the PrtS activity. Second, a 15-kb PCR product encompassing the prtS genomic island was transferred by natural transformation using the competence-inducing peptide in three distinct prtS-defective genetic backgrounds having or not a truncated sortase A gene. We showed that in all cases the milk acidification rate of transformants was significantly increased, reaching a level similar to that of wild-type fast acidifying strains. Furthermore, it appeared that the prtS-encoded activity does not depend on the prtS copy number or on its chromosomal integration locus. We have successfully used natural competence to transfer the prtS locus encoding the cell-envelope proteinase in three slow acidifying strains of S. thermophilus, allowing their conversion into fast acidifying derivatives. The efficient protocol developed in this article will provide the dairy industry with novel and optimised S. thermophilus starter strains.

Compensating for Language Deficits in Amnesia II: H.M.’s Spared versus Impaired Encoding Categories

PubMed Central

MacKay, Donald G.; Johnson, Laura W.; Hadley, Chris

2013-01-01

Although amnesic H.M. typically could not recall where or when he met someone, he could recall their topics of conversation after long interference-filled delays, suggesting impaired encoding for some categories of novel events but not others. Similarly, H.M. successfully encoded into internal representations (sentence plans) some novel linguistic structures but not others in the present language production studies. For example, on the Test of Language Competence (TLC), H.M. produced uncorrected errors when encoding a wide range of novel linguistic structures, e.g., violating reliably more gender constraints than memory-normal controls when encoding referent-noun, pronoun-antecedent, and referent-pronoun anaphora, as when he erroneously and without correction used the gender-inappropriate pronoun “her” to refer to a man. In contrast, H.M. never violated corresponding referent-gender constraints for proper names, suggesting that his mechanisms for encoding proper name gender-agreement were intact. However, H.M. produced no more dysfluencies, off-topic comments, false starts, neologisms, or word and phonological sequencing errors than controls on the TLC. Present results suggest that: (a) frontal mechanisms for retrieving and sequencing word, phrase, and phonological categories are intact in H.M., unlike in category-specific aphasia; (b) encoding mechanisms in the hippocampal region are category-specific rather than item-specific, applying to, e.g., proper names rather than words; (c) H.M.’s category-specific mechanisms for encoding referents into words, phrases, and propositions are impaired, with the exception of referent gender, person, and number for encoding proper names; and (d) H.M. overuses his intact proper name encoding mechanisms to compensate for his impaired mechanisms for encoding other functionally equivalent linguistic information. PMID:24961410

Compensating for Language Deficits in Amnesia II: H.M.'s Spared versus Impaired Encoding Categories.

PubMed

MacKay, Donald G; Johnson, Laura W; Hadley, Chris

2013-03-27

Although amnesic H.M. typically could not recall where or when he met someone, he could recall their topics of conversation after long interference-filled delays, suggesting impaired encoding for some categories of novel events but not others. Similarly, H.M. successfully encoded into internal representations (sentence plans) some novel linguistic structures but not others in the present language production studies. For example, on the Test of Language Competence (TLC), H.M. produced uncorrected errors when encoding a wide range of novel linguistic structures, e.g., violating reliably more gender constraints than memory-normal controls when encoding referent-noun, pronoun-antecedent, and referent-pronoun anaphora, as when he erroneously and without correction used the gender-inappropriate pronoun "her" to refer to a man. In contrast, H.M. never violated corresponding referent-gender constraints for proper names, suggesting that his mechanisms for encoding proper name gender-agreement were intact. However, H.M. produced no more dysfluencies, off-topic comments, false starts, neologisms, or word and phonological sequencing errors than controls on the TLC. Present results suggest that: (a) frontal mechanisms for retrieving and sequencing word, phrase, and phonological categories are intact in H.M., unlike in category-specific aphasia; (b) encoding mechanisms in the hippocampal region are category-specific rather than item-specific, applying to, e.g., proper names rather than words; (c) H.M.'s category-specific mechanisms for encoding referents into words, phrases, and propositions are impaired, with the exception of referent gender, person, and number for encoding proper names; and (d) H.M. overuses his intact proper name encoding mechanisms to compensate for his impaired mechanisms for encoding other functionally equivalent linguistic information.

Disruption of the psbA gene by the copy correction mechanism reveals that the expression of plastid-encoded genes is regulated by photosynthesis activity.

PubMed

Khan, Muhammad Sarwar; Hameed, Waqar; Nozoe, Mikio; Shiina, Takashi

2007-05-01

The functional analysis of genes encoded by the chloroplast genome of tobacco by reverse genetics is routine. Nevertheless, for a small number of genes their deletion generates heteroplasmic genotypes, complicating their analysis. There is thus the need for additional strategies to develop deletion mutants for these genes. We have developed a homologous copy correction-based strategy for deleting/mutating genes encoded on the chloroplast genome. This system was used to produce psbA knockouts. The resulting plants are homoplasmic and lack photosystem II (PSII) activity. Further, the deletion mutants exhibit a distinct phenotype; young leaves are green, whereas older leaves are bleached, irrespective of light conditions. This suggests that senescence is promoted by the absence of psbA. Analysis of the transcript levels indicates that NEP (nuclear-encoded plastid RNA polymerase)-dependent plastid genes are up regulated in the psbA deletion mutants, whereas the bleached leaves retain plastid-encoded plastid RNA polymerase activity. Hence, the expression of NEP-dependent plastid genes may be regulated by photosynthesis, either directly or indirectly.

Regulatory Shifts in Plastid Transcription Play a Key Role in Morphological Conversions of Plastids during Plant Development.

PubMed

Liebers, Monique; Grübler, Björn; Chevalier, Fabien; Lerbs-Mache, Silva; Merendino, Livia; Blanvillain, Robert; Pfannschmidt, Thomas

2017-01-01

Plastids display a high morphological and functional diversity. Starting from an undifferentiated small proplastid, these plant cell organelles can develop into four major forms: etioplasts in the dark, chloroplasts in green tissues, chromoplasts in colored flowers and fruits and amyloplasts in roots. The various forms are interconvertible into each other depending on tissue context and respective environmental condition. Research of the last two decades uncovered that each plastid type contains its own specific proteome that can be highly different from that of the other types. Composition of these proteomes largely defines the enzymatic functionality of the respective plastid. The vast majority of plastid proteins is encoded in the nucleus and must be imported from the cytosol. However, a subset of proteins of the photosynthetic and gene expression machineries are encoded on the plastid genome and are transcribed by a complex transcriptional apparatus consisting of phage-type nuclear-encoded RNA polymerases and a bacterial-type plastid-encoded RNA polymerase. Both types recognize specific sets of promoters and transcribe partly over-lapping as well as specific sets of genes. Here we summarize the current knowledge about the sequential activity of these plastid RNA polymerases and their relative activities in different types of plastids. Based on published plastid gene expression profiles we hypothesize that each conversion from one plastid type into another is either accompanied or even preceded by significant changes in plastid transcription suggesting that these changes represent important determinants of plastid morphology and protein composition and, hence, the plastid type.

Regulatory Shifts in Plastid Transcription Play a Key Role in Morphological Conversions of Plastids during Plant Development

PubMed Central

Liebers, Monique; Grübler, Björn; Chevalier, Fabien; Lerbs-Mache, Silva; Merendino, Livia; Blanvillain, Robert; Pfannschmidt, Thomas

2017-01-01

Plastids display a high morphological and functional diversity. Starting from an undifferentiated small proplastid, these plant cell organelles can develop into four major forms: etioplasts in the dark, chloroplasts in green tissues, chromoplasts in colored flowers and fruits and amyloplasts in roots. The various forms are interconvertible into each other depending on tissue context and respective environmental condition. Research of the last two decades uncovered that each plastid type contains its own specific proteome that can be highly different from that of the other types. Composition of these proteomes largely defines the enzymatic functionality of the respective plastid. The vast majority of plastid proteins is encoded in the nucleus and must be imported from the cytosol. However, a subset of proteins of the photosynthetic and gene expression machineries are encoded on the plastid genome and are transcribed by a complex transcriptional apparatus consisting of phage-type nuclear-encoded RNA polymerases and a bacterial-type plastid-encoded RNA polymerase. Both types recognize specific sets of promoters and transcribe partly over-lapping as well as specific sets of genes. Here we summarize the current knowledge about the sequential activity of these plastid RNA polymerases and their relative activities in different types of plastids. Based on published plastid gene expression profiles we hypothesize that each conversion from one plastid type into another is either accompanied or even preceded by significant changes in plastid transcription suggesting that these changes represent important determinants of plastid morphology and protein composition and, hence, the plastid type. PMID:28154576

The Generality-Specificity of Encoding and Decoding Skills with Spontaneous and Deliberate Nonverbal Behavior. Technical Report No. 443.

ERIC Educational Resources Information Center

Atkinson, Michael L.; Allen, Vernon L.

This experiment was designed to investigate the generality-specificity of the accuracy of both encoders and decoders across different types of nonverbal behavior. It was expected that encoders and decoders would exhibit generality in their behavior--i.e., the same level of accuracy--on the dimension of behavior content…

Memory for emotional words: The role of semantic relatedness, encoding task and affective valence.

PubMed

Ferré, Pilar; Fraga, Isabel; Comesaña, Montserrat; Sánchez-Casas, Rosa

2015-01-01

Emotional stimuli have been repeatedly demonstrated to be better remembered than neutral ones. The aim of the present study was to test whether this advantage in memory is mainly produced by the affective content of the stimuli or it can be rather accounted for by factors such as semantic relatedness or type of encoding task. The valence of the stimuli (positive, negative and neutral words that could be either semantically related or unrelated) as well as the type of encoding task (focused on either familiarity or emotionality) was manipulated. The results revealed an advantage in memory for emotional words (either positive or negative) regardless of semantic relatedness. Importantly, this advantage was modulated by the encoding task, as it was reliable only in the task which focused on emotionality. These findings suggest that congruity with the dimension attended at encoding might contribute to the superiority in memory for emotional words, thus offering us a more complex picture of the underlying mechanisms behind the advantage for emotional information in memory.

A Cognitive Framework for Battlefield Commanders’ Situation Assessment

DTIC Science & Technology

1994-07-01

27 , • -- i--i i i•’ i i • -i• ’• i i i 1 i • |_!_! Stressors (1) j7- Actake space In ~ (2) maike la, demands onr ~ retrival a Increase I - ctual...structures to encode new data. A key point is that retrieval cues are associated with the new material in the encoding stage. With practice in a specific...that match the retrieval cues even after long periods of time. We suspect that aspects of skilled memory characterize expertise in a variety of

The genome of Brucella melitensis.

PubMed

DelVecchio, Vito G; Kapatral, Vinayak; Elzer, Philip; Patra, Guy; Mujer, Cesar V

2002-12-20

The genome of Brucella melitensis strain 16M was sequenced and contained 3,294,931 bp distributed over two circular chromosomes. Chromosome I was composed of 2,117,144 bp and chromosome II has 1,177,787 bp. A total of 3,198 ORFs were predicted. The origins of replication of the chromosomes are similar to each other and to those of other alpha-proteobacteria. Housekeeping genes such as those that encode for DNA replication, protein synthesis, core metabolism, and cell-wall biosynthesis were found on both chromosomes. Genes encoding adhesins, invasins, and hemolysins were also identified.

Cloning and Expression of Genes for Dengue Virus Type-2 Encoded-Antigens for Rapid Diagnosis and Vaccine Development

DTIC Science & Technology

1988-10-31

00 0 Cloning and Expression of Genes for Dengue Virus (Type-2 Encoded-Antigens for Rapid ODiagnosis and Vaccine DevelopmentN| ANNUAL PROGRESS REPORT...11. TITLE (include Security Classification) Cloning and Expression of Genes f or Dengue Virus Type 2 Fncoded Antigens for Rapid Diagnosis and Vaccine ...epidemics in Central and South Americas and the Caribbean is a cause of major concern. An effective vaccine is not available to protect individuals

A Single Sfp-Type Phosphopantetheinyl Transferase Plays a Major Role in the Biosynthesis of PKS and NRPS Derived Metabolites in Streptomyces ambofaciens ATCC23877

PubMed Central

Bunet, Robert; Riclea, Ramona; Laureti, Luisa; Hôtel, Laurence; Paris, Cédric; Girardet, Jean-Michel; Spiteller, Dieter; Dickschat, Jeroen S.; Leblond, Pierre; Aigle, Bertrand

2014-01-01

The phosphopantetheinyl transferases (PPTases) are responsible for the activation of the carrier protein domains of the polyketide synthases (PKS), non ribosomal peptide synthases (NRPS) and fatty acid synthases (FAS). The analysis of the Streptomyces ambofaciens ATCC23877 genome has revealed the presence of four putative PPTase encoding genes. One of these genes appears to be essential and is likely involved in fatty acid biosynthesis. Two other PPTase genes, samT0172 (alpN) and samL0372, are located within a type II PKS gene cluster responsible for the kinamycin production and an hybrid NRPS-PKS cluster involved in antimycin production, respectively, and their products were shown to be specifically involved in the biosynthesis of these secondary metabolites. Surprisingly, the fourth PPTase gene, which is not located within a secondary metabolite gene cluster, appears to play a pleiotropic role. Its product is likely involved in the activation of the acyl- and peptidyl-carrier protein domains within all the other PKS and NRPS complexes encoded by S. ambofaciens. Indeed, the deletion of this gene affects the production of the spiramycin and stambomycin macrolide antibiotics and of the grey spore pigment, all three being PKS-derived metabolites, as well as the production of the nonribosomally produced compounds, the hydroxamate siderophore coelichelin and the pyrrolamide antibiotic congocidine. In addition, this PPTase seems to act in concert with the product of samL0372 to activate the ACP and/or PCP domains of the antimycin biosynthesis cluster which is also responsible for the production of volatile lactones. PMID:24498152

p21Waf1/Cip1/Sdi1 Prevents Apoptosis as Well as Stimulates Growth in Cells Transformed or Immortalized by Human T-Cell Leukemia Virus Type 1-Encoded Tax

PubMed Central

Kawata, Sanae; Ariumi, Yasuo; Shimotohno, Kunitada

2003-01-01

Human T-cell leukemia virus type 1 (HTLV-1) Tax regulates the expression of virally encoded genes, as well as various endogenous host genes in trans. Tax-mediated regulation of gene expression is important for the immortalization of normal human T lymphocytes and the transformation of fibroblast cells, such as Rat-1 cells. Tax has the ability to transactivate p21Waf1/Cip1/Sdi1, resulting in high expression levels in HTLV-1-immortalized cells. Since p21 expression is suppressed due to methylation of the promoter region in Rat-l cell line, p21 may not be critical for the transformation of this cell line by Tax. To further understand the role of p21 for the proliferation of Tax-transformed Rat-1 cells, we examined the effect of ectopic expression of p21 in these cells. Here, we observed that p21 expression enhanced the transformation of this cell line via at least two mechanisms: (i) the enhancement of NF-κB activation and/or CREB signaling and (ii) the excitation of antiapoptotic machinery. To analyze the role of p21 that is overexpressed in HTLV-1-immortalized lymphocytes, p21 expression was suppressed by using an antisense oligonucleotide specific for p21 mRNA; these cells then became sensitive to apoptotic induction. These results suggest that p21 plays an important role in the proliferation of Tax-expressing cells through the regulation of at least two independent mechanisms. PMID:12805427

Host-Associated Genomic Features of the Novel Uncultured Intracellular Pathogen Ca. Ichthyocystis Revealed by Direct Sequencing of Epitheliocysts

PubMed Central

Qi, Weihong; Vaughan, Lloyd; Katharios, Pantelis; Schlapbach, Ralph; Seth-Smith, Helena M.B.

2016-01-01

Advances in single-cell and mini-metagenome sequencing have enabled important investigations into uncultured bacteria. In this study, we applied the mini-metagenome sequencing method to assemble genome drafts of the uncultured causative agents of epitheliocystis, an emerging infectious disease in the Mediterranean aquaculture species gilthead seabream. We sequenced multiple cyst samples and constructed 11 genome drafts from a novel beta-proteobacterial lineage, Candidatus Ichthyocystis. The draft genomes demonstrate features typical of pathogenic bacteria with an obligate intracellular lifestyle: a reduced genome of up to 2.6 Mb, reduced G + C content, and reduced metabolic capacity. Reconstruction of metabolic pathways reveals that Ca. Ichthyocystis genomes lack all amino acid synthesis pathways, compelling them to scavenge from the fish host. All genomes encode type II, III, and IV secretion systems, a large repertoire of predicted effectors, and a type IV pilus. These are all considered to be virulence factors, required for adherence, invasion, and host manipulation. However, no evidence of lipopolysaccharide synthesis could be found. Beyond the core functions shared within the genus, alignments showed distinction into different species, characterized by alternative large gene families. These comprise up to a third of each genome, appear to have arisen through duplication and diversification, encode many effector proteins, and are seemingly critical for virulence. Thus, Ca. Ichthyocystis represents a novel obligatory intracellular pathogenic beta-proteobacterial lineage. The methods used: mini-metagenome analysis and manual annotation, have generated important insights into the lifestyle and evolution of the novel, uncultured pathogens, elucidating many putative virulence factors including an unprecedented array of novel gene families. PMID:27190004

Simulation Experiment Description Markup Language (SED-ML) Level 1 Version 3 (L1V3).

PubMed

Bergmann, Frank T; Cooper, Jonathan; König, Matthias; Moraru, Ion; Nickerson, David; Le Novère, Nicolas; Olivier, Brett G; Sahle, Sven; Smith, Lucian; Waltemath, Dagmar

2018-03-19

The creation of computational simulation experiments to inform modern biological research poses challenges to reproduce, annotate, archive, and share such experiments. Efforts such as SBML or CellML standardize the formal representation of computational models in various areas of biology. The Simulation Experiment Description Markup Language (SED-ML) describes what procedures the models are subjected to, and the details of those procedures. These standards, together with further COMBINE standards, describe models sufficiently well for the reproduction of simulation studies among users and software tools. The Simulation Experiment Description Markup Language (SED-ML) is an XML-based format that encodes, for a given simulation experiment, (i) which models to use; (ii) which modifications to apply to models before simulation; (iii) which simulation procedures to run on each model; (iv) how to post-process the data; and (v) how these results should be plotted and reported. SED-ML Level 1 Version 1 (L1V1) implemented support for the encoding of basic time course simulations. SED-ML L1V2 added support for more complex types of simulations, specifically repeated tasks and chained simulation procedures. SED-ML L1V3 extends L1V2 by means to describe which datasets and subsets thereof to use within a simulation experiment.

Repression of YdaS Toxin Is Mediated by Transcriptional Repressor RacR in the Cryptic rac Prophage of Escherichia coli K-12.

PubMed

Krishnamurthi, Revathy; Ghosh, Swagatha; Khedkar, Supriya; Seshasayee, Aswin Sai Narain

2017-01-01

Horizontal gene transfer is a major driving force behind the genomic diversity seen in prokaryotes. The cryptic rac prophage in Escherichia coli K-12 carries the gene for a putative transcription factor RacR, whose deletion is lethal. We have shown that the essentiality of racR in E. coli K-12 is attributed to its role in transcriptionally repressing toxin gene(s) called ydaS and ydaT , which are adjacent to and coded divergently to racR . IMPORTANCE Transcription factors in the bacterium E. coli are rarely essential, and when they are essential, they are largely toxin-antitoxin systems. While studying transcription factors encoded in horizontally acquired regions in E. coli , we realized that the protein RacR, a putative transcription factor encoded by a gene on the rac prophage, is an essential protein. Here, using genetics, biochemistry, and bioinformatics, we show that its essentiality derives from its role as a transcriptional repressor of the ydaS and ydaT genes, whose products are toxic to the cell. Unlike type II toxin-antitoxin systems in which transcriptional regulation involves complexes of the toxin and antitoxin, repression by RacR is sufficient to keep ydaS transcriptionally silent.

Identification and characterization of a phospholipid scramblase encoded by planarian Dugesia japonica.

PubMed

Han, Yu; Li, Ao; Gao, Lili; Wu, Weiwei; Deng, Hongkuan; Hu, Wenjing; Li, Na; Sun, Shimin; Zhang, Xiufang; Zhao, Bosheng; Liu, Baohua; Pang, Qiuxiang

2017-02-20

Phospholipid scramblases (PLSCRs) are the conserved calcium-binding, type II transmembrane proteins synthesized in all eukaryotic organisms. In mammals, these proteins play essential roles in various physiological processes, especially in the immune responses. However, the existence of PLSCRs and their biological functions in planarian are still unknown at present. In this study, a new member of PLSCRs was identified in planarian Dugesia japonica (D. japonica), named DjPLSCR. The sequence analysis revealed that it contains an opening reading frame consisting of 726bp encoding a putative protein of 241 amino acids with a predicted molecular mass of ~28.7kDa and an isoelectric point of 6.21. Whole-mount in situ hybridization showed that mRNAs of DjPLSCR are predominantly expressed in adult and regenerative pharynx which is an important organ of immune system in planarians. Importantly, we found that the transcription level of DjPLSCR was significantly upregulated when planarians were stimulated with the pathogen-associated molecular patterns [polyinosinic-polycytidylic acid, lipopolysaccharide, peptidoglycan and β-glucan], suggesting that DjPLSCR is involved in the immune response upon pathogen invasion. Our findings provide the first experimental insights into the characteristics and potential functions of PLSCR in planarians. Copyright © 2016 Elsevier B.V. All rights reserved.

Treating a novel plasticity defect rescues episodic memory in Fragile X model mice

PubMed Central

Wang, Weisheng; Cox, Brittney M.; Jia, Yousheng; Le, Aliza A.; Cox, Conor D.; Jung, Kwang M.; Hou, Bowen; Piomelli, Daniele; Gall, Christine M.; Lynch, Gary

2017-01-01

Episodic memory, a fundamental component of human cognition, is significantly impaired in autism. We report the first evidence for this problem in the Fmr1-knockout (KO) mouse model of Fragile X syndrome and describe potentially treatable underlying causes. The hippocampus is critical for the formation and use of episodes, with semantic (cue identity) information relayed to the structure via the lateral perforant path (LPP). The unusual form of synaptic plasticity expressed by the LPP (lppLTP) was profoundly impaired in Fmr1-KOs relative to wild type mice. Two factors contributed to this defect: i) reduced GluN1 subunit levels in synaptic NMDA receptors and related currents, and ii) impaired retrograde synaptic signaling by the endocannabinoid 2-archadonolglycerol (2-AG). Studies using a novel serial cue paradigm showed that episodic encoding is dependent on both the LPP and the endocannabinoid receptor CB1, and is strikingly impaired in Fmr1-KOs. Enhancing 2-AG signaling rescued both lppLTP and learning in the mutants. Thus, two consequences of the Fragile-X mutation converge on plasticity at one site in hippocampus to prevent encoding of a basic element of cognitive memory. Collectively, the results suggest a clinically plausible approach to treatment. PMID:29133950

Purification, Reconstitution, and Inhibition of Cytochrome P-450 Sterol Δ22-Desaturase from the Pathogenic Fungus Candida glabrata

PubMed Central

Lamb, David C.; Maspahy, Segula; Kelly, Diane E.; Manning, Nigel J.; Geber, Antonia; Bennett, John E.; Kelly, Steven L.

1999-01-01

Sterol Δ22-desaturase has been purified from a strain of Candida glabrata with a disruption in the gene encoding sterol 14α-demethylase (cytochrome P-45051; CYP51). The purified cytochrome P-450 exhibited sterol Δ22-desaturase activity in a reconstituted system with NADPH–cytochrome P-450 reductase in dilaurylphosphatidylcholine, with the enzyme kinetic studies revealing a Km for ergosta-5,7-dienol of 12.5 μM and a Vmax of 0.59 nmol of this substrate metabolized/min/nmol of P-450. This enzyme is encoded by CYP61 (ERG5) in Saccharomyces cerevisiae, and homologues have been shown in the Candida albicans and Schizosaccharomyces pombe genome projects. Ketoconazole, itraconazole, and fluconazole formed low-spin complexes with the ferric cytochrome and exhibited type II spectra, which are indicative of an interaction between the azole moiety and the cytochrome heme. The azole antifungal compounds inhibited reconstituted sterol Δ22-desaturase activity by binding to the cytochrome with a one-to-one stoichiometry, with total inhibition of enzyme activity occurring when equimolar amounts of azole and cytochrome P-450 were added. These results reveal the potential for sterol Δ22-desaturase to be an antifungal target and to contribute to the binding of drugs within the fungal cell. PMID:10390230

Amygdala activity at encoding corresponds with memory vividness and with memory for select episodic details.

PubMed

Kensinger, Elizabeth A; Addis, Donna Rose; Atapattu, Ranga K

2011-03-01

It is well known that amygdala activity during encoding corresponds with subsequent memory for emotional information. It is less clear how amygdala activity relates to the subjective and objective qualities of a memory. In the present study, participants viewed emotional and neutral objects while undergoing a functional magnetic resonance imaging scan. Participants then took a memory test, identifying which verbal labels named a studied object and indicating the vividness of their memory for that object. They then retrieved episodic details associated with each object's presentation, selecting which object exemplar had been studied and indicating in which screen quadrant, study list, and with which encoding question the exemplar had been studied. Parametric analysis of the encoding data allowed examination of the processes that tracked with increasing memory vividness or with an increase in the diversity of episodic details remembered. Dissociable networks tracked these two increases, and amygdala activity corresponded with the former but not the latter. Subsequent-memory analyses revealed that amygdala activity corresponded with memory for exemplar type but not for other episodic features. These results emphasize that amygdala activity does not ensure accurate encoding of all types of episodic detail, yet it does support encoding of some item-specific details and leads to the retention of a memory that will feel subjectively vivid. The types of episodic details tied to amygdala engagement may be those that are most important for creating a subjectively vivid memory. Copyright © 2011 Elsevier Ltd. All rights reserved.

Amygdala Activity at Encoding Corresponds with Memory Vividness and with Memory for Select Episodic Details

PubMed Central

Kensinger, Elizabeth A.; Addis, Donna Rose; Atapattu, Ranga K.

2011-01-01

It is well known that amygdala activity during encoding corresponds with subsequent memory for emotional information. It is less clear how amygdala activity relates to the subjective and objective qualities of a memory. In the present study, participants viewed emotional and neutral objects while undergoing a functional magnetic resonance imaging scan. Participants then took a memory test, identifying which verbal labels named a studied object and indicating the vividness of their memory for that object. They then retrieved episodic details associated with each object’s presentation, selecting which object exemplar had been studied and indicating in which screen quadrant, study list, and with which encoding question the exemplar had been studied. Parametric analysis of the encoding data allowed examination of the processes that tracked with increasing memory vividness or with an increase in the diversity of episodic details remembered. Dissociable networks tracked these two increases, and amygdala activity corresponded with the former but not the latter. Subsequent-memory analyses revealed that amygdala activity corresponded with memory for exemplar type but not for other episodic features. These results emphasize that amygdala activity does not ensure accurate encoding of all types of episodic detail, yet it does support encoding of some item-specific details and leads to the retention of a memory that will feel subjectively vivid. The types of episodic details tied to amygdala engagement may be those that are most important for creating a subjectively vivid memory. PMID:21262244

H-NOX Regulation of c-di-GMP Metabolism and Biofilm Formation in Legionella pneumophila

PubMed Central

Carlson, Hans K.; Vance, Russell E.; Marletta, Michael A.

2010-01-01

Summary Heme Nitric oxide/OXygen (H-NOX) domains are a family of hemoprotein sensors that are widespread in bacterial genomes, but limited information is available on their function. Legionella pneumophila is the only prokaryote found thus far to encode two H-NOX proteins. This paper presents data supporting a role for one of the Legionella pneumophila H-NOXs in the regulation of biofilm formation. In summary: (i) unmarked deletions in the hnox1 gene do not affect growth rate in liquid culture or replication in permissive macrophages; (ii) theΔhnox1 strain displays a hyper-biofilm phenotype; (iii) the gene adjacent to hnox1 is a GGDEF-EAL protein, lpg1057, and overexpression in Legionella pneumophila of this protein, or the well-studied diguanylate cyclase, vca0956, results in a hyper-biofilm phenotype; (iv) the Lpg1057 protein displays diguanylate cyclase activity in vitro and this activity is inhibited by the Hnox1 protein in the Fe(II)-NO ligation state, but not the Fe(II) unligated state; (v) consistent with the Hnox1 regulation of Lpg1057, unmarked deletions of lpg1057 in theΔhnox1 background results in reversion of the hyper-biofilm phenotype back to wild-type biofilm levels. Taken together, these results suggest a role for hnox1 in regulating c-di-GMP production by lpg1057 and biofilm formation in response to NO. PMID:20572940

Chromosomal DNA Deletions Explain Phenotypic Characteristics of Two Antigenic Variants, Phase II and RSA 514 (Crazy), of the Coxiella burnetii Nine Mile Strain†

PubMed Central

Hoover, T. A.; Culp, D. W.; Vodkin, M. H.; Williams, J. C.; Thompson, H. A.

2002-01-01

After repeated passages through embyronated eggs, the Nine Mile strain of Coxiella burnetii exhibits antigenic variation, a loss of virulence characteristics, and transition to a truncated lipopolysaccharide (LPS) structure. In two independently derived strains, Nine Mile phase II and RSA 514, these phenotypic changes were accompanied by a large chromosomal deletion (M. H. Vodkin and J. C. Williams, J. Gen. Microbiol. 132:2587-2594, 1986). In the work reported here, additional screening of a cosmid bank prepared from the wild-type strain was used to map the deletion termini of both mutant strains and to accumulate all the segments of DNA that comprise the two deletions. The corresponding DNAs were then sequenced and annotated. The Nine Mile phase II deletion was completely nested within the deletion of the RSA 514 strain. Basic alignment and homology studies indicated that a large group of LPS biosynthetic genes, arranged in an apparent O-antigen cluster, was deleted in both variants. Database homologies identified, in particular, mannose pathway genes and genes encoding sugar methylases and nucleotide sugar epimerase-dehydratase proteins. Candidate genes for addition of sugar units to the core oligosaccharide for synthesis of the rare sugar 6-deoxy-3-C-methylgulose (virenose) were identified in the deleted region. Repeats, redundancies, paralogous genes, and two regions with reduced G+C contents were found within the deletions. PMID:12438347

[Methanotrophic bacteria in cold seeps of the floodplains of northern rivers].

PubMed

Belova, S É; Oshkin, I Iu; Glagolev, M V; Lapshina, E D; Maksiutov, Sh Sh; Dedysh, S N

2013-01-01

Small mud volcanoes (cold seeps), which are common in the floodplains of northern rivers, are a potentially important, although poorly studied sources of atmospheric methane. Field research on the cold seeps of the Mukhrina River (Khanty-Mansiysk Autonomous okrug, Russia) revealed methane fluxes from these structures to be orders of magnitude higher than from equivalent areas of the mid-taiga bogs. Microbial communities developing around the seeps were formed under conditions of high methane concentrations, low temperatures (3-5 degrees C), and near-neutral pH. Molecular identification of methane-oxidizing bacteria from this community by analysis of the pmoA gene encoding particulate methane monooxygenase revealed both type I and type II methanotrophs (classes Gammaproteobacteria and Alphaproteobacteria, respectively), with predomination of type I methanotrophs. Among the latter, microorganisms related to Methylobacterpsychrophilus and Methylobacter tundripaludum, Crenothrix polyspora (a stagnant water dweller), and a number of methanotrophs belonging to unknown taxa were detected. Growth characteristics of two isolates were determined. Methylobactersp. CMS7 exhibited active growth at 4-10 degrees C, while Methylocystis sp. SB12 grew better at 20 degrees C. Experimental results confirmed the major role ofmethanotrophic gammaproteobacteria in controlling the methane emission from cold river seeps.

Correlating levels of type III secretion and secreted proteins with fecal shedding of Escherichia coli O157:H7 in cattle

USDA-ARS?s Scientific Manuscript database

The locus of enterocyte effacement (LEE) encodes a type III secretion system (T3SS) for secreting factors that enable Escherichia coli O157:H7 to produce attaching and effacing lesions (A/E) on epithelial cells. The importance of LEE-encoded proteins in intestinal colonization of cattle is well-stud...

Complete Genome Sequence of the Methanococcus maripaludis Type Strain JJ (DSM 2067), a Model for Selenoprotein Synthesis in Archaea.

PubMed

Poehlein, Anja; Heym, Daniel; Quitzke, Vivien; Fersch, Julia; Daniel, Rolf; Rother, Michael

2018-04-05

Methanococcus maripaludis type strain JJ (DSM 2067) is an important organism because it serves as a model for primary energy metabolism and hydrogenotrophic methanogenesis and is amenable to genetic manipulation. The complete genome (1.7 Mb) harbors 1,815 predicted protein-encoding genes, including 9 encoding selenoproteins. Copyright © 2018 Poehlein et al.

Eisenstein series for infinite-dimensional U-duality groups

NASA Astrophysics Data System (ADS)

Fleig, Philipp; Kleinschmidt, Axel

2012-06-01

We consider Eisenstein series appearing as coefficients of curvature corrections in the low-energy expansion of type II string theory four-graviton scattering amplitudes. We define these Eisenstein series over all groups in the E n series of string duality groups, and in particular for the infinite-dimensional Kac-Moody groups E 9, E 10 and E 11. We show that, remarkably, the so-called constant term of Kac-Moody-Eisenstein series contains only a finite number of terms for particular choices of a parameter appearing in the definition of the series. This resonates with the idea that the constant term of the Eisenstein series encodes perturbative string corrections in BPS-protected sectors allowing only a finite number of corrections. We underpin our findings with an extensive discussion of physical degeneration limits in D < 3 space-time dimensions.

Mechanisms and pathways of growth failure in primordial dwarfism.

PubMed

Klingseisen, Anna; Jackson, Andrew P

2011-10-01

The greatest difference between species is size; however, the developmental mechanisms determining organism growth remain poorly understood. Primordial dwarfism is a group of human single-gene disorders with extreme global growth failure (which includes Seckel syndrome, microcephalic osteodysplastic primordial dwarfism I [MOPD] types I and II, and Meier-Gorlin syndrome). Ten genes have now been identified for microcephalic primordial dwarfism, encoding proteins involved in fundamental cellular processes including genome replication (ORC1 [origin recognition complex 1], ORC4, ORC6, CDT1, and CDC6), DNA damage response (ATR [ataxia-telangiectasia and Rad3-related]), mRNA splicing (U4atac), and centrosome function (CEP152, PCNT, and CPAP). Here, we review the cellular and developmental mechanisms underlying the pathogenesis of these conditions and address whether further study of these genes could provide novel insight into the physiological regulation of organism growth.

Mass spectrometric characterization of membrane integral low molecular weight proteins from photosystem II in barley etioplasts.

PubMed

Plöscher, Matthias; Granvogl, Bernhard; Zoryan, Mikael; Reisinger, Veronika; Eichacker, Lutz Andreas

2009-02-01

In Photosystem II (PSII), a high number of plastid encoded and membrane integral low molecular weight proteins smaller than 10 kDa, the proteins PsbE, F, H, I, J, K, L, M, N, Tc, Z and the nuclear encoded PsbW, X, Y1, Y2 proteins have been described. Here we show that all low molecular weight proteins of PSII already accumulate in the etioplast membrane fraction in darkness, whereas PsaI and PsaJ of photosystem I (PSI) represent the only low molecular weight proteins that do not accumulate in darkness. We found by BN-PAGE separation of membrane protein complexes and selective MS that the accumulation of one-helix proteins from PSII is light independent and occurs in etioplasts. In contrast, in chloroplasts isolated from light-grown plants, low molecular weight proteins were found to specifically accumulate in PSI and II complexes. Our results demonstrate how plants grown in darkness prepare for the induction of chlorophyll dependent photosystem assembly upon light perception. We anticipate that our investigation will provide the essential means for the analysis of protein assembly in any membrane utilizing low molecular weight protein subunits.

An intronic open reading frame was released from one of group II introns in the mitochondrial genome of the haptophyte Chrysochromulina sp. NIES-1333

PubMed Central

Nishimura, Yuki; Kamikawa, Ryoma; Hashimoto, Tetsuo; Inagaki, Yuji

2014-01-01

Mitochondrial (mt) genome sequences, which often bear introns, have been sampled from phylogenetically diverse eukaryotes. Thus, we can anticipate novel insights into intron evolution from previously unstudied mt genomes. We here investigated the origins and evolution of three introns in the mt genome of the haptophyte Chrysochromulina sp. NIES-1333, which was sequenced completely in this study. All the three introns were characterized as group II, on the basis of predicted secondary structure, and the conserved sequence motifs at the 5′ and 3′ termini. Our comparative studies on diverse mt genomes prompt us to propose that the Chrysochromulina mt genome laterally acquired the introns from mt genomes in distantly related eukaryotes. Many group II introns harbor intronic open reading frames for the proteins (intron-encoded proteins or IEPs), which likely facilitate the splicing of their host introns. However, we propose that a “free-standing,” IEP-like protein, which is not encoded within any introns in the Chrysochromulina mt genome, is involved in the splicing of the first cox1 intron that lacks any open reading frames. PMID:25054084

Two tropinone reductases with different stereospecificities are short-chain dehydrogenases evolved from a common ancestor.

PubMed Central

Nakajima, K; Hashimoto, T; Yamada, Y

1993-01-01

In the biosynthetic pathway of tropane alkaloids, tropinone reductase (EC 1.1.1.236) (TR)-I and TR-II, respectively, reduce a common substrate, tropinone, stereospecifically to the stereoisomeric alkamines tropine and pseudotropine (psi-tropine). cDNA clones coding for TR-I and TR-II, as well as a structurally related cDNA clone with an unknown function, were isolated from the solanaceous plant Datura stramonium. The cDNA clones for TR-I and TR-II encode polypeptides containing 273 and 260 amino acids, respectively, and when these clones were expressed in Escherichia coli, the recombinant TRs showed the same strict stereospecificity as that observed for the native TRs that had been isolated from plants. The deduced amino acid sequences of the two clones showed an overall identity of 64% in 260-amino acid residues and also shared significant similarities with enzymes in the short-chain, nonmetal dehydrogenase family. Genomic DNA-blot analysis detected the TR-encoding genes in three tropane alkaloid-producing solanaceous species but did not detect them in tobacco. We discuss how the two TRs may have evolved to catalyze the opposite stereospecific reductions. Images Fig. 4 Fig. 5 PMID:8415746

A Zn(II)2Cys6 DNA binding protein regulates the sirodesmin PL biosynthetic gene cluster in Leptosphaeria maculans

PubMed Central

Fox, Ellen M.; Gardiner, Donald M.; Keller, Nancy P.; Howlett, Barbara J.

2008-01-01

A gene, sirZ, encoding a Zn(II)2Cys6 DNA binding protein is present in a cluster of genes responsible for the biosynthesis of the epipolythiodioxopiperazine (ETP) toxin, sirodesmin PL in the ascomycete plant pathogen, Leptosphaeria maculans. RNA-mediated silencing of sirZ gives rise to transformants that produce only residual amounts of sirodesmin PL and display a decrease in the transcription of several sirodesmin PL biosynthetic genes. This indicates that SirZ is a major regulator of this gene cluster. Proteins similar to SirZ are encoded in the gliotoxin biosynthetic gene cluster of Aspergillus fumigatus (gliZ) and in an ETP-like cluster in Penicillium lilacinoechinulatum (PlgliZ). Despite its high level of sequence similarity to gliZ, PlgliZ is unable to complement the gliotoxin-deficiency of a mutant of gliZ in A. fumigatus. Putative binding sites for these regulatory proteins in the promoters of genes in these clusters were predicted using bioinformatic analysis. These sites are similar to those commonly bound by other proteins with Zn(II)2Cys6 DNA binding domains. PMID:18023597

The human immune response to tuberculosis and its treatment: a view from the blood

PubMed Central

Cliff, Jacqueline M; Kaufmann, Stefan H E; McShane, Helen; van Helden, Paul; O'Garra, Anne

2015-01-01

The immune response upon infection with the pathogen Mycobacterium tuberculosis is poorly understood, hampering the discovery of new treatments and the improvements in diagnosis. In the last years, a blood transcriptional signature in tuberculosis has provided knowledge on the immune response occurring during active tuberculosis disease. This signature was absent in the majority of asymptomatic individuals who are latently infected with M. tuberculosis (referred to as latent). Using modular and pathway analyses of the complex data has shown, now in multiple studies, that the signature of active tuberculosis is dominated by overexpression of interferon-inducible genes (consisting of both type I and type II interferon signaling), myeloid genes, and inflammatory genes. There is also downregulation of genes encoding B and T-cell function. The blood signature of tuberculosis correlates with the extent of radiographic disease and is diminished upon effective treatment suggesting the possibility of new improved strategies to support diagnostic assays and methods for drug treatment monitoring. The signature suggested a previously under-appreciated role for type I interferons in development of active tuberculosis disease, and numerous mechanisms have now been uncovered to explain how type I interferon impedes the protective response to M. tuberculosis infection. PMID:25703554

Is junk DNA bunk? A critique of ENCODE

PubMed Central

Doolittle, W. Ford

2013-01-01

Do data from the Encyclopedia Of DNA Elements (ENCODE) project render the notion of junk DNA obsolete? Here, I review older arguments for junk grounded in the C-value paradox and propose a thought experiment to challenge ENCODE’s ontology. Specifically, what would we expect for the number of functional elements (as ENCODE defines them) in genomes much larger than our own genome? If the number were to stay more or less constant, it would seem sensible to consider the rest of the DNA of larger genomes to be junk or, at least, assign it a different sort of role (structural rather than informational). If, however, the number of functional elements were to rise significantly with C-value then, (i) organisms with genomes larger than our genome are more complex phenotypically than we are, (ii) ENCODE’s definition of functional element identifies many sites that would not be considered functional or phenotype-determining by standard uses in biology, or (iii) the same phenotypic functions are often determined in a more diffuse fashion in larger-genomed organisms. Good cases can be made for propositions ii and iii. A larger theoretical framework, embracing informational and structural roles for DNA, neutral as well as adaptive causes of complexity, and selection as a multilevel phenomenon, is needed. PMID:23479647

Only One of the Five Ralstonia solanacearum Long-Chain 3-Ketoacyl-Acyl Carrier Protein Synthase Homologues Functions in Fatty Acid Synthesis

PubMed Central

Cheng, Juanli; Ma, Jincheng; Lin, Jinshui; Fan, Zhen-Chuan; Cronan, John E.

2012-01-01

Ralstonia solanacearum, a major phytopathogenic bacterium, causes a bacterial wilt disease in diverse plants. Although fatty acid analyses of total membranes of R. solanacearum showed that they contain primarily palmitic (C16:0), palmitoleic (C16:1) and cis-vaccenic (C18:1) acids, little is known regarding R. solanacearum fatty acid synthesis. The R. solanacearum GMI1000 genome is unusual in that it contains four genes (fabF1, fabF2, fabF3, and fabF4) annotated as encoding 3-ketoacyl-acyl carrier protein synthase II homologues and one gene (fabB) annotated as encoding 3-ketoacyl-acyl carrier protein synthase I. We have analyzed this puzzling apparent redundancy and found that only one of these genes, fabF1, encoded a long-chain 3-ketoacyl-acyl carrier protein synthase, whereas the other homologues did not play roles in R. solanacearum fatty acid synthesis. Mutant strains lacking fabF1 are nonviable, and thus, FabF1 is essential for R. solanacearum fatty acid biosynthesis. Moreover, R. solanacearum FabF1 has the activities of both 3-ketoacyl-acyl carrier protein synthase II and 3-ketoacyl-acyl carrier protein synthase I. PMID:22194290

Identification and Manipulation of a Novel Signaling Mechanism to Improve Articular Cartilage Restoration After Posttraumatic Joint Injury

DTIC Science & Technology

2014-10-01

collagen II (COL II). As a result, the newly-formed fibrocartilage has poor biomechanical properties and often evidences poor integration with the... fibrocartilage generation at the site of injury defines a new potential avenue of intervention in the field of cartilage restoration. We defined the...lysophosphatidic acid (LPA)-autotaxin (ATX; encoded by the ENPP2 gene) signaling axis as an important mediator of fibrocartilage formation both in vitro and in

Unlocking Barriers to DNA Vaccine Immunogenicity: A Cross-Species Analysis of Cytosolic DNA Sensing in Skeletal Muscle Myocytes

DTIC Science & Technology

2017-10-01

CRISPR Subtask 1A: i) design and produce mammalian expression plasmids encoding the Cas9 protein and specially...duration in SOW: 2017 Q4 – 2018 Q1 Subtask 2A: i) produce mouse myocyte cell lines that have undergone gene disruption via a technique named CRISPR ii...named CRISPR ii) confirm gene disruption and GFP expression iii) select multiple individual clones characterized with quantitative gene

Nonstructural Protein NSs of Schmallenberg Virus Is Targeted to the Nucleolus and Induces Nucleolar Disorganization

PubMed Central

Gouzil, Julie; Fablet, Aurore; Lara, Estelle; Caignard, Grégory; Cochet, Marielle; Kundlacz, Cindy; Palmarini, Massimo; Varela, Mariana; Breard, Emmanuel; Sailleau, Corinne; Viarouge, Cyril; Coulpier, Muriel; Zientara, Stéphan

2016-01-01

ABSTRACT Schmallenberg virus (SBV) was discovered in Germany in late 2011 and then spread rapidly to many European countries. SBV is an orthobunyavirus that causes abortion and congenital abnormalities in ruminants. A virus-encoded nonstructural protein, termed NSs, is a major virulence factor of SBV, and it is known to promote the degradation of Rpb1, a subunit of the RNA polymerase II (Pol II) complex, and therefore hampers global cellular transcription. In this study, we found that NSs is mainly localized in the nucleus of infected cells and specifically appears to target the nucleolus through a nucleolar localization signal (NoLS) localized between residues 33 and 51 of the protein. NSs colocalizes with nucleolar markers such as B23 (nucleophosmin) and fibrillarin. We observed that in SBV-infected cells, B23 undergoes a nucleolus-to-nucleoplasm redistribution, evocative of virus-induced nucleolar disruption. In contrast, the nucleolar pattern of B23 was unchanged upon infection with an SBV recombinant mutant with NSs lacking the NoLS motif (SBVΔNoLS). Interestingly, unlike wild-type SBV, the inhibitory activity of SBVΔNoLS toward RNA Pol II transcription is impaired. Overall, our results suggest that a putative link exists between NSs-induced nucleolar disruption and its inhibitory function on cellular transcription, which consequently precludes the cellular antiviral response and/or induces cell death. IMPORTANCE Schmallenberg virus (SBV) is an emerging arbovirus of ruminants that spread in Europe between 2011 and 2013. SBV induces fetal abnormalities during gestation, with the central nervous system being one of the most affected organs. The virus-encoded NSs protein acts as a virulence factor by impairing host cell transcription. Here, we show that NSs contains a nucleolar localization signal (NoLS) and induces disorganization of the nucleolus. The NoLS motif in the SBV NSs is absolutely necessary for virus-induced inhibition of cellular transcription. To our knowledge, this is the first report of nucleolar functions for NSs within the Bunyaviridae family. PMID:27795408

Nonstructural Protein NSs of Schmallenberg Virus Is Targeted to the Nucleolus and Induces Nucleolar Disorganization.

PubMed

Gouzil, Julie; Fablet, Aurore; Lara, Estelle; Caignard, Grégory; Cochet, Marielle; Kundlacz, Cindy; Palmarini, Massimo; Varela, Mariana; Breard, Emmanuel; Sailleau, Corinne; Viarouge, Cyril; Coulpier, Muriel; Zientara, Stéphan; Vitour, Damien

2017-01-01

Schmallenberg virus (SBV) was discovered in Germany in late 2011 and then spread rapidly to many European countries. SBV is an orthobunyavirus that causes abortion and congenital abnormalities in ruminants. A virus-encoded nonstructural protein, termed NSs, is a major virulence factor of SBV, and it is known to promote the degradation of Rpb1, a subunit of the RNA polymerase II (Pol II) complex, and therefore hampers global cellular transcription. In this study, we found that NSs is mainly localized in the nucleus of infected cells and specifically appears to target the nucleolus through a nucleolar localization signal (NoLS) localized between residues 33 and 51 of the protein. NSs colocalizes with nucleolar markers such as B23 (nucleophosmin) and fibrillarin. We observed that in SBV-infected cells, B23 undergoes a nucleolus-to-nucleoplasm redistribution, evocative of virus-induced nucleolar disruption. In contrast, the nucleolar pattern of B23 was unchanged upon infection with an SBV recombinant mutant with NSs lacking the NoLS motif (SBVΔNoLS). Interestingly, unlike wild-type SBV, the inhibitory activity of SBVΔNoLS toward RNA Pol II transcription is impaired. Overall, our results suggest that a putative link exists between NSs-induced nucleolar disruption and its inhibitory function on cellular transcription, which consequently precludes the cellular antiviral response and/or induces cell death. Schmallenberg virus (SBV) is an emerging arbovirus of ruminants that spread in Europe between 2011 and 2013. SBV induces fetal abnormalities during gestation, with the central nervous system being one of the most affected organs. The virus-encoded NSs protein acts as a virulence factor by impairing host cell transcription. Here, we show that NSs contains a nucleolar localization signal (NoLS) and induces disorganization of the nucleolus. The NoLS motif in the SBV NSs is absolutely necessary for virus-induced inhibition of cellular transcription. To our knowledge, this is the first report of nucleolar functions for NSs within the Bunyaviridae family. Copyright © 2016 Gouzil et al.

Cloning, characterization, expression analysis and inhibition studies of a novel gene encoding Bowman-Birk type protease inhibitor from rice bean

USDA-ARS?s Scientific Manuscript database

This paper presents the first study describing the isolation, cloning and characterization of a full length gene encoding Bowman-Birk protease inhibitor (RbTI) from rice bean (Vigna umbellata). A full-length protease inhibitor gene with complete open reading frame of 327bp encoding 109 amino acids w...

When Does Memory Monitoring Succeed versus Fail? Comparing Item-Specific and Relational Encoding in the DRM Paradigm

ERIC Educational Resources Information Center

Huff, Mark J.; Bodner, Glen E.

2013-01-01

We compared the effects of item-specific versus relational encoding on recognition memory in the Deese-Roediger-McDermott paradigm. In Experiment 1, we directly compared item-specific and relational encoding instructions, whereas in Experiments 2 and 3 we biased pleasantness and generation tasks, respectively, toward one or the other type of…

ICP22 and the UL13 Protein Kinase Are both Required for Herpes Simplex Virus-Induced Modification of the Large Subunit of RNA Polymerase II

PubMed Central

Long, Melissa C.; Leong, Vivian; Schaffer, Priscilla A.; Spencer, Charlotte A.; Rice, Stephen A.

1999-01-01

Herpes simplex virus type 1 (HSV-1) infection alters the phosphorylation of the large subunit of RNA polymerase II (RNAP II), resulting in the depletion of the hypophosphorylated and hyperphosphorylated forms of this polypeptide (known as IIa and IIo, respectively) and induction of a novel, alternatively phosphorylated form (designated IIi). We previously showed that the HSV-1 immediate-early protein ICP22 is involved in this phenomenon, since induction of IIi and depletion of IIa are deficient in cells infected with 22/n199, an HSV-1 ICP22 nonsense mutant (S. A. Rice, M. C. Long, V. Lam, P. A. Schaffer, and C. A. Spencer, J. Virol. 69:5550–5559, 1995). However, depletion of IIo still occurs in 22/n199-infected cells. This suggests either that another viral gene product affects the RNAP II large subunit or that the truncated ICP22 polypeptide encoded by 22/n199 retains residual activity which leads to IIo depletion. To distinguish between these possibilities, we engineered an HSV-1 ICP22 null mutant, d22-lacZ, and compared it to 22/n199. The two mutants are indistinguishable in their effects on the RNAP II large subunit, suggesting that an additional viral gene product is involved in altering RNAP II. Two candidates are UL13, a protein kinase which has been implicated in ICP22 phosphorylation, and the virion host shutoff (Vhs) factor, the expression of which is positively regulated by ICP22 and UL13. To test whether UL13 is involved, a UL13-deficient viral mutant, d13-lacZ, was engineered. This mutant was defective in IIi induction and IIa depletion, displaying a phenotype very similar to that of d22-lacZ. In contrast, a Vhs mutant had effects that were indistinguishable from wild-type HSV-1. Therefore, UL13 but not the Vhs function plays a role in modifying the RNAP II large subunit. To study the potential role of UL13 in viral transcription, we carried out nuclear run-on transcription analyses in infected human embryonic lung cells. Infections with either UL13 or ICP22 mutants led to significantly reduced amounts of viral genome transcription at late times after infection. Together, our results suggest that ICP22 and UL13 are involved in a common pathway that alters RNAP II phosphorylation and that in some cell lines this change promotes viral late transcription. PMID:10364308

The molecular genetics of Usher syndrome.

PubMed

Ahmed, Z M; Riazuddin, S; Riazuddin, S; Wilcox, E R

2003-06-01

Association of sensorineural deafness and progressive retinitis pigmentosa with and without a vestibular abnormality is the hallmark of Usher syndrome and involves at least 12 loci among three different clinical subtypes. Genes identified for the more commonly inherited loci are USH2A (encoding usherin), MYO7A (encoding myosin VIIa), CDH23 (encoding cadherin 23), PCDH15 (encoding protocadherin 15), USH1C (encoding harmonin), USH3A (encoding clarin 1), and USH1G (encoding SANS). Transcripts from all these genes are found in many tissues/cell types other than the inner ear and retina, but all are uniquely critical for retinal and cochlear cell function. Many of these protein products have been demonstrated to have direct interactions with each other and perform an essential role in stereocilia homeostasis.

Aroma compounds generation in citrate metabolism of Enterococcus faecium: Genetic characterization of type I citrate gene cluster.

PubMed

Martino, Gabriela P; Quintana, Ingrid M; Espariz, Martín; Blancato, Victor S; Magni, Christian

2016-02-02

Enterococcus is one of the most controversial genera belonging to Lactic Acid Bacteria. Research involving this microorganism reflects its dual behavior as regards its safety. Although it has also been associated to nosocomial infections, natural occurrence of Enterococcus faecium in food contributes to the final quality of cheese. This bacterium is capable of fermenting citrate, which is metabolized to pyruvate and finally derives in the production of the aroma compounds diacetyl, acetoin and 2,3 butanediol. Citrate metabolism was studied in E. faecium but no data about genes related to these pathways have been described. A bioinformatic approach allowed us to differentiate cit(-) (no citrate metabolism genes) from cit(+) strains in E. faecium. Furthermore, we could classify them according to genes encoding for the transcriptional regulator, the oxaloacetate decarboxylase and the citrate transporter. Thus we defined type I organization having CitI regulator (DeoR family), CitM cytoplasmic soluble oxaloacetate decarboxylase (Malic Enzyme family) and CitP citrate transporter (2-hydroxy-carboxylate transporter family) and type II organization with CitO regulator (GntR family), OAD membrane oxaloacetate decarboxylase complex (Na(+)-transport decarboxylase enzyme family) and CitH citrate transporter (CitMHS family). We isolated and identified 17 E. faecium strains from regional cheeses. PCR analyses allowed us to classify them as cit(-) or cit(+). Within the latter classification we could differentiate type I but no type II organization. Remarkably, we came upon E. faecium GM75 strain which carries the insertion sequence IS256, involved in adaptative and evolution processes of bacteria related to Staphylococcus and Enterococcus genera. In this work we describe the differential behavior in citrate transport, metabolism and aroma generation of three strains and we present results that link citrate metabolism and genetic organizations in E. faecium for the first time. Copyright © 2015 Elsevier B.V. All rights reserved.

Copper-free Sonogashira cross-coupling for functionalization of alkyne-encoded proteins in aqueous medium and in bacterial cells.

PubMed

Li, Nan; Lim, Reyna K V; Edwardraja, Selvakumar; Lin, Qing

2011-10-05

Bioorthogonal reactions suitable for functionalization of genetically or metabolically encoded alkynes, for example, copper-catalyzed azide-alkyne cycloaddition reaction ("click chemistry"), have provided chemical tools to study biomolecular dynamics and function in living systems. Despite its prominence in organic synthesis, copper-free Sonogashira cross-coupling reaction suitable for biological applications has not been reported. In this work, we report the discovery of a robust aminopyrimidine-palladium(II) complex for copper-free Sonogashira cross-coupling that enables selective functionalization of a homopropargylglycine (HPG)-encoded ubiquitin protein in aqueous medium. A wide range of aromatic groups including fluorophores and fluorinated aromatic compounds can be readily introduced into the HPG-containing ubiquitin under mild conditions with good to excellent yields. The suitability of this reaction for functionalization of HPG-encoded ubiquitin in Escherichia coli was also demonstrated. The high efficiency of this new catalytic system should greatly enhance the utility of Sonogashira cross-coupling in bioorthogonal chemistry.

Staphylococcal SCCmec elements encode an active MCM-like helicase and thus may be replicative

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

Mir-Sanchis, Ignacio; Roman, Christina A.; Misiura, Agnieszka

2016-08-29

Methicillin-resistant Staphylococcus aureus (MRSA) is a public-health threat worldwide. Although the mobile genomic island responsible for this phenotype, staphylococcal cassette chromosome (SCC), has been thought to be nonreplicative, we predicted DNA-replication-related functions for some of the conserved proteins encoded by SCC. We show that one of these, Cch, is homologous to the self-loading initiator helicases of an unrelated family of genomic islands, that it is an active 3'-to-5' helicase and that the adjacent ORF encodes a single-stranded DNA–binding protein. Our 2.9-Å crystal structure of intact Cch shows that it forms a hexameric ring. Cch, like the archaeal and eukaryotic MCM-familymore » replicative helicases, belongs to the pre–sensor II insert clade of AAA+ ATPases. Additionally, we found that SCC elements are part of a broader family of mobile elements, all of which encode a replication initiator upstream of their recombinases. Replication after excision would enhance the efficiency of horizontal gene transfer.« less