scholarly journals Molecular Characterization of Bovine Enteric Caliciviruses: a Distinct Third Genogroup of Noroviruses (Norwalk-Like Viruses) Unlikely To Be of Risk to Humans

2003 ◽  
Vol 77 (4) ◽  
pp. 2789-2798 ◽  
Author(s):  
S. L. Oliver ◽  
A. M. Dastjerdi ◽  
S. Wong ◽  
L. El-Attar ◽  
C. Gallimore ◽  
...  
Keyword(s):  
Gene Sequence ◽  
Orf3 Gene ◽  
Sequence Analyses ◽  
Animal Reservoir ◽  
Reading Frame ◽  
Reverse Transcription Pcr ◽  
The United Kingdom ◽  
Enteric Caliciviruses ◽  
Relationship Of

ABSTRACT Bovine enteric caliciviruses (BoCVs) have been classified in the Norovirus (Norwalk-like virus) genus of the Caliciviridae, raising questions about zoonotic transmission and an animal reservoir for the human Norwalk-like viruses (NLVs), an important cause of nonbacterial gastroenteritis in humans. We examined the genetic relationship of human NLVs to BoCVs that were identified by using reverse transcription-PCR with primer pairs originally designed to detect human NLVs. Polymerase, capsid, and open reading frame 3 (ORF3) gene sequence analyses of BoCVs that were identified from 1976 to 2000 from throughout the United Kingdom showed that BoCVs formed a distinct third genogroup of closely related viruses distinct from the human genogroup I and II NLVs. Evidence was not obtained to support the concept that BoCVs are circulating in humans and pose a threat to human health.

scholarly journals Molecular Characterization of a Flagellar Export Locus of Helicobacter pylori

1999 ◽  
Vol 67 (5) ◽  
pp. 2060-2070 ◽  
Author(s):  
Steffen Porwollik ◽  
Brian Noonan ◽  
Paul W. O’Toole
Keyword(s):  
Helicobacter Pylori ◽  
Virulence Factor ◽  
Housekeeping Genes ◽  
Reading Frame ◽  
Reverse Transcription Pcr ◽  
Mutant Strains ◽  
H Pylori ◽  
Export Protein ◽  
Successful Colonization

ABSTRACT Motility of Helicobacter species has been shown to be essential for successful colonization of the host. We have investigated the organization of a flagellar export locus in Helicobacter pylori. A 7-kb fragment of the H. pylori CCUG 17874 genome was cloned and sequenced, revealing an operon comprising an open reading frame of unknown function (ORF03), essential housekeeping genes (ileS and murB), flagellar export genes (fliI and fliQ), and a homolog to a gene implicated in virulence factor transport in other pathogens (virB11). A promoter for this operon, showing similarity to the Escherichia coli ς70 consensus, was identified by primer extension. Cotranscription of the genes in the operon was demonstrated by reverse transcription-PCR, and transcription of virB11, fliI, fliQ, andmurB was detected in human or mouse biopsies obtained from infected hosts. The genetic organization of this locus was conserved in a panel of H. pylori clinical isolates. EngineeredfliI and fliQ mutant strains were completely aflagellate and nonmotile, whereas a virB11 mutant still produced flagella. The fliI and fliQ mutant strains produced reduced levels of flagellin and the hook protein FlgE. Production of OMP4, a member of the outer membrane protein family identified in H. pylori 26695, was reduced in both thevirB11 mutant and the fliI mutant, suggesting related functions of the virulence factor export protein (VirB11) and the flagellar export component (FliI).

scholarly journals Identification and Preliminary Characterization of Two cDNAs Encoding Unique Carbonic Anhydrases from the Marine Alga Emiliania huxleyi

2006 ◽  
Vol 72 (8) ◽  
pp. 5500-5511 ◽  
Author(s):  
Amelia R. Soto ◽  
Hong Zheng ◽  
Dorinda Shoemaker ◽  
Jason Rodriguez ◽  
Betsy A. Read ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Time Course ◽  
Dissolved Inorganic Carbon ◽  
Emiliania Huxleyi ◽  
Carbonic Anhydrases ◽  
Reading Frame ◽  
Preliminary Characterization ◽  
Reverse Transcription Pcr ◽  
Ecological Importance

ABSTRACT Marine coccolithophorid algae are thought to play a significant role in carbon cycling due to their ability to incorporate dissolved inorganic carbon (DIC) into both calcite and photosynthetic products. Among coccolithophorids, Emiliania huxleyi is the most prolific, forming massive blooms that affect the global environment. In addition to its ecological importance, the elaborate calcite structures (coccoliths) are being investigated for the design of potential materials for science and biotechnological devices. To date, most of the research focus in this organism has involved the partitioning of DIC between calcification and photosynthesis, primarily using measurements of an external versus internal carbonic anhydrase (CA) activity under defined conditions. The actual genes, proteins, and pathways employed in these processes have not been identified and characterized (see the work of Quinn et al. in this issue [P. Quinn, R. M. Bowers, X. Zhang, T. M. Wahlund, M. A. Fanelli, D. Olszova, and B. A. Read, Appl. Environ. Microbiol. 72:5512-5526, 2006]). In this study, the cloning and preliminary characterization of two genetically distinct carbonic anhydrase cDNAs are described. Phylogenetic analysis indicated that these two genes belonged to the gamma (γ-EhCA2) and delta (δ-EhCA1) classes of carbonic anhydrases. The deduced amino acid sequence of δ-EhCA1 revealed that it encodes a protein of 702 amino acids (aa) (ca. 77.3 kDa), with a transmembrane N-terminal region of 373 aa and an in-frame C-terminal open reading frame of 329 aa that defines the CA region. The γ-EhCA2 protein was 235 aa in length (ca. 24.9 kDa) and was successfully expressed in Escherichia coli BL21(DE3) and purified as an active recombinant CA. The expression levels of each transcript from quantitative reverse transcription-PCR experiments under bicarbonate limitation and over a 24-h time course suggest that these isozymes perform different functions in E. huxleyi.

scholarly journals Characterization of Strains unlike Mesorhizobium loti That Nodulate Lotus spp. in Saline Soils of Granada, Spain

2010 ◽  
Vol 76 (12) ◽  
pp. 4019-4026 ◽  
Author(s):  
María J. Lorite ◽  
Socorro Muñoz ◽  
José Olivares ◽  
María J. Soto ◽  
Juan Sanjuán
Keyword(s):  
Gene Sequence ◽  
Low Fertility ◽  
Salt Tolerant ◽  
Forage Legumes ◽  
Sequence Analyses ◽  
Mesorhizobium Loti ◽  
Mesorhizobium Tianshanense ◽  
High Salt Tolerance ◽  
Repetitive Extragenic Palindromic Pcr

ABSTRACT Lotus species are forage legumes with potential as pastures in low-fertility and environmentally constrained soils, owing to their high persistence and yield under those conditions. The aim of this work was the characterization of phenetic and genetic diversity of salt-tolerant bacteria able to establish efficient symbiosis with Lotus spp. A total of 180 isolates able to nodulate Lotus corniculatus and Lotus tenuis from two locations in Granada, Spain, were characterized. Molecular identification of the isolates was performed by repetitive extragenic palindromic PCR (REP-PCR) and 16S rRNA, atpD, and recA gene sequence analyses, showing the presence of bacteria related to different species of the genus Mesorhizobium: Mesorhizobium tarimense/Mesorhizobium tianshanense, Mesorhizobium chacoense/Mesorhizobium albiziae, and the recently described species, Mesorhizobium alhagi. No Mesorhizobium loti-like bacteria were found, although most isolates carried nodC and nifH symbiotic genes closely related to those of M. loti, considered the type species of bacteria nodulating Lotus, and other Lotus rhizobia. A significant portion of the isolates showed both high salt tolerance and good symbiotic performance with L. corniculatus, and many behaved like salt-dependent bacteria, showing faster growth and better symbiotic performance when media were supplemented with Na or Ca salts.

scholarly journals Molecular cloning and characterization of CIDE-3, a novel member of the cell-death-inducing DNA-fragmentation-factor (DFF45)-like effector family

2003 ◽  
Vol 370 (1) ◽  
pp. 195-203 ◽  
Author(s):  
Liang LIANG ◽  
Mujun ZHAO ◽  
Zhenhua XU ◽  
Kazunari K. YOKOYAMA ◽  
Tsaiping LI
Keyword(s):  
Amino Acids ◽  
Cell Death ◽  
Small Intestine ◽  
Dna Fragmentation ◽  
Nuclear Dna ◽  
Fluorescent Protein ◽  
Reading Frame ◽  
Reverse Transcription Pcr ◽  
Green Fluorescent

DNA fragmentation is one of the critical steps in apoptosis, which is induced by DNA fragmentation factor (DFF). DFF is composed of two subunits, a 40kDa caspase-activated nuclease (DFF40) and a 45kDa inhibitor (DFF45). Recently a novel family of cell-death-inducing DFF45-like effectors (CIDEs) has been identified. Among CIDEs, two from human (CIDE-A and CIDE-B) and three from mouse (CIDE-A, CIDE-B and FSP27) have been reported. In this study human CIDE-3, a novel member of CIDEs, was identified upon sequence analysis of a previously unidentified cDNA that encoded a protein of 238 amino acids. It was shown to be a human homologue of mouse FSP27, and shared homology with the CIDE-N and CIDE-C domains of CIDEs. Apoptosis-inducing activity was clearly shown by DNA-fragmentation assay of the nuclear DNA of CIDE-3 transfected 293T cells. The expression pattern of CIDE-3 was different from that of CIDE-B. As shown by Northern-blot analysis, CIDE-3 was expressed mainly in human small intestine, heart, colon and stomach, while CIDE-B showed strong expression in liver and small intestine and at a lower level in colon, kidney and spleen. Green-fluorescent-protein-tagged CIDE-3 was revealed in some cytosolic corpuscles. Alternative splicing of the CIDE-3 gene was also identified by reverse transcription PCR, revealing that two transcripts, CIDE-3 and CIDE-3α, were present in HepG2 and A375 cells. CIDE-3 comprised a full-length open reading frame with 238 amino acids; in CIDE-3α exon 3 was deleted and it encoded a protein of 164 amino acids. Interestingly the CIDE-3α isoform still kept the apoptosis-inducing activity and showed the same pattern of subcellular localization as CIDE-3. Consistent with its chromosome localization at 3p25, a region associated with high frequency loss of heterozygosity in many tumours, CIDE-3 may play an important role in prevention of tumorigenesis.

scholarly journals Cloning and Expression of the algL Gene, Encoding the Azotobacter chroococcum Alginate Lyase: Purification and Characterization of the Enzyme

1999 ◽  
Vol 181 (5) ◽  
pp. 1409-1414 ◽  
Author(s):  
Ana Peciña ◽  
Alberto Pascual ◽  
Antonio Paneque
Keyword(s):  
Gene Sequence ◽  
Azotobacter Vinelandii ◽  
Alginate Lyase ◽  
Azotobacter Chroococcum ◽  
Open Reading Frame ◽  
Cloning And Expression ◽  
Gene Encoding ◽  
Reading Frame ◽  
Encoding Gene

ABSTRACT The alginate lyase-encoding gene (algL) ofAzotobacter chroococcum was localized to a 3.1-kbEcoRI DNA fragment that revealed an open reading frame of 1,116 bp. This open reading frame encodes a protein of 42.98 kDa, in agreement with the value previously reported by us for this protein. The deduced protein has a potential N-terminal signal peptide that is consistent with its proposed periplasmic location. The analysis of the deduced amino acid sequence indicated that the gene sequence has a high homology (90% identity) to the Azotobacter vinelandii gene sequence, which has very recently been deposited in the GenBank database, and that it has 64% identity to the Pseudomonas aeruginosa gene sequence but that it has rather low homology (15 to 22% identity) to the gene sequences encoding alginate lyase in other bacteria. The A. chroococcum AlgL protein was overproduced in Escherichia coli and purified to electrophoretic homogeneity in a two-step chromatography procedure on hydroxyapatite and phenyl-Sepharose. The kinetic and molecular parameters of the recombinant alginate lyase are similar to those found for the native enzyme.

scholarly journals Characterization of Ti Ringspot-Associated Virus, a Novel Emaravirus Associated with an Emerging Ringspot Disease of Cordyline fruticosa

Plant Disease ◽  
2019 ◽  
Vol 103 (9) ◽  
pp. 2345-2352 ◽  
Author(s):  
Alejandro Olmedo-Velarde ◽  
Adam C. Park ◽  
Jari Sugano ◽  
Janice Y. Uchida ◽  
Michael Kawate ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
High Plains ◽  
Virus Species ◽  
Sequence Analyses ◽  
Sequence Identity ◽  
Reverse Transcription Pcr ◽  
Eriophyid Mites ◽  
Cordyline Fruticosa ◽  
Novel Virus

Ti ringspot is an emerging foliar disease of the ti plant (Cordyline fruticosa) in Hawaii that is quickly spreading throughout the islands. Symptoms include small chlorotic ringspots on leaves that often coalesce to form larger lesions. Although several virus species have been discovered in symptomatic plants, none have been associated with these symptoms. Here, we report and characterize a novel virus closely associated with ti ringspot symptoms in Hawaii. The presence of double membrane bodies approximately 85 nm in diameter in symptomatic cells and sequence analyses of five genomic RNA segments obtained by high-throughput sequencing indicate that this virus is most closely related to members of the plant virus genus Emaravirus. Phylogenetic and sequence homology analyses place this virus on a distinct clade within the Emaravirus genus along with High Plains wheat mosaic emaravirus, blue palo verde broom virus, and Raspberry leaf blotch emaravirus. Sequence identity values with taxonomically relevant proteins indicate that this represents a new virus species, which we are tentatively naming ti ringspot-associated virus (TiRSaV). TiRSaV-specific reverse transcription PCR assays detected the virus in several experimental herbaceous host species following mechanical inoculation. TiRSaV was also detected in eriophyid mites collected from symptomatic ti plants, which may represent a putative arthropod vector of the virus.

Characterization of an Operon Encoding Two c-Type Cytochromes, an aa3-Type Cytochrome Oxidase, and Rusticyanin in Thiobacillus ferrooxidansATCC 33020

1999 ◽  
Vol 65 (11) ◽  
pp. 4781-4787 ◽  
Author(s):  
Corinne Appia-Ayme ◽  
Nicolas Guiliani ◽  
Jeanine Ratouchniak ◽  
Violaine Bonnefoy
Keyword(s):  
Electron Transfer ◽  
Thiobacillus Ferrooxidans ◽  
Energetic Metabolism ◽  
Reading Frame ◽  
Electron Transfer Proteins ◽  
Reverse Transcription Pcr ◽  
Genes Encoding ◽  
Transcriptional Start Sites ◽  
Transfer Chain

ABSTRACT Despite the importance of Thiobacillus ferrooxidans in bioremediation and bioleaching, little is known about the genes encoding electron transfer proteins implicated in its energetic metabolism. This paper reports the sequences of the fourcox genes encoding the subunits of anaa 3-type cytochrome c oxidase. These genes are in a locus containing four other genes:cyc2, which encodes a high-molecular-weight cytochromec; cyc1, which encodes ac 4-type cytochrome (c 552); open reading frame 1, which encodes a putative periplasmic protein of unknown function; and rus, which encodes rusticyanin. The results of Northern and reverse transcription-PCR analyses indicated that these eight genes are cotranscribed. Two transcriptional start sites were identified for this operon. Upstream from each of the start sites was a ς70-type promoter recognized in Escherichia coli. While transcription in sulfur-grown T. ferrooxidans cells was detected from the two promoters, transcription in ferrous-iron-grown T. ferrooxidans cells was detected only from the downstream promoter. The cotranscription of seven genes encoding redox proteins suggests that all these proteins are involved in the same electron transfer chain; a model taking into account the biochemistry and the genetic data is discussed.

Characterization of a Second Ornithine Decarboxylase Isolated from Morganella morganii

2008 ◽  
Vol 71 (3) ◽  
pp. 657-661 ◽  
Author(s):  
BLANCA DE LAS RIVAS ◽  
RAMÓN GONZÁLEZ ◽  
JOSÉ MARÍA LANDETE ◽  
ROSARIO MUÑOZ
Keyword(s):  
Ornithine Decarboxylase ◽  
Growth Conditions ◽  
Open Reading Frame ◽  
Morganella Morganii ◽  
Reading Frame ◽  
Reverse Transcription Pcr ◽  
Acid Protein ◽  
New Gene ◽  
Encoding Genes

The genes involved in the putrescine formation by Morganella morganii were investigated because putrescine is an indicator of food process deterioration. We report here on the existence of a new gene for ornithine decarboxylase (ODC) in M. morganii. The sequenced 5,311-bp DNA region showed the presence of four complete and one partial open reading frame. Putative functions have been assigned to several gene products by sequence comparison with the proteins included in the databases. The third open reading frame (speC ) encoded a 722–amino acid protein showing 70.9% identity to the M. morganii ODC previously characterized (SpeF ).The speC gene has been expressed in Escherichia coli, resulting in ODC activity. The presence of a functional promoter (PspeC ) located upstream of speC has been demonstrated. Quantitative real-time reverse transcription PCR assay was used to quantify expression of both M. morganii ODC-encoding genes, speC and speF, under different growth conditions. This assay allows us to identify SpeF as the inducible M. morganii ODC, since it was highly expressed in the presence of ornithine.

scholarly journals Thermostable Chitosanase from Bacillus sp. Strain CK4: Cloning and Expression of the Gene and Characterization of the Enzyme

2000 ◽  
Vol 66 (9) ◽  
pp. 3727-3734 ◽  
Author(s):  
Ho-Geun Yoon ◽  
Hee-Yun Kim ◽  
Young-Hee Lim ◽  
Hye-Kyung Kim ◽  
Dong-Hoon Shin ◽  
...  
Keyword(s):  
Gene Sequence ◽  
Specific Activity ◽  
High Specific Activity ◽  
Industrial Applications ◽  
Cloning And Expression ◽  
Rrna Gene ◽  
Phenotypic Analysis ◽  
Reading Frame ◽  
The 16S Rrna Gene

ABSTRACT A thermostable chitosanase gene from the environmental isolateBacillus sp. strain CK4, which was identified on the basis of phylogenetic analysis of the 16S rRNA gene sequence and phenotypic analysis, was cloned, and its complete DNA sequence was determined. The thermostable chitosanase gene was composed of an 822-bp open reading frame which encodes a protein of 242 amino acids and a signal peptide corresponding to a 30-kDa enzyme. The deduced amino acid sequence of the chitosanase from Bacillus sp. strain CK4 exhibits 76.6, 15.3, and 14.2% similarities to those from Bacillus subtilis, Bacillus ehemensis, and Bacillus circulans, respectively. C-terminal homology analysis shows thatBacillus sp. strain CK4 belongs to cluster III withB. subtilis. The gene was similar in size to that of the mesophile B. subtilis but showed a higher preference for codons ending in G or C. The enzyme contains 2 additional cysteine residues at positions 49 and 211. The recombinant chitosanase has been purified to homogeneity by using only two steps with column chromatography. The half-life of the enzyme was 90 min at 80�C, which indicates its usefulness for industrial applications. The enzyme had a useful reactivity and a high specific activity for producing functional oligosaccharides as well, with trimers through hexamers as the major products.

scholarly journals Molecular characterization of Eurytrema coelomaticum in cattle from Paraná, Brazil

2014 ◽  
Vol 23 (3) ◽  
pp. 383-386 ◽  
Author(s):  
Gustavo Freire Figueira ◽  
Victor Henrique Silva de Oliveira ◽  
Alessandra Taroda ◽  
Amauri Alcindo Alfieri ◽  
Selwyn Arlington Headley
Keyword(s):  
Molecular Characterization ◽  
Gene Sequence ◽  
Domestic Animals ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
Sequence Analyses ◽  
Pcr Assay ◽  
Bovine Pancreas

This study investigated the occurrence of Eurytremaspp. in cattle by analysis of the partial 18S rRNA gene sequence. Trematodes from 44 bovine pancreas were collected and classified based on typical morphological features. PCR assay and sequence analyses of amplified products confirmed that the trematodes classified as Eurytrema coelomaticum were phylogenetically distinct from those identified as E. pancreaticum. The results of this study represent the first molecular characterization of E. coelomaticum within the Americas, and provide an efficient method to differentiate digenean trematodes of domestic animals.

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