scholarly journals d-Xylose (d-glucose) isomerase from Arthrobacter strain N.R.R.L. B3728. Gene cloning, sequence and expression

1991 ◽  
Vol 277 (1) ◽  
pp. 263-271 ◽  
Author(s):  
T Loviny-Anderton ◽  
P C Shaw ◽  
M K Shin ◽  
B S Hartley
Keyword(s):  
Sequence Similarity ◽  
Shuttle Vector ◽  
Xylose Isomerase ◽  
Glucose Isomerase ◽  
Open Reading Frame ◽  
Reading Frame ◽  
E Coli ◽  
Ribosome Binding ◽  
Arthrobacter Strain ◽  
Cloned Gene

Arthrobacter strain N.R.R.L. B3728 superproduces a D-xylose isomerase that is also a useful industrial D-glucose isomerase. The gene (xylA) that encodes it has been cloned by complementing a xylA mutant of the ancestral strain, with the use of a shuttle vector. The 5′ region shows strong sequence similarity to Escherichia coli consensus promoters and ribosome-binding sequences and allows high levels of expression in E. coli. The coding sequence shows similarity to those for other D-xylose isomerases and is followed by 22 nucleotide residues with stop codons in each reading frame, a good ‘consensus’ ribosome-binding site and an open reading frame showing similarity to those of known D-xylulokinases (xylB). Studies on the expression of the cloned gene in Arthrobacter and in E. coli suggest that the two genes are part of a xyl operon regulated by a repressor that is defective in strain B3728. Codon usage in these two genes, and in another open reading frame (nxi) that was adventitiously isolated during early cloning attempts, shows some characteristic omissions and a strong G + C preference in redundant positions.

scholarly journals Oligoribonuclease Is Encoded by a Highly Conserved Gene in the 3′-5′ Exonuclease Superfamily

1998 ◽  
Vol 180 (10) ◽  
pp. 2779-2781 ◽  
Author(s):  
Xiaolan Zhang ◽  
Liuqin Zhu ◽  
Murray P. Deutscher
Keyword(s):  
Kanamycin Resistance ◽  
Open Reading Frame ◽  
Sequence Information ◽  
Gene Encoding ◽  
Reading Frame ◽  
E Coli ◽  
Kanamycin Resistance Cassette ◽  
Wide Range ◽  
Conserved Gene ◽  
Cloned Gene

ABSTRACT Oligoribonuclease, a 3′-to-5′ exoribonuclease specific for small oligoribonucleotides, was purified to homogeneity from extracts ofEscherichia coli. The purified protein is an α2 dimer of 40 kDa. NH2-terminal sequence analysis of the protein identified the gene encoding oligoribonuclease as yjeR(o204a), a previously reported open reading frame located at 94 min on the E. coli chromosome. However, as a consequence of the sequence information, the translation start site of this open reading frame has been revised. Cloning of yjeRled to overexpression of oligoribonuclease activity, and interruption of the cloned gene with a kanamycin resistance cassette eliminated the overexpression. On the basis of these data, we propose thatyjeR be renamed orn. Orthologs of oligoribonuclease are present in a wide range of organisms, extending up to humans.

scholarly journals Antigenic Characterization of ORF2 and ORF3 Proteins of Hepatitis E Virus (HEV)

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1385
Author(s):  
Giulia Pezzoni ◽  
Lidia Stercoli ◽  
Eleonora Pegoiani ◽  
Emiliana Brocchi
Keyword(s):  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Recombinant Antigen ◽  
Open Reading Frame ◽  
Reading Frame ◽  
E Coli ◽  
Antigenic Characterization ◽  
Antigenic Properties ◽  
Open Reading Frame 2

To evaluate the antigenic properties of Hepatitis E Virus (HEV) Open Reading Frame 2 and 3 (ORF2 and ORF3) codified proteins, we expressed different portions of ORF2 and the entire ORF3 in E. coli, a truncated ORF2, was also expressed in baculovirus. A panel of 37 monoclonal antibodies (MAbs) was raised against ORF2 (1–660 amino acids) and MAbs were mapped and characterized using the ORF2 expressed portions. Selected HEV positive and negative swine sera were used to evaluate ORF2 and ORF3 antigens’ immunogenicity. The MAbs were clustered in six groups identifying six antigenic regions along the ORF2. Only MAbs binding to the sixth ORF2 antigenic region (394–608 aa) were found to compete with HEV positive sera and efficiently catch the recombinant antigen expressed in baculovirus. The ORF2 portion from 394–608 aa demonstrated to include most immunogenic epitopes with 85% of HEV positive swine sera reacting against the region from 461–544 aa. Only 5% of the selected HEV sera reacted against the ORF3 antigen.

scholarly journals Gene Cloning and Molecular Characterization of a Two-Enzyme System Catalyzing the Oxidative Detoxification of β-Endosulfan

2002 ◽  
Vol 68 (12) ◽  
pp. 6237-6245 ◽  
Author(s):  
Tara D. Sutherland ◽  
Irene Horne ◽  
Robyn J. Russell ◽  
John G. Oakeshott
Keyword(s):  
Shuttle Vector ◽  
Open Reading Frame ◽  
Cosmid Library ◽  
Flavin Mononucleotide ◽  
Flavin Reductase ◽  
Reading Frame ◽  
Reductase Gene ◽  
Degradation Activity ◽  
Layer Chromatography

ABSTRACT The gram-positive bacterium Mycobacterium sp. strain ESD is able to use the cyclodiene insecticide endosulfan as a source of sulfur for growth. This activity is dependent on the absence of sulfite or sulfate in the growth medium. A cosmid library of strain ESD DNA was constructed in a Mycobacterium-Escherichia coli shuttle vector and screened for endosulfan-degrading activity in Mycobacterium smegmatis, a species that does not degrade endosulfan. Using this method, we identified a single cosmid that conferred sulfur-dependent endosulfan-degrading activity on the host strain. An open reading frame (esd) was identified within this cosmid that, when expressed behind a constitutive promoter in a mycobacterial expression vector, conferred sulfite- and sulfate-independent β-endosulfan degradation activity on the recombinant strain. The translation product of this gene (Esd) had up to 50% sequence identity with an unusual family of monooxygenase enzymes that use reduced flavins, provided by a separate flavin reductase enzyme, as cosubstrates. An additional partial open reading frame was located upstream of the Esd gene that had sequence homology to the same monooxygenase family. A flavin reductase gene, identified in the M. smegmatis genome, was cloned, expressed, and used to provide reduced flavin mononucleotide for Esd in enzyme assays. Thin-layer chromatography and gas chromatography analyses of the enzyme assay mixtures revealed the disappearance of β-endosulfan and the appearance of the endosulfan metabolites, endosulfan monoaldehyde and endosulfan hydroxyether. This suggests that Esd catalyzes the oxygenation of β-endosulfan to endosulfan monoaldehyde and endosulfan hydroxyether. Esd did not degrade either α-endosulfan or the metabolite of endosulfan, endosulfan sulfate.

scholarly journals Cloning and characterization of α-pinene synthase from Chamaecyparis formosensis Matsum

Holzforschung ◽  
2009 ◽  
Vol 63 (1) ◽  
Author(s):  
Fang-Hua Chu ◽  
Pei-Min Kuo ◽  
Yu-Rong Chen ◽  
Sheng-Yang Wang
Keyword(s):  
Major Product ◽  
Open Reading Frame ◽  
Terpene Synthase ◽  
Geranyl Diphosphate ◽  
Reading Frame ◽  
E Coli ◽  
Phylogenetic Taxonomy ◽  
Chamaecyparis Formosensis ◽  
Pcr Method

AbstractAnalyzing the gene sequences of terpene synthase (TPS) may contribute to a better understanding of terpenes biosynthesis and evolution of phylogenetic taxonomy.Chamaecyparis formosensisis an endemic and precious conifer of Taiwan. To understand the biosynthesis mechanism of terpenes in this tree, a full length of putative mono-TPS, named asCf-Pin(GeneBank accession no. EU099434), was obtained by PCR method and RACE extension. TheCf-Pinhas an 1887-bp open reading frame and encodes 628 amino acids. To identify the function ofCf-Pin,the recombinant protein fromEscherichia coliwas incubated with geranyl diphosphate, produced one major product, the structure of which was elucidated. GC/MS analysis and matching of retention time and mass spectrum with authentic standards revealed that this product isα-pinene. This is the first report of cloning of a mono-TPS and functionally expressed inE. coliand which could be identified asα-pinene synthase from a Cupressaceae conifer.

scholarly journals Cloning, Nucleotide Sequence, and Expression of the Gene Encoding the Bacteriocin Boticin B from Clostridium botulinum Strain 213B

2000 ◽  
Vol 66 (12) ◽  
pp. 5480-5483 ◽  
Author(s):  
Sean S. Dineen ◽  
Marite Bradshaw ◽  
Eric A. Johnson
Keyword(s):  
Amino Acids ◽  
Nucleotide Sequence ◽  
Clostridium Botulinum ◽  
Shuttle Vector ◽  
Open Reading Frame ◽  
Gene Encoding ◽  
Reading Frame ◽  
Heat Stable ◽  
Group I

ABSTRACT Boticin B is a heat-stable bacteriocin produced byClostridium botulinum strain 213B that has inhibitory activity against various strains of C. botulinum and related clostridia. The gene encoding the bacteriocin was localized to a 3.0-kb HindIII fragment of an 18.8-kb plasmid, cloned, and sequenced. DNA sequencing revealed the boticin B structural gene,btcB, to be an open reading frame encoding 50 amino acids. A C. botulinum strain 62A transconjugant containing theHindIII fragment inserted into a clostridial shuttle vector expressed boticin B, although at much lower levels than those observed in C. botulinum 213B. To our knowledge, this is the first demonstration and characterization of a bacteriocin from toxigenic group I C. botulinum.

Molecular and genetic analysis of the gene encoding the Saccharomyces cerevisiae strand exchange protein Sep1

1991 ◽  
Vol 11 (5) ◽  
pp. 2593-2608 ◽  
Author(s):  
D X Tishkoff ◽  
A W Johnson ◽  
R D Kolodner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Open Reading Frame ◽  
Strand Exchange ◽  
Wild Type ◽  
Homologous Pairing ◽  
Gene Encoding ◽  
Reading Frame ◽  
Cloned Gene ◽  
Diploid Cells ◽  
Exchange Protein

Vegetatively grown Saccharomyces cerevisiae cells contain an activity that promotes a number of homologous pairing reactions. A major portion of this activity is due to strand exchange protein 1 (Sep1), which was originally purified as a 132,000-Mr species (R. Kolodner, D. H. Evans, and P. T. Morrison, Proc. Natl. Acad. Sci. USA 84:5560-5564, 1987). The gene encoding Sep1 was cloned, and analysis of the cloned gene revealed a 4,587-bp open reading frame capable of encoding a 175,000-Mr protein. The protein encoded by this open reading frame was overproduced and purified and had a relative molecular weight of approximately 160,000. The 160,000-Mr protein was at least as active in promoting homologous pairing as the original 132,000-Mr species, which has been shown to be a fragment of the intact 160,000-Mr Sep1 protein. The SEP1 gene mapped to chromosome VII within 20 kbp of RAD54. Three Tn10LUK insertion mutations in the SEP1 gene were characterized. sep1 mutants grew more slowly than wild-type cells, showed a two- to fivefold decrease in the rate of spontaneous mitotic recombination between his4 heteroalleles, and were delayed in their ability to return to growth after UV or gamma irradiation. Sporulation of sep1/sep1 diploids was defective, as indicated by both a 10- to 40-fold reduction in spore formation and reduced spore viability of approximately 50%. The majority of sep1/sep1 diploid cells arrested in meiosis after commitment to recombination but prior to the meiosis I cell division. Return-to-growth experiments showed that sep1/sep1 his4X/his4B diploids exhibited a five- to sixfold greater meiotic induction of His+ recombinants than did isogenic SEP1/SEP1 strains. sep1/sep1 mutants also showed an increased frequency of exchange between HIS4, LEU2, and MAT and a lack of positive interference between these markers compared with wild-type controls. The interaction between sep1, rad50, and spo13 mutations suggested that SEP1 acts in meiosis in a pathway that is parallel to the RAD50 pathway.

scholarly journals Characterization of the gene celD and its encoded product 1,4-β-d-glucan glucohydrolase D from Pseudomonas fluorescens subsp. cellulosa

1992 ◽  
Vol 285 (3) ◽  
pp. 947-955 ◽  
Author(s):  
J E Rixon ◽  
L M A Ferreira ◽  
A J Durrant ◽  
J I Laurie ◽  
G P Hazlewood ◽  
...  
Keyword(s):  
Pseudomonas Fluorescens ◽  
Plant Cell Wall ◽  
Genomic Library ◽  
Cell Envelope ◽  
Sequence Similarity ◽  
Significant Proportion ◽  
Structural Similarity ◽  
Reading Frame ◽  
E Coli ◽  
Beta Glucosidase

A genomic library of Pseudomonas fluorescens subsp. cellulosa DNA constructed in pUC18 and expressed in Escherichia coli was screened for recombinants expressing 4-methylumbelliferyl beta-D-glucoside hydrolysing activity (MUGase). A single MUGase-positive clone was isolated. The MUGase hydrolysed cellobiose, cellotriose, cellotetraose, cellopentaose and cellohexaose to glucose, by sequentially cleaving glucose residues from the non-reducing end of the cello-oligosaccharides. The Km values for cellobiose and cellohexaose hydrolysis were 1.2 mM and 28 microM respectively. The enzyme exhibited no activity against soluble or insoluble cellulose, xylan and xylobiose. Thus the MUGase is classified as a 1,4-beta-D-glucan glucohydrolase (EC 3.2.1.74) and is designated 1,4-beta-D-glucan glucohydrolase D (CELD). When expressed by E. coli, CELD was located in the cell-envelope fraction; a significant proportion of the native enzyme was also associated with the cell envelope when synthesized by its endogenous host. The nucleotide sequence of the gene, celD, which encodes CELD, revealed an open reading frame of 2607 bp, encoding a protein of M(r) 92,000. The deduced primary structure of CELD was confirmed by the M(r) of CELD (85,000) expressed by E. coli and P. fluorescens subsp. cellulosa, and by the experimentally determined N-terminus of the enzyme purified from E. coli, which showed identity with residues 52-67 of the celD translated sequence. The structure of the N-terminal region of full-length CELD was similar to the signal peptides of P. fluorescens subsp. cellulosa plant-cell-wall hydrolases. Deletion of the N-terminal 47 residues of CELD solubilized MUGase activity in E. coli. CELD exhibited sequence similarity with beta-glucosidase B of Clostridium thermocellum, particularly in the vicinity of the active-site aspartate residue, but did not display structural similarity with the mature forms of cellulases and xylanases expressed by P. fluorescens subsp. cellulosa.

scholarly journals Identification of a Pneumococcal Glycosidase That Modifies O-Linked Glycans

2009 ◽  
Vol 77 (4) ◽  
pp. 1389-1396 ◽  
Author(s):  
Carolyn Marion ◽  
Dominique H. Limoli ◽  
Gregory S. Bobulsky ◽  
Jessica L. Abraham ◽  
Amanda M. Burnaugh ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Bifidobacterium Longum ◽  
Airway Epithelial Cells ◽  
Open Reading Frame ◽  
Glycan Structure ◽  
Dependent Manner ◽  
Upper Respiratory Tract ◽  
Reading Frame ◽  
Human Airway ◽  
Published Evidence

ABSTRACT Colonization of the airway by Streptococcus pneumoniae is typically asymptomatic; however, progression of bacteria beyond the oronasopharynx can cause diseases including otitis media and pneumonia. The mechanisms by which S. pneumoniae establishes and maintains colonization remain poorly understood. Both N-linked and O-linked glycans are abundant in the airway. Our previous research demonstrated that S. pneumoniae can sequentially deglycosylate N-linked glycans and suggested that this modification of sugar structures may aid in colonization. There is published evidence that S. pneumoniae expresses a secreted O-glycosidase that cleaves galactose β1-3 N-acetylgalactosamine (Galβ1-3GalNAc) from core-1 O-linked glycans; however, the biological function of this enzyme has not previously been determined. We established that the activity is not secreted but is instead surface associated in a sortase-dependent manner. Genome analysis revealed an open reading frame predicted to encode a sortase-dependent surface protein with sequence similarity to the O-glycosidase of Bifidobacterium longum. Deletion of this pneumococcal open reading frame confirmed that this gene encodes an O-glycosidase. Experiments using a model glycoconjugate demonstrated that this O-glycosidase, together with the neuraminidase NanA, is required for S. pneumoniae to cleave sialylated core-1 O-linked glycans. The ability of the O-glycosidase mutant to cleave this glycan structure was restored by both genetic complementation and the addition of O-glycosidase. The mutant showed a reduction in adherence to human airway epithelial cells and a significantly decreased ability to colonize the upper respiratory tract, suggesting that cleavage of core-1 O-linked glycans enhances the ability of S. pneumoniae to colonize the human airway.

Molecular Cloning and Characterization of a cDNA Encoding a Transferrin Homolog from Bombyx mori

1999 ◽  
Vol 380 (12) ◽  
pp. 1455-1459 ◽  
Author(s):  
Eun Young Yun ◽  
Seok Woo Kang ◽  
Jae Sam Hwang ◽  
Tae Won Goo ◽  
Sang Hyun Kim ◽  
...  
Keyword(s):  
Bombyx Mori ◽  
Cdna Sequence ◽  
The Self ◽  
Open Reading Frame ◽  
Iron Binding ◽  
Defense System ◽  
Reading Frame ◽  
E Coli ◽  
Self Defense

Abstract We isolated a cDNA representing a message that was strongly induced by injection with E. coli in Bombyx mori. The 2160 bp cDNA has an open reading frame of 644 amino acids and the deduced product a predicted molecular mass of 71 kDa. The cDNA sequence shared high homology with the transferrins known so far, and its deduced peptide had unique features of transferrins, that is, sites of cystein residues and iron binding. We suggest that the B. mori transferrin plays an important role in the self-defense system.

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