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At least 721 records · Page 40Linked to original sources
PubMed1988

Molecular cloning of multiple xylanase genes from Pseudomonas fluorescens subsp. cellulosa.

Pseudomonas fluorescens subsp. cellulosa was shown to express extracellular xylanases. Genes encoding these enzymes were isolated from a gene library of P. fluorescens subsp. cellulosa DNA, constructed in bacteriophage lambda 47.1. One of the phages (PXC) that expressed xylanase also conferred the ability to hydrolyse carboxymethylcellulose. An 11.8 kb HindIII DNA restriction fragment and a 6.2 kb EcoRI DNA fragment were subcloned from two distinct xylanase-expressing phages, into pUC18, to yield recombinant plasmids pGHJ4 and pGHJ5 respectively. Cells of Escherichia coli harbouring either of these two plasmids, or plasmid pJHH1 (comprising the cellulase gene from PXC, previously cloned on a 7.3 kb partial EcoRI DNA fragment in pUC18), expressed xylanase activity. The positions of the xylanase genes in the recombinant plasmids were elucidated by subcloning and transposon mutagenesis. In pJHH1 the xylanase gene was adjacent to the DNA region encoding the endoglucanase. The polysaccharide-degrading genes in pJHH1 were transcribed from different promotors. Hybridization studies revealed that the xylanase genes encoded by pGHJ4 and pGHJ5 showed strong homology. All three cloned enzymes cleaved p-nitrophenyl beta-D-glucopyranoside and 4-methylumbelliferyl beta-D-cellobioside. Xylan and glucose did not affect expression of xylanase in E. coli strains harbouring pJHH1, pGHJ4 or pGHJ5.

Cloning, Molecular↗
PubMed1986

Construction of a host-vector system in Candida maltosa by using an ARS site isolated from its genome.

To construct a host-vector system in an n-alkane-assimilating yeast, Candida maltosa, the isolation of an ARS site from its genome which replicates autonomously in C. maltosa was attempted. Leu- mutants of C. maltosa were transformed with a gene library prepared by using YEp13 (LEU2+) as a vector, and Leu+ transformants were obtained at a high frequency. A plasmid named pCS1 was isolated from the recipient cells. pCS1 contained a 6.3-kilobase (kb) fragment of the C. maltosa genome, and a 3.8-kb fragment with ARS activity was subcloned and designated the TRA (transformation ability) region. Vectors (pTRA1 and pTRA11) for C. maltosa J288 were constructed that contained this 3.8-kb fragment, pBR322, and the LEU2 gene of Saccharomyces cerevisiae. Transformation of C. maltosa J288 with these plasmids was successful by both spheroplast and lithium acetate methods. Southern blot analysis suggested that the copy number of pTRA1 in C. maltosa was between 10 and 20, and it was stably maintained during growth without selective pressure in the medium. It was also found that these vectors could transform S. cerevisiae leu2- to LEU2+, suggesting that the TRA region contained an ARS site(s) that was specific not only for C. maltosa but also for S. cerevisiae.

Candida↗
PubMed1986

Isolation and identification of the Aspergillus nidulans gdhA gene encoding NADP-linked glutamate dehydrogenase.

The Neurospora crassa am gene was used as a heterologous probe to identify clones from two independently constructed Aspergillus nidulans gene libraries. These clones have a common HindIII 1.85 kb fragment. This A. nidulans nucleotide stretch hybridises to a N. crassa 2.7 kb BamHI fragment of wild type DNA but not to a co-migrating fragment from the DNA of the N. crassa am132 deletion mutant. One A. nidulans clone was shown to complement the N. crasse am132 deletion strain. The N. crassa transformants show low levels (approximately 5%) of heterologous glutamate dehydrogenase activity. The A. nidulans gdhA gene was found to locate in N. crassa at both the "homologous" (i.e. am) site as well as non-homologous sites. Partial nucleotide analysis of the fragment has revealed the 5' end of the locus and considerable homology with the N. crassa am gene. We concluded that we have cloned the A. nidulans gdhA gene.

Aspergillus nidulans↗
PubMed2004

Systematic 16S rRNA gene sequencing of atypical clinical isolates identified 27 new bacterial species associated with humans.

Clinical microorganisms isolated during a 5-year study in our hospital that could not be identified by conventional criteria were studied by 16S rRNA gene sequence analysis. Each isolate yielded a > or =1,400-bp sequence containing <5 ambiguities which was compared with the GenBank 16S rRNA gene library; 1,404 such isolates were tested, and 120 were considered unique (27 isolates) or rare (< or =10 cases reported in the literature) human pathogens. Eleven new species, "Actinobaculum massiliae," "Candidatus Actinobaculum timonae," Paenibacillus sanguinis, "Candidatus Bacteroides massiliae," Chryseobacterium massiliae, "Candidatus Chryseobacterium timonae," Paenibacillus massiliensis, "Candidatus Peptostreptococcus massiliae," "Candidatus Prevotella massiliensis," Rhodobacter massiliensis, and "Candidatus Veillonella atypica" were identified. Sixteen species were obtained from humans for the first time. Our results show the important role that 16S rRNA gene sequence-based bacterial identification currently plays in recognizing unusual and emerging bacterial diseases.

Adolescent↗
PubMed1998

Properties of the P-type ATPases encoded by the copAP operons of Helicobacter pylori and Helicobacter felis.

The cop operons of Helicobacter pylori and Helicobacter felis were cloned by gene library screening. Both operons contain open reading frames for a P-type ion pump (CopA) with homology to Cd2+ and Cu2+ ATPases and a putative ion binding protein (CopP), the latter representing a CopZ homolog of the copYZAB operon of Enterococcus hirae. The predicted CopA ATPases contained an N-terminal GMXCXXC ion binding motif and a membrane-associated CPC sequence. A synthetic N-terminal peptide of the H. pylori CopA ATPase bound to Cu2+ specifically, and gene disruption mutagenesis of CopA resulted in an enhanced growth sensitivity of H. pylori to Cu2+ but not to other divalent cations. As determined experimentally, H. pylori CopA contains four pairs of transmembrane segments (H1 to H8), with the ATP binding and phosphorylation domains lying between H6 and H7, as found for another putative transition metal pump of H. pylori (K. Melchers, T. Weitzenegger, A. Buhmann, W. Steinhilber, G. Sachs, and K. P. Schäfer, J. Biol. Chem. 271:446-457, 1996). The corresponding transmembrane segments of the H. felis CopA pump were identified by hydrophobicity analysis and via sequence similarity. To define functional domains, similarly oriented regions of the two enzymes were examined for sequence identity. Regions with high degrees of identity included the N-terminal Cu2+ binding domain, the regions of ATP binding and phosphorylation in the energy transduction domain, and a transport domain consisting of the last six transmembrane segments with conserved cysteines in H4, H6, and H7. The data suggest that H. pylori and H. felis employ conserved mechanisms of ATPase-dependent copper resistance.

Adenosine Triphosphatases↗
PubMed1991

Molecular cloning and nucleotide sequencing of the Arthrobacter dextranase gene and its expression in Escherichia coli and Streptococcus sanguis.

A bacterial strain, which assimilated dextran and water-insoluble glucan produced by Streptococcus mutans, was isolated from soil. The bacterium produced and secreted potent dextranase activity, which was identified as Arthrobacter sp. and named CB-8. The dextranase was purified and some enzymatic properties were characterized. The enzyme efficiently decomposed the water-insoluble glucan as well as dextran. A gene library from the bacteria was constructed with Escherichia coli, using plasmid pUC19, and clones producing dextranase activity were selected. Based on the result of nucleotide sequencing analysis, it was deduced that the dextranase was synthesized in CB-8 cells as a polypeptide precursor consisting of 640 amino acid residues, including 49 N-terminal amino acid residues which could be regarded as a signal peptide. In the E. coli transformant, the dextranase activity was detected mostly in the periplasmic space. The gene for the dextranase was introduced into Streptococcus sanguis, using an E. coli-S. sanguis shuttle vector that contained the promoter sequence of a gene for glucosyltransferase derived from a strain of S. mutans. The active dextranase was also expressed and accumulated in S. sanguis cells.

Amino Acid Sequence↗
PubMed2002

[Tn4560 transposition in Streptomyces ansochromogenes and its application for cloning of genes involved in nikkomycin biosynthesis].

No transformant was obtained when pUC1169(pMT660::Tn4556::vph) from S. coelicolor J1501 was used to transform the protoplast of S. ansochromogenes by general transformation for many times. Transformants were obtained when the protoplast of S. ansochromogenes was prepared at 50 degrees C for 30 min by heat attenuation of restriction. But the transformation frequency is very low, only 0.4 transformant per microgram DNA. Transformation frequency increased 10(3)-10(4) times when the protoplast of S. ansochromogenes was transformed by pUC1169 from itself. With Tn4560 transposition, 8 mutants lost ability to produce nikkomycin were screened from 4068 colonies with VioR-ThioS. Southern blotting suggested that Tn4560 was inserted in at least 4 positions of chromosomal DNA in S. ansochromogenes. By using the cloned 30 kb DNA fragment involved in nikkomycin biosynthesis as a probe, southern hybridization was carried out with 8 mutant total DNA digested with different enzymes. Only total DNA from Nik5 displayed signals, which were the same as those of the wild type. With HPLC analysis, 8 mutants could be classified into two types for nikkomycin production, one is Nik5, the other is Nik1, 2, 3, 4, 6, 7 and Nik8. As deduced preliminarily, the deletion of large chromosomal DNA fragment involved in nikkomycin biosynthesis took place in the 7 mutants. The partial gene library of Nik5 was constructed, the 4.2 kb and the 0.6 kb chromosomal DNA fragment flanking Tn4560 were cloned. Sequence analysis showed that the 4.8 kb DNA fragment (GenBank accession number:AF349618) contains two complete ORFs and one partial ORF.

Aminoglycosides↗
PubMed1998

Isolation and analysis of metA, a methionine biosynthetic gene encoding homoserine acetyltransferase in corynebacterium glutamicum.

The metA gene encoding homoserine acetyltransferase, the first enzyme of the methionine biosynthetic pathway, was isolated from a pMT1-based corynebacterium glutamicum gene library via complementation of an Escherichia coli metA mutant. A DNA-sequence analysis of the cloned DNA is identified an open-reading frame of 1,137 bp which encodes a protein with the molecular weight of 41,380 comprising 379 amino acids. The putative protein product showed good amino acid-sequence homology to its counterpart in other organisms. The internal fragment of the cloned DNA was successfully used to disrupt chromosomal metA, demonstrating the identity of the cloned gene. The C. glutamicum metA mutant lost the ability to grow on glucose minimal medium supplemented with homoserine. However, the mutant could grow on a minimal medium supplemented with cystathionine, demonstrating that C. glutamicum uses the cystathionine route to synthesize methionine. Introduction of a plasmid carrying cloned metA into C. glutamicum resulted in a 10-fold increase in enzyme activities and expression of a protein product of M(r) 41,000, which agrees with the sequence data and is similar in size to those of other homoserine acetyltransferases. Unlike E. coli whose metA product uses succinyl coenzyme A as a substrate, the cloned metA gene produced homoserine acetyltransferase which uses only acetyl coenzyme A as the acyl donor.

Acetyltransferases↗
PubMed1985

Expression of Actinomyces viscosus antigens in Escherichia coli: cloning of a structural gene (fimA) for type 2 fimbriae.

A cosmid gene library of Actinomyces viscosus T14V was prepared in Escherichia coli to examine the expression of A. viscosus antigens and to gain insight into the structure of A. viscosus type 1 and type 2 fimbriae. Out of this library of 550 clones, 28 reacted in a colony immunoassay with antibodies against A. viscosus cells. The proteins responsible for these reactions were identified in three clones. Clones AV1209 and AV2009 displayed nonfimbrial antigens with subunits of 40 and 58 kilodaltons, respectively. Clone AV1402 showed a 59-kilodalton protein that reacted with monospecific antibody against type 2 fimbriae and that comigrated with a subunit of type 2 fimbriae during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This indicates that AV1402 expresses a gene (fimA) for a subunit of A. viscosus type 2 fimbriae.

Actinomyces↗
PubMed1985

Molecular cloning and characterization of feline cellular genetic sequences homologous to the oncogene of the McDonough strain of feline sarcoma virus.

The organization within the cat genome of cellular genetic sequences homologous to the viral oncogene v-fms of the McDonough strain of feline sarcoma virus (SM-FeSV) was determined. Four cosmid clones containing overlapping v-fms homologous cellular DNA inserts representing a contiguous region of cellular DNA of approximately 80 kbp in length have been isolated from a feline cosmid gene library. Within this region of the cat genome, the c-fms genetic sequences are dispersed over a region of around 30 kbp and are interspersed with at least three intervening sequences.

Animals↗
PubMed1986

[Cloning of the genes of hemolytic factors of Bordetella pertussis in Escherichia coli].

The gene library of B. pertussis strain No. 475 (serovar 1. 2. 3.) was obtained on plasmid pUC19 in E. coli strain JM107. The average size of the cloned fragments of chromosomal DNA was 4.9 Kb. Out of 1,700 tested recombinant clones, six caused visible hemolysis on petri dishes with agar containing 4% of sheep red blood cells after 16-hour incubation at 37 degrees C. Only two out of four isolated plasmids were similar in Pst I restriction. Consequently, the existence of several different genes responsible for the hemolytic factors of B. pertussis may be assumed. None of the cloned fragments had Hind III sites. Clones harboring hybrid plasmids possessed different modes of hemolysis, which may be indicative of gene expression in E. coli.

Bordetella pertussis↗
PubMed2000

The GacS sensor kinase regulates alginate and poly-beta-hydroxybutyrate production in Azotobacter vinelandii.

Azotobacter vinelandii produces two polymers: the extracellular polysaccharide alginate and the intracellular polyester poly-beta-hydroxybutyrate (PHB). A cosmid clone (pSMU588) from an A. vinelandii gene library diminished alginate production by A. vinelandii mucoid strain ATCC 9046. The nucleotide sequence and predicted amino acid sequence of the locus responsible for the mucoidy suppression revealed 65% identity to Pseudomonas GacS, a transmembrane sensor kinase of the two-component regulators, whose cognate response regulator, GacA, is a global activator regulating several products and virulence factors. Plasmid pMC15, harboring gacS, and a strain carrying a gacS nonpolar mutation were constructed. Either pMC15 or the gacS mutation significantly reduced alginate production and transcription of algD, the gene coding for the key enzyme GDP-mannose dehydrogenase of the alginate biosynthetic pathway. We found that the gacS mutation also reduced PHB accumulation and impaired encystment. Taken together, these data indicate that in A. vinelandii the gacSA global system regulates polymer synthesis.

Alginates↗
PubMed1991

A highly thermostable neutral protease from Bacillus caldolyticus: cloning and expression of the gene in Bacillus subtilis and characterization of the gene product.

By using a gene library of Bacillus caldolyticus constructed in phage lambda EMBL12 and selecting for proteolytically active phages on plates supplemented with 0.8% skim milk, chromosomal B. caldolyticus DNA fragments that specified proteolytic activity were obtained. Subcloning of one of these fragments in a protease-deficient Bacillus subtilis strain resulted in protease proficiency of the host. The nucleotide sequence of a 2-kb HinfI-MluI fragment contained an open reading frame (ORF) that specified a protein of 544 amino acids. This ORF was denoted as the B. caldolyticus npr gene, because the nucleotide and amino acid sequences of the ORF were highly similar to that of the Bacillus stearothermophilus npr gene. Additionally, the size, pH optimum, and sensitivity to the specific Npr inhibitor phosphoramidon of the secreted enzyme indicated that the B. caldolyticus enzyme was a neutral protease. The B. sterothermophilus and B. caldolyticus enzymes differed at only three amino acid positions. Nevertheless, the thermostability and optimum temperature of the B. caldolyticus enzyme were 7 to 8 degrees C higher than those of the B. stearothermophilus enzyme. In a three-dimensional model of the B. stearothermophilus Npr the three substitutions (Ala-4 to Thr, Thr-59 to Ala, and Thr-66 to Phe) were present at solvent-exposed positions. The role of these residues in thermostability was analyzed by using site-directed mutagenesis. It was shown that all three amino acid substitutions contributed to the observed difference in thermostability between the neutral proteases from B. stearothermophilus and B. caldolyticus.

Amino Acid Sequence↗
PubMed1992

Molecular structure of the multifunctional fatty acid synthetase gene of Brevibacterium ammoniagenes: its sequence of catalytic domains is formally consistent with a head-to-tail fusion of the two yeast genes FAS1 and FAS2.

The Brevibacterium ammoniagenes fatty acid synthetase (FAS) gene was isolated from a series of overlapping clones by both immunological and plaque hybridization screening of two independent gene libraries. From the isolated DNA a contiguous segment of 10,549 bp was sequenced in both directions. The sequenced DNA contained a very long (9312 nucleotides) open reading frame coding for a protein of 3104 amino acids and with a molecular mass of 327,466 daltons. Based on characteristic sequence motifs known from other FAS systems, seven different FAS active centres were identified at distinct locations within the polypeptide chain. Only one component enzyme, the 3-hydroxydecanoyl beta, gamma-dehydratase, has not yet been localized definitively within the gene. Translation is presumed to start from a GUG triplet located 25 nucleotides downstream of the transcriptional initiation site. There is a canonical Shine-Dalgarno sequence just before this start codon. Comparison of the B. ammoniagenes FAS sequence with those of other known fatty acid synthetases revealed a particularly high degree of similarity to the products of the two yeast genes, FAS1 and FAS2 (30% identical and 46% identical plus closely related amino acids). This similarity extends over the entire length of the genes and involves not only the primary sequences of individual component enzymes but also their sequential order within the multifunctional proteins. These data, together with those on the structure of other fatty acid synthetases, are interpreted in terms of a contribution of both primary structure and subunit cooperation to a conserved topology of functional domains common to all type I FAS complexes.

Amino Acid Sequence↗
PubMed2003

Photosynthetic acclimation is reflected in specific patterns of gene expression in drought-stressed loblolly pine.

Because the product of a single gene can influence many aspects of plant growth and development, it is necessary to understand how gene products act in concert and upon each other to effect adaptive changes to stressful conditions. We conducted experiments to improve our understanding of the responses of loblolly pine (Pinus taeda) to drought stress. Water was withheld from rooted plantlets of to a measured water potential of -1 MPa for mild stress and -1.5 MPa for severe stress. Net photosynthesis was measured for each level of stress. RNA was isolated from needles and used in hybridizations against a microarray consisting of 2173 cDNA clones from five pine expressed sequence tag libraries. Gene expression was estimated using a two-stage mixed linear model. Subsequently, data mining via inductive logic programming identified rules (relationships) among gene expression, treatments, and functional categories. Changes in RNA transcript profiles of loblolly pine due to drought stress were correlated with physiological data reflecting photosynthetic acclimation to mild stress or photosynthetic failure during severe stress. Analysis of transcript profiles indicated that there are distinct patterns of expression related to the two levels of stress. Genes encoding heat shock proteins, late embryogenic-abundant proteins, enzymes from the aromatic acid and flavonoid biosynthetic pathways, and from carbon metabolism showed distinctive responses associated with acclimation. Five genes shown to have different transcript levels in response to either mild or severe stress were chosen for further analysis using real-time polymerase chain reaction. The real-time polymerase chain reaction results were in good agreement with those obtained on microarrays.

Acclimatization↗
PubMed1989

Cloning and sequencing of two tandem genes involved in degradation of 2,3-dihydroxybiphenyl to benzoic acid in the polychlorinated biphenyl-degrading soil bacterium Pseudomonas sp. strain KKS102.

Two genes involved in the degradation of biphenyl were isolated from a gene library of a polychlorinated biphenyl-degrading soil bacterium, Pseudomonas sp. strain KKS102, by using a broad-host-range cosmid vector, pKS13. When a 3.2-kilobase (kb) PstI fragment of a 29-kb cosmid DNA insert was subcloned into pUC18 at the PstI site downstream of the lacZ promoter, Escherichia coli cells carrying this recombinant plasmid expressed 2,3-dihydroxybiphenyl dioxygenase activity. Nucleotide sequencing of the 3.2-kb PstI fragment revealed that there were two open reading frames (ORFI [882 base pairs] and ORFII [834 base pairs], in this gene order). Results of analysis of Tn5 insertion mutants and unidirectional deletion mutants suggested that the ORFI coded for 2,3-dihydroxybiphenyl dioxygenase. When the sequence of ORFI was compared with that of bphC of Pseudomonas pseudoalcaligenes KF707 (K. Furukawa, N. Arima, and T. Miyazaki, J. Bacteriol. 169:427-429, 1987), the homology was 68%, with both strains having the same Shine-Dalgarno sequence. The result of gas chromatography-mass spectrometry analysis of the metabolic product suggested that the ORFII had meta cleavage compound hydrolase activity to produce benzoic acid. DNA sequencing suggested that these two genes were contained in one operon.

Amino Acid Sequence↗
PubMed1999

Analysis of Corynebacterium glutamicum methionine biosynthetic pathway: isolation and analysis of metB encoding cystathionine gamma-synthase.

The metB gene encoding cystathionine y-synthase, the second enzyme of methionine biosynthetic pathway, was isolated from a pSL109-based Corynebacterium glutamicum gene library via complementation of an Escherichia coli metB mutant. A DNA-sequence analysis of the cloned DNA identified an open-reading frame of 1161 bp which encodes a protein with the molecular weight of 41,655 comprising of 386 amino acids. The putative protein product showed good amino acid-sequence homology to its counterpart in other organisms. Introduction of a plasmid carrying the cloned metB into the C. glutamicum resulted in a 10-fold increase in cystathionine gamma-synthase activities, demonstrating the identity of the cloned gene. The C. glutamicum metB mutant which was generated by the site-specific integration of the cloned DNA into its chromosome did not lose the ability to grow on glucose minimal medium lacking supplemental methionine. The growth rate of the mutant strain was also comparable to that of the parental strain. These data indicate that, in addition to the transsulfuration pathway, other methionine biosynthetic pathways may be present in C. glutamicum.

Amino Acid Sequence↗