bluuebunny/crossref_metadata_2025_split huggingface dataset:
Micro Review Bending the rules: the 2μ plasmid of yeast
Authors: Murray J.A.H. 
Published: October 2006

SummaryThe replication of euckaryotic DNA is normally initiated at each origin only once per ceil cycle. Yet, In spite of this restriction, the 2μ plasmid of yeast has evolved an elegant mechanism which can allow it to rapidly amplify its copy number without initiating multiple rounds of replication. It achieves this by exploiting a plasmid‐en coded site‐specific recombination system in a way that is apparently unique to this plasmid. The 2μ plasmid has also evolved a mechanism that allows effective partition of itself between mother and daughter cells. Together these processes ensure the persistence of the 2μ plasmid within a population, even though retention of the plasmid Is of no advantage to the host cell and causes a slightly slower growth rate. The success of this survival strategy is illustrated by the near ubiquity of the 2μ. plasmid in both wild‐type and laboratory strains of yeast.
Translation of galE and coordination of galactose operon expression in Escherichia coli: effects of insertions and deletions in the non‐translated leader sequence
Authors: Bingham A. H. A. , Busby S. J. W. 
Published: October 2006

SummaryUsing gene‐manipulation techniques, we made a set of short insertions and deletions in the Escherichia coli galactose operon between the transcription start site and the Shine‐Dalgarno sequence of the first gene of the operon, galE. Translation initiation is severely reduced when the distance between the 5′ end of the message and the Shine‐Daigarno sequence drops below 15 bases.Transcription of the gal operon can start at two distinct sites, S1 and S2, separated by 5 bp, situated 16 and 21 bp upstream of the galE Shine‐Dalgarno sequence, respectively. When transcription starts at S2, gal operon expression is discoordinate as the galE gene is better translated than promoter‐distal genes. Here we report that gal operon expression is discoordinate even when message starting at S2 is shortened. This shortened. that the better translation of galE from transcripts starting at S2 is not simply due to the fact that they are longer than transcripts starting at S1.
Evolutionary relationships in the genus Bordetella
Authors: Arico B. , Gross R. , Smida J. , Rappuoli R. 
Published: October 2006

SummaryThe nucleotide sequence of the pertussis toxin operon of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica, has shown that the last two species contain many common mutations and are likely to derive from a common ancestor (Arico and Rappuoli, 1987). To elucidate further the evolutionary relationships between the Bordetella species, we have cloned and sequenced the promoter region and the gene coding for the S1 subunit of pertussis toxin from additional B. pertussis strains, such as the type strain BP 18323 and two recent clinical isolates, namely strain BP 13456 from Sweden and strain BP SA1 from Italy. While the strains BP SA1 and BP 13456 are shown to differ from the published B. pertussis sequences by only one base pair, the type strain BP 18323 contains a total of 11 base‐pair substitutions. Remarkably, 9 of the 11 substitutions found in BP 18323 are also common to B. parapertussis and B. bronchiseptica, strongly suggesting that this strain derives from the same ancestor as B. parapertussis and B. bronchiseptica.Computer analysis of the sequence data allows the construction of an evolutionary ‘tree’ showing that the B. pertussis strains are very homogeneous and significantly distant from B. parapertussis and B. bronchiseptica. Therefore the proposed conversion from B. parapertussis to B. pertussis appears highly improbable.
Molecular characterization of the resolvase gene, res, carried by a multicopy plasmid from Clostridium perfringens: common evolutionary origin for prokaryotic site‐specific recombinases
Authors: Garnier T. , Saurin W. , Cole S. T. 
Published: October 2006

Summary Clostridium perfringens strain CPN50 harbours a 10.2 kb plasmid known as plP404 which, in addition to a set of UV‐inducible genes involved in bacteriocin production, carries res, a gene probably encoding a site‐specific recombinase. The RES protein is highly homologous to the resolvases of transposons from both Gram‐negative and Gram‐positive bacteria as well as enzymes involved in site‐specific DNA inversion. A likely role for the RES protein would be to stabilize plP404 by reducing the number of plasmid multimers resulting from homologous recombination. A putative resolution site for RES action was found overlapping the res promoter. Phylogenetic analysis of the primary structures of ten site‐specific recombinases suggested a common descent and showed the RES protein to be closest to the resolvase encoded by Tn917 from Strepfococcus faecalis.
The calmodulin‐sensitive adenylate cyclase of Bordetella pertussis: cloning and expression in Escherichia col
Authors: Glaser P. , Ladant D. , Sezer O. , Pichot F. , Ullmann A. , Danchin A. 
Published: October 2006

SummaryThe adenylate cyclase toxin of the prokaryote Bordetella pertussis is stimulated by the eukaryotic regulatory protein, calmodulin. A general strategy, using the adenylate‐cyclase‐calmodulin interaction as a tool, has permitted cloning and expression of the toxin in Escherichia coli in the absence of any B. pertussis trans‐activating factor. We show that the protein is synthesized in a large precursor form composed of 1706 amino acids. The calmodulin‐stimulated catalytic activity resides in the amino‐terminal 450 amino acids of the adenylate cyclase. The enzyme expressed in E. coli is recognized in Western blots by antibodies directed against purified B. pertussis adenylate cyclase, and its activity is inhibited by these antibodies.
The three‐dimensional structures of S‐layers of two novel Eubacterium species isolated from inflammatory human processes
Authors: Sjögren A. , Wang D. N. , Hovmöller S. , Haapasalo M. , Ranta H. , Kerosuo E. , Jousimies‐Somer H. , Lounatmaa K. 
Published: October 2006

SummaryThe three‐dimensional structures of the crystalline surface layers of two species of Eubacteria have been determined by electron microscopy and computerized image processing. The S‐layer of Eubacterium sp. ES4C has tetragonal symmetry, with a unit cell spacing of 10.6 nm and a thickness of 9.5 nm, while that of Eubacterium sp. AHN 990 has hexagonal symmetry a = b = 15.7 nm and a thickness of 13 nm. The resolutions in the reconstructions were 2.5 nm and 1.8 nm, respectively. The reconstruction of the S‐layer of strain ES4C reveals a distinct domain structure: a major tetramer, arms connecting adjacent unit cells, and a minor tetramer. The S‐layer of strain AHN 990, on the other hand, has a rather complex arrangement, centred around the six‐fold axis.
The virulence protein Yop5 of Yersinia pseudotuberculosis is regulated at transcriptional level by plasmid‐plB1 ‐encoded trans‐acting elements controlled by temperature and calcium
Authors: Forsberg A. , Wolf‐Watz H. 
Published: October 2006

SummaryThe DNA sequence of the structural gene (yopE) of one of the Yersinia pseudotuberculosis virulence plas‐mid‐plB1‐encoded proteins, Yop5, is presented. The deduced protein showed a molecular weight of 22971 Daltons. A specific mutant, having a kanamycin‐resistance fragment inserted within the yopE gene was no longer virulent for mice. The expression of the Yop5 protein is regulated at the level of transcription by temperature as well as by the Ca2+ ‐concentration of the medium. A significant increase in the level of transcription was not detected until 45 min after a temperature shift from 26°C to 37°C in the absence of calcium; addition of Ca2+ inhibited the expression. The yopE promoter is under positive, as well as negative, plB1‐encoded control. The positive function is solely regulated by temperature, while the regulation of the negative function involves at least five different plasmid‐encoded gene loci; one of these genes encodes the V‐antigen.
Differential specificity of two insecticidal toxins from Bacillus thuringiensis var. aizawai
Authors: Knowles B. H. , Ellar D. J. 
Published: October 2006

Summary Bacillus thuringlensis var. aizawai HD–249 produces more than one protein of 130–135 kD in its insecticidal crystal δ‐endotoxin. We describe an indirect method of assessing the relative contribution to toxicity of two of these protoxins using monospecific antibodies directed against their active proteolytic products. Our results show that one toxin is active against Spodoptera frugiperda but not Choristoneura fumiferana cells in vitro, while the other lyses C. fumiferana but not S. frugiperda cells. There is no indication of synergism between these toxins in vitro.
The expression of Pseudomonas aeruginosa PAK pilin gene mutants in Escherichia coli
Authors: Pasloske B. L. , Carpenter M. R. , Frost L. S. , Finlay B. B. , Paranchych† W. 
Published: October 2006

SummaryPrevious work has demonstrated the expression of the cloned pilin gene of Pseudomonas aeruginosa PAK within Escherichia coli and has pinpointed this protein's localization exclusively to the cytoplasmic membrane (Finlay et al., 1986). To define regions of the pilin subunit necessary for its stability and transport within E. coli, we constructed six mutants of the pilin gene and studied their expression and localization using a T7 promoter system. Two of the mutants have either a 4‐ or 8‐amino‐acid deletion at the N‐terminus and both were stably expressed and transported primarily to the cytoplasmic membrane of E. coli. The other four mutants are C‐terminal truncations having between 36 and 56 amino acids of the C‐terminal region of the unprocessed pilin. Studies with these truncated mutants revealed that only the first 36 residues of the unprocessed pilin subunit were required for insertion into the E. coli membrane.
Cloning, characterization and regulation of an α‐amylase gene from Streptomyces limosus
Authors: Virolle M.‐J. , Bibb M. J. 
Published: October 2006

SummaryAn α‐amylase gene (aml) of Streptomyces limosus ATCC 19778 was cloned in Streptomyces lividans 66. S1 mapping experiments identified an aml transcript 1.8 kb in length and the extracellular enzyme was estimated to be 59 kD in size, suggesting that aml was transcribed as a monocistronic mRNA species. Expression of the gene was induced by maltose (or maltodextrins) in S. Iimosus and when aml was cloned in S. lividans or Streptomyces coelicolor A3(2). In S. Iimosus, mannitol repressed aml expression while glucose had little or no effect; in S. lividans and S. coelicolor the relative effects of the two sugars were reversed. Both induction and carbon‐source repression of aml expression appeared to occur at the level of transcriptional initiation. Glucose repression in S. coelicolor was dependent upon a functional glucose kinase gene.
Recombination among Protein II genes of Neisseria gonorrhoeae generates new coding sequences and increases structural variability in the Protein II family
Authors: Connell T. D. , Black W. J. , Kawula T. H. , Barritt D. S. , Dempsey J. A. , Kverneland K. , Stephenson A. , Schepart B. S. , Murphy G. L. , Cannon J. G. 
Published: October 2006

SummaryExpression of Neisseria gonorrhoeae Protein II (P.II) is subject to phase variation and antigenic variation. The P.II proteins made by one strain possess both unique and conserved antigenic determinants. To study the mechanism of antigenic variation, we cloned several P.II genes, using as probes a panel of monoclonal antibodies (MAbs) specific for unique determinants. The DNA sequences of three P.II genes showed that they shared a conserved framework, with two short hypervariable (HV) regions being responsible for most of the differences among them. We demonstrated that unique epitopes recognized by the MAbs were at least partially encoded by one of the HV regions. Moreover, we found that reassortment of the two HV regions among P.II genes occurs, generating increased structural and antigenic variability in the P.II protein family.
Proteins from the prokaryotic nucleoid: primary and quaternary structure of the 15‐kD Escherichia coli DNA binding protein H‐NS
Authors: Falconl M. , Gualtierl M. T. , La Teana A. , Losso M. A. , Pon C. L. 
Published: October 2006

SummaryThe primary sequence of H‐NS (136 amino acid residues, Mr = 15402), an abundant Escherichia coli DNA‐binding protein, has been elucidated and its quaternary structure has been investigated by protein‐protein cross‐linking reactions. It was found that H‐NS exists predominantly as a dimer, even at very low concentrations, but may form tetramers at higher concentrations and that the protein‐protein interaction responsible for the dimerization is chiefly hydrophobic.
Expression of a biologically active diphtheria toxin fragment B in Escherichia coli
Authors: Cabiaux V. , Phalipon A. , Wattiez R. , Falmagne P. , Ruysschaert J. M. , Kaczorek M. 
Published: October 2006

SummaryThe toxB gene of Corynebacterium diphtheriae bacteriophage β encoding the B fragment of diphtheria toxin was cloned into an inducible expression vector. When expressed In Escherichia coli, fragment B was not proteolysed and was indistinguishable, by immunological criteria, from wild‐type C. diphthsriae derived fragment B. Soluble fragment B was partially purified from the cytoplasm by saline precipitation steps and was shown to compete with the wild‐type diphtheria toxin for binding to receptors of sensitive eukaryotic cells. A complete diphtheria toxin was reconstituted by formation of the disulphide bridge between purified fragment A and recombinant fragment B, which migrates at the expected Mr on Western blots and which was able to block protein synthesis by ADP‐ribosylation of elongation factor–2, thereby indicating that the recombinant fragment B had retained its biological activity.
Catabolite control of the elevation of PGK mRNA levels by heat shock in Saccharomyces cerevisiae
Authors: Piper P. W. , Curran B. , Davies M. W. , Hirst K. , Lockheart A. , Seward K. 
Published: October 2006

SummaryHeat shock enhances the very high level of transcription of the phosphoglycerate kinase (PGK) gene in fermentative cultures of Saccharomyces cerevisiae. This response of PGK mRNA levels was not found on gluconeogenic carbon sources, and could be switched on or off subject to availability of fermentable carbon source. The addition of glucose to yeast growing on glycerol resulted in acquisition, within 30–60 min, of the ability to elevate PGK mRNA levels after heat shock. In addition, in aerobic cultures growing on glucose the exhaustion of the medium glucose coincided with a loss of the heat‐shock effect on PGK mRNA and a switch‐over to slower growth by aerobic respiration. Levels of hsp26 mRNA were analysed during these experiments.Contrasting with this requirement for fermentable catabolite for manifestation of a heat‐shock response of PGK mRNA levels, the PGK enzyme was not synthesized at a greater level in heat‐shocked fermentative than in gluconeogenic cultures. PGK is one of only a few proteins made efficiently after mild heat shock of yeast. Thus, heat‐stress‐induced elevation of PGK mRNA levels does not appreciably increase PGK synthesis during exposure to high temperatures and so its role may be to assist cells repressed in mitochondrial function during recovery following a heat shock.
virA and virG are the Ti‐plasmid functions required for chemotaxis of Agrobacterium tumefaciens towards acetosyringone
Authors: Shaw C. H. , Ashby A. M. , Brown A. , Royal C. , Loake G. J. , Shaw C. M. 
Published: October 2006

SummaryOctopine and nopaline Ti‐plasmids confer upon Agrobacterium tumefaciens C58C1 the ability to respond chemotactically to the vir‐inducing phenolic wound exudate, acetosyringone. A. tumefaciens C58C1 containing Ti‐plasmids with Tn5 insertions in virB, C., D or E exhibited marked chemotaxis towards acetosyringone. However, Ti‐plasmids with mutations in virA or VirG were unable to confer the responsive phenotype. Of the cosmid clones pVK219 (virAB) pVK221 (virBGC) pVK225 (virGCDE) and pVK257 (virABGC) mobilized to cured A. tumefaciens C58C1, only pVK257 bestowed acetosyringone chemotaxis. virA and virG are thus required for chemotaxis of A. tumefaciens towards acetosyringone. This suggests a multifunctional role for virA and virG: at low concentrations of acetosyringone they mediate chemotaxis and at higher concentrations they effect vir‐induction.
Nucleotide sequence and transcriptional analysis of the aerCaerA region of Aeromonas sobria encoding aerolysin and its regulatory region
Authors: Husslein V. , Huhle B. , Jarchau T. , Lurz R. , Goebel W. , Chakraborty T. 
Published: October 2006

SummaryThe nucleotide sequence of a 2510 base pair chromosomal fragment containing the aerolysin gene aerA, and its regulatory region aerC, from a clinical isolate of Aeromonas sobria was determined. The aerolysin gene coded for a 54.5 kD polypeptide and had a G + C content of 59%, indicating that it is endogenous to the genus Aeromonas. In contrast, the aerC region was characterized by its high A + T content (61%) and the presence of a core motif, aATAAAa, repeated eight times within 300 base pairs. A 12 base pair repeat, 5′ AATAAAACCGGG3′, present within this region occurred as a direct repeat 544 base pairs away, within the coding region of aerolysin. RNA polymerase binding studies and S1 mapping allowed the detection of two divergent non‐overlapping promoters within aerC. Despite having identical transcriptional start sites in both A. sobria and Escherichia coli, the amount of aerolysin transcript produced in E. coli is 30–40 times less than that found in A. sobria. The signal peptide of preproaerolysin was shown by deletion to be essential for export of the toxin to the external medium. The mature toxin is a hydrophilic protein with no hydrophobic stretches long enough to cross a membrane. A search for similarities to the primary sequence of aerolysin revealed that the toxin may share a functional similarity to haemolysin (hly A) of E. coli.
Overexpression, solubilization and refolding of a genetically engineered derivative of the penicillin‐binding protein 3 of Escherichia coli K12
Authors: Belder J. Bartholomé‐De , Nguyen‐Disteche M. , Houba‐Herin N. , Ghuysen J. M. , Maruyama I. N. , Hara H. , Hirota Y. , Inouye M. 
Published: October 2006

SummaryReplacement of the amino‐terminal 40‐amino‐acid region of the 588‐ammo‐acid precursor of the membrane‐bound penicillin‐binding protein 3 (P6P3) by the decapeptide MKGKEFQAWI was carried out by altering the amino‐coding end of the ftsl gene. Insertion of the modified gene into a runaway‐replication plasmid under the control of a fused Ipp promoter and lac promoter/operator, resulted in the overexpression by Escherichia coli of the modified PBP3 (designated PBP3**) in the cytoplasm. About 80% of the accumulated PBP3** underwent sequestration in the form of insoluble protein granules that were isolated by cell breakage or cell lysis. After selective removal of contaminants by an EDTA‐lysozyme/DNase (de‐oxyribonuclease)/Nonidet extraction, treatment of the granules with guanidinium chloride followed by dialysis against buffer containing 0.5 M NaCI yielded a refolded, water‐soluble PBP3**, which, upon chromatography on Superose 12, exhibited the expected 60000 molecular mass. The refolded PBP3** bound benzytpenicillin in a 1 to 1 molar ratio, was highly sensitive to aztreonam and showed the same degree of thermostability, in terms of penicillin‐binding capacity, as the parent, membrane‐bound PBP3, suggesting that protein refolding occurred with formation of the correct intramolecular interactions. Two to three mg of refolded PBP** can be obtained from 1 litre of culture of the overproducing strain.
The protein encoded by gvpC is a minor component of gas vesicles isolated from the cyanobacteria Anabaena flos‐aquae and Microcyctis sp.
Authors: Hayes P. K. , Lazarus C. M. , Bees A. , Walker J. E. , Walsby A. E. 
Published: October 2006

SummaryThe proteins present in gas vesicles of the cyanobacteria Anabaena flos‐aquae and Microcystis sp. Were separated by SDS‐polyacrylamide gel electrophoresis. Each contained a protein of Mr 22 K whose N‐terminal amino acid sequences showed homology with that of the Calothrix sp. PCC 7601 gvpC gene product. The gvpC gene from A. flos‐aquae was cloned and sequenced. The derived amino acid sequence for the gene product indicated a protein, GVPc, of 193 residues and Mr 21985 containing five highly conserved 33 amino acid repeats. The sequence was identical at the N‐terminus to that of the Mr 22K protein present in gas vesicles and showed correspondence to seven tryptic peptides isolated from gas vesicles. This establishes that GVPc forms a second protein component of the gas vesicle, in addition to the main constituent, the 70 residue GVPa. Quantitative amino acid analysis of entire gas vesicles reveals that GVPc accounts for only 2.9% of the protein molecules and 8.2% of the mass present: this is insufficient to form the conical end caps of the gas vesicles. It is suggested that GVPc provides the hydrophilic outer surface of the gas vesicle wall; the 33 amino acid repeats may interact with the periodic structure provided by GVPa.
Molecular cloning, identification and ranscriptional analysis of genes involved in acetate utilization in Neurospora crassa
Authors: Thomas C. M. , Connerton I. F. , Fincham J. R. S. 
Published: October 2006

SummaryFour Neurospora crassa genomic clones have been selected as hybridizing much more strongly to labeled mRNA isolated from acetate‐grown mycelium than to mRNA from sucrose‐grown mycelium. Hybridization of restriction fragments with acetate‐specific mRNA or cDNA has been used to delimit the transcribed region(s) of each clone. The transcription of all four clones is strongly induced by transfer of growing mycelium from sucrose to acetate as sole carbon source. In wild‐type mycelium, mRNAs corresponding to the four clones reach maximum levels after four hours of induction. They accumulate more rapidly and reach higher levels in an acetate non‐utilizing mutant, acu‐7, which has been found to overproduce enzymes of the glyoxylate cycle and to have a partial block in the TCA cycle. Molecular transformation of a Neurosporaacu‐5 mutant and of an Aspergillus nidulans acuE mutant by DNA of clone 2 and clone 1, respectively, strongly suggests that clone 2 codes for acetyl‐coenzyme A synthetase and that clone 1 codes for malate synthase. The transcribed segments of clones 1 and 2each hybridize to corresponding clones from Aspergillusnidulans (R. A. Sandeman and M. J. Hynes, personal communication).
Studies of UV‐inducible promoters from Clostridium perfringens in vivo and in vitro
Authors: Gamier T. , Cole S. T. 
Published: October 2006

SummaryExpression of a 4kb segment of the bacteriocinogenic plasmid, plP404, from Clostridium perfringens inducible by UV‐irradiation. DNA sequence analysis revealed that this region contains three genes: uviA, uviB and bcn encoding the bacteriocin BCN5. Biochemical studies with mRNAs showed that expression was controlled at the transcriptional level and that the genes were organized in two independent transcriptional units, uviAB and ben both directed by tandem promoters inducible by UV light. The bcn gene is transcribed from three promoters (P1, P2, P3) while transcription of uviAB is directed by two promoters (P4, P5). With the exception of P4, which bears some resemblance to the consensus eubacterial promoter sequence, none of these promoters was recognized invitro by the major forms of RNA polymerase from C. perfringens, Bacillus subtilis or Escherichia coli, Promoters P1, P3 and P5, which show striking homology with each other, contain unusual sequences in the ‘−35’ and ‘−10’ regions known to be recognized by RNA polymerase and this might indicate positive control.