- Volume 139, Issue 10, 1993
Volume 139, Issue 10, 1993
- Review Article
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- Biochemistry
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Identification of two distinct NADH oxidases corresponding to H2O2-forming oxidase and H2O-forming oxidase induced in Streptococcus mutans
SUMMARY: Two distinct NADH oxidases, corresponding to H2O2-forming and H2O-forming enzymes were purified to homogeneity from Streptococcus mutans and their basic properties determined. The H2O2-forming enzyme was a tetramer with a subunit molecular mass of about 56 kDa and required flavin adenine dinucleotide (FAD) for full activity. The enzyme had an isoelectric point of 6.6 and exhibited optimal activity at pH 6.0 The H2O-forming enzyme was a monomer with a molecular mass of 50 kDa and activity independent of exogenously added flavin. The enzyme had an isoelectric point of 4.8 and exhibited optimal activity between pH 7.0 and 7.5 Both enzymes oxidized NADH (Km 0.05 and 0.025 mM for the H2O2- and H2O-forming enzyme, respectively) but not NADPH and contained 1 mol of FAD per monomer. Spectra of the oxidized enzymes exhibited maxima at 271, 383 and 449 nm for the H2O2-forming enzyme and 271, 375 and 447 nm for the H2O-forming enzyme. Antibodies raised against the H2O2-forming enzyme or the H2O-forming enzyme reacted with their corresponding antigen, but did not cross-react. The amino-terminal regions of the two enzymes had completely different amino acid sequences.
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The distribution of the outer gas vesicle protein, GvpC, on the Anabaena gas vesicle, and its ratio to GvpA
More LessSUMMARY: Previous studies have shown that gas vesicles isolated from the cyanobacterium Anabaena flos-aquae contain two types of protein, GvpA, a small hydrophobic protein that forms the main ribbed structure, and GvpC, a protein comprising five repeats of a 33-amino-acid-residue motif, which is located on the outer surface of the GvpA shell. GvpC was shown to increase the critical collapse pressure of the gas vesicles; it was thought to do this by forming a series of molecular ties that bind the ribs together. We now show that antibodies raised against GvpC label both the central cylinders and the conical end caps of native gas vesicles but fail to bind to gas vesicles that have been stripped of GvpC. The molar ratio of GvpA to GvpC has been calculated from amino acid analyses of gas vesicle hydrolysates by reference to the abundance of amino acids that occur predominantly or exclusively in one protein or the other; the molar ratio was found to be 25:1 in freshly isolated gas vesicles and 23:1 in gas vesicles saturated with GvpC. We have considered three ways in which the 33-residue repeats of GvpC might interact with the crystallographic unit cell of GvpA molecules in the ribs. The Anabaena GvpC will bind to and restore the strength of gas vesicles isolated from Aphanizomenon and Microcystis that lack their native GvpC.
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- Biotechnology
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Targeting of interleukin-2 to the periplasm of Escherichia coli
SUMMARY: A synthetic gene coding for interleukin-2 (IL-2) was used to produce large amounts of recombinant IL-2 (met-IL-2) in Escherichia coli. Met-IL-2 was found to accumulate in the cytoplasm in an insoluble, aggregated form. Inclusion bodies located at the pole caps of cells were detected using immunogold labelling. Constructs were designed to fuse the IL-2 gene to DNA fragments encoding signal peptides for an outer-membrane protein (OmpA) or for a periplasmic protein (PhoA) of E. coli. No significant maturation was observed with these fusion proteins which were found in an insoluble form in the cytoplasm. The influence of charge disposition at the N-terminus of the mature portion of the protein was investigated by replacing positively charged amino acids with glutamic acid. None of the introduced substitutions had any effect. Various factors that might affect expression, secretion and folding were examined in an attempt to obtain secretion. By fusing IL-2 to the precursor maltose-binding protein (preMBP) a large fraction of the preMBP-IL-2 protein was correctly processed and transported to the periplasmic space. IL-2 derived from MBP-IL-2 after FXa cleavage possessed similar specific activity to recombinant IL-2 produced in Chinese Hamster ovary cells.
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Cloning, nucleotide sequence and characterization of the mannitol dehydrogenase gene from Rhodobacter sphaeroides
More LessSUMMARY: Transposon mutagenesis and antibiotic enrichment were employed to isolate a mutant of Rhodobacter sphaeroides Si4 designated strain M22, that had lost the ability to grow on D-mannitol and to produce the enzyme mannitol dehydrogenase (MDH). DNA flanking the transposon in the mutant strain was used as a probe for the identification and cloning of the MDH gene (mtlK). A 5.5 kb EcoRI/Bg/II fragment from R. sphaeroides Si4 was isolated and shown to complement the mutation in R. sphaeroides M22. Successful complementation required that a promoter of the vector-plasmid pRK415 be present, suggesting that the mtlK gene is part of a larger operon. Using oligonucleotides derived from the N-terminal sequence of MDH as probes mtlK was located on the complementing fragment and the gene was sequenced. The mtlK open reading frame encodes a protein of 51 404 Da with an N-terminal sequence identical to that obtained from amino acid analysis of the purified MDH. The MDH of R. sphaeroides Si4 exhibits distant similarity to the mannitol-1-phosphate dehydrogenases from Escherichia coli and Enterococcus faecalis, with 28.1% and 26.3% identity, respectively. Mutant strains deficient in MtlK displayed substantial levels of sorbitol dehydrogenase activity, originally thought to be only a minor activity associated with the MDH enzyme. It is likely that we have uncovered an additional polyol dehydrogenase with activity for sorbitol. The mtlK gene can be used for overexpression of MDH in E. coli in order to obtain sufficient amounts of enzyme for further investigations and applications.
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- Environmental Microbiology
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Control analysis of microbial interactions in continuous culture: a simulation study
More LessSUMMARY: Metabolic Control Analysis (MCA) has been applied to flux of substrates and products during the growth of a single species and of two interacting species in a chemostat. Single-species growth was described by classical chemostat kinetics and the two-species interaction was commensalism, the first species converting the inflowing limiting substrate to a product which provided the limiting substrate for the second species. For the single species situation, control of flux to product is shared by the dilution rate and bacterial specific growth rate, and control can be quantified in terms of two flux control coefficients CJ D and CJμum1representing the fractional changes in flux resulting from fractional changes in the dilution rate and maximum specific growth rate, respectively. At low dilution rates, dilution rate exerts greater control, whilst CJμm1exceeds CJ D at high dilution rates. In the two-species commensal interaction, additional control on flux to product is exerted by species 2 and may be quantified in a further flux control coefficient CJμm2. Control exerted by a particular species in this interaction increases as factors, e.g. maximum specific growth rate and saturation constant for growth, change to decrease its specific growth rate. Control over flux by a species is also increased by addition of an inhibitor specific to that species and a method is proposed for determining experimentally the flux control coefficient for a species which can be inhibited in this manner.
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- Genetics And Molecular Biology
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A chromosome integrative vector system utilizing DNA fragments of a lysogenic phage of Rhizobium leguminosarum
More LessThe attachment site (attP) of phage øU, a lysogenic phage of Rhizohium leguminosarum biovar trifolii, was identified on a 6.0 kb EcoRI fragment of the phage DNA. Plasmid pCI6 was constructed by cloning this EroRI fragment into the EcoRI site of suicide plasmid vector pSUP202. Escherichia coli S17-1 harbouring plasmid pCI6 was mated with wild-type R. leguminosarum biovar trifolii strain 4S and its lysogenic strain 4S(øU) using tetracycline resistance (Tcr) as a selection marker. The Tcr R.leguminosarum biovar trifolii transconjugants appeared at high frequency (10-3-10-4 per recipient cell in both matings). Southern hybridization with the attP fragment and pSUP202 as probes indicated that plasmid pCI6 integrated into the chromosome of all these transconjugants in the same manner as phage øU.
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New derivatives of TOL plasmid pWWO
More LessSUMMARY: Two new segregants, PPW1-1 and PPW161-1, of Pseudomonas putida were isolated from the stock cultures PaW85(pWW0) and PaW85(pWW0-161). Strain PPW1-1 had lost its ability to grow on m-xylene but was able to grow on m-toluate. A deletion of the left-hand of transposon Tn4651, including the upper-operon genes, had taken place in plasmid pWWOmut11, isolated from strain PPW1-1. Additional deletions were observed in pWWOmut1 after ‘benzoate-curing’ (plasmids pWW0mut15, pWWOmut19, pWW0mut27). The genes of the upper-operon and beginning of the meta-operon were deleted from pWW0-161mut1, isolated from strain PPW161-1. Despite this deletion, cells of PPW161-1 grew on all normal TOL plasmid substrates. The Tol+ phenotype was stable in cells of PPW161-1 growing on benzoate. We propose that this is because in cells of strain PPW161-1 the catabolic genes deleted from pWW0-161mut1 were integrated into the chromosome at the site where the (chromosomally encoded) ortho-pathway genes are located, resulting in the inability of the cells to use this pathway.
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Formation of linear plasmid multimers promoted by the phage lambda Red-system in lon mutants of Escherichia coli
More LessSUMMARY: We report here the formation of plasmid linear multimers promoted by the Red-system of phage lambda using a multicopy plasmid comprised of lambda reda, and red genes, under the control of the lambda cl 857 repressor. Our observations have revealed that the multimerization of plasmid DNA is dependent on the red and recA genes, suggesting a concerted role for these functions in the formation of plasmid multimers. The formation of multimers occurred in a recBCD + sbcB + xthA + lon genetic background at a higher frequency than in the isogenic lon + host cells. The multimers comprised tandem repeats of monomer plasmid DNA. Treatment of purified plasmid DNA with exonuclease III revealed the presence of free double-chain ends in the molecules. Determination of the size of multimeric DNA, by pulse field gel electrophoresis, revealed that the bulk of the DNA was in the range 50240 kb, representing approximately 524 unit lengths of monomeric plasmid DNA. We provide a conceptual framework for Red-system-promoted formation and enhanced accumulation of plasmid linear multimers in lon mutants of E. coli.
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Cloning and sequencing of a gene encoding acidophilic amylase from Bacillus acidocaldarius
More LessSUMMARY: Two starch-degrading enzymes produced by Bacillus acidocaldavius (renamed as Alicyclobacillus acidocaldarius) were identified. According to SDS-PAGE, the apparent molecular masses of the enzymes were 90 and 160 kDa. Eight peptide fragments and the N-terminal end of the 90 kDa polypeptide were sequenced. An oligonucleotide, based on the amino acid sequence of a peptide fragment of the 90 kDa protein, was used to screen a lDgt10 bank of B. acidocaldarius, and the region encoding the 90 kDa protein was cloned. Unexpectedly, the ORF continued upstream of the N terminus of the 90 kDa protein. The entire ORF was 1301 amino acids (aa) long (calculated molecular mass 140 kDa) and it was preceded by a putative ribosomal binding site and a promoter. Computer analysis showed that the 1301 aa protein was closely related to an a-amylase-pullulanase of Clostridium thermohydrosulfuricum. We suggest that the starch-degrading 160 kDa protein of B. acidocaldarius is an a-amylase-pullulanase, and the 90 kDa protein is a cleavage product of the 160 kDa protein. Another ORF, apparently in the same transcription unit, was found downstream from the amylase gene. It encoded a protein that was closely related to the maltose-binding protein of Escherichia coli.
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Genotypic typing and phylogenetic analysis of Salmonella paratyphi B and S. Java with IS200
More LessSUMMARY: Salmonella paratyphi B and Salmonella java are biovars of common serotype 1,4,[5],12:b:1,2 which respectively cause human paratyphoid fever and gastroenteritis. In order to define genotypes and phylogenetic relationships in this group, we examined representative strains for restriction fragment length polymorphisms (RFLPs) in and around the 16S ribosomal RNA (rrn) genes, and the five to eleven insertion sites of the Salmonella-specific DNA insertion sequence IS200. One of four 16S rrn profiles was predominant, and was shared by the majority of strains, irrespective of their designation as S. paratyphi B or S. Java. On the other hand, thirteen unique IS200 profiles were found and this technique was able to distinguish, for the first time, distinct genotypes for S. paratyphi B and S.java. One of the S. paratyphi B profiles, Spj-IP1.0, represented a globally-distributed clone. Greater diversity was detected within IS200 profiles of S. java than within those of S. paratyphi B. IS200 profiles described a phylogenetic complex in which strains of both biovars could be placed. They constituted reproducible molecular fingerprints, which could be compared in a band-matching database suitable for molecular epidemiological typing.
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fliU and fliV: two flagellar genes essential for biosynthesis of Salmonella and Escherichia coli flagella
More LessSUMMARY: The possible functions of two recently described flagellar genes, fliU and fliV, have been examined. Introduction of gene fliC, encoding the bacterial flagellin protein, into a number of flagellin-deficient Salmonella and Escherichia coli strains failed to complement the mutations in these strains, and the FliC flagellin was accumulated in the bacterial cytoplasm. Complementation with fliU and fliV, which map downstream of fliC, restored motility to some of the mutants which became flagellated. After inactivation of either fliU or fliV, such complementation no longer occurred and the flagellin protein accumulated in the cytoplasm, which suggested that both genes are required for the secretion of flagellin and expression of motility. Expression of these genes from high copy number plasmids resulted in the synthesis of exceptionally long flagella and in detection of the FliV protein on polyacrylamide gels.
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Nucleic acid sequence-based amplification (NASBA) for the identification of mycobacteria
More LessSUMMARY: Nucleic acid sequence-based amplification (NASBA), an isothermal amplification technique for nucleic acids (NA), was investigated for the species-specific identification of mycobacteria. A set of primers was selected from a highly conserved region of the 16S rRNA sequence of mycobacteria sandwiching a variable sequence to perform amplification of mycobacterial RNA. Species-specific probes for the M. tuberculosis complex, M. avium-paratuberculosis, M. intracellulare and M. leprae were hybridized in-solution with the amplified nucleic acids of 10 pathogenic mycobacteria and 11 closely related bacteria, as well as with human-derived NA in an enzyme-linked gel assay (ELGA). Each probe was shown to hybridize specifically to the amplified single-stranded RNA of the corresponding species. Thirty-two clinical isolates of M. tuberculosis strains from different parts of the world were correctly identified by NASBA using the M. tuberculosi-complex-specificprobe. In combination with the ELGA, NASBA could identify mycobacteria rapidly, i.e. in less than 6 h. The relative simplicity and rapidity of this technique makes it an attractive tool for species-specific identification of mycobacteria.
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Use of a triplex polymerase chain reaction for the detection and differentiation of Mycoplasma pneumoniae and Mycoplasma genitalium in the presence of human DNA
More LessSUMMARY: PCR primers corresponding to the adhesin genes of Mycoplasma pneumoniae and Mycoplasma genitalium were shown to detect the corresponding organisms specifically. Absence of cross-reaction with seven other mollicute species and six unrelated bacterial species commonly found in humans was demonstrated. Positive control primers directed against human mitochondrial DNA could be mixed with the Mycoplasma primers without loss of specificity or sensitivity. A detection level of 10 c.f.u. of either Mycoplasma species could be readily obtained, even in the presence of 104 human cells. The triplex PCR method developed is very simple and does not require hybridization or the use of radioisotopes and allows detection and differentiation of these mycoplasmas against the background of human DNA found in clinical specimens.
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Construction of Bacillus anthracis mutant strains producing a single toxin component
More LessSUMMARY: The two protein exotoxins secreted by Bacillus anthvacis are composed of three distinct components: protective antigen (PA), lethal factor (LF), and (o)edema factor (EF). We have developed a genetic strategy that permits us selectively to inactivate each of the genes coding for PA, EF or LF. This strategy involved the deletion of a portion of the structural gene and the insertion of an antibiotic resistance cassette. With this technique, double mutant strains of B. anthracis producing only one toxin component have been constructed. Characterization of the mutant strains indicated that they produced the expected single toxin protein. Using a simple, two-step protocol, we have purified PA, LF and EF to homogeneity from culture supernatants. These three mutant strains are potentially powerful tools for studying the individual effect of each toxin component in vitro and in vivo.
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φ F1 and φF3, two novel virulent, archaeal phages infecting different thermophilic strains of the genus Methanobacterium
More LessSUMMARY: Two virulent, archaeal phages, øF1 and øF3, were isolated that were capable of infecting different thermophilic members of the genus Methanobacterium. Both phages exhibited a similar morphology consisting of a polyhedral head and a tail but differed considerably in their host specificities and the size and topology of their genomes. Phage øF1 contained a linear, double-stranded DNA genome of 85 + 5 kb in size and showed a broad host range including M. thermoformicicum strains Z-245, FTF, FF1, FF3 and CSM3, and M. thermoautotrophicum strain ΔH. In contrast, øF3 phage particles contained a circular or terminally redundant linear genome, comprising approximately 36 + 2 kb double-stranded DNA, and could only be propagated on M. thermoformicicum strain FF3. Hybridization experiments did not reveal similarity between the genomes of øF1 and øF3 nor between both phages and genomic DNA from different thermophilic members of the genus Methanobacterium or DNA from phage øM1 of M. thermoautotrophicum Marburg. A physical map of both phage genomes was constructed. The DNA of phage øF1 was found to contain multiple GGCC sites which form the target for the restriction-modification (R/M) system MthTI of M. thermoformicicum THF. In contrast, the DNA of øF1 contained only a single CTAG site recognized by the R/M systems MthZI and MthFI of M. thermoformicicum Z-245 and FTF, respectively. The distribution of these sites correlates well with the capacity of øF1 to infect M. thermoformicicum strains Z-245 and FTF but not strain THF.
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The temperate phages RP2 and RP3 of Streptomyces rimosus
SUMMARY: The oxytetracycline-producing Streptomyces rimosus strains R6-65 and R7 (ATCC 10970) are lysogenic for the two narrow-host-range phages RP2 and RP3. Both phages are released at low frequency from the lysogenic strains and form plaques on ‘cured’ S. rimosus strains. RP2 and RP3 are of similar shape with flexible tails and contain double-stranded DNA of about 70% G + C with cohesive ends (group B1 of bacteriophage classification). The two phages also have identical, very slow, growth kinetics in S. rimosus, with a latent phase of about 6 h and a rise period of about 4 h. RP2 and RP3 are heteroimmune and they differ slightly in their size of phage particles and length of DNA (64.7 and 62.4 kb for RP2 and RP3, respectively). The restriction maps of the two phages are completely different, and hybridization experiments showed only one short region of sequence similarity (less than 430 bp); the two phages are thus essentially unrelated. Both phages lysogenize their hosts by recombination via defined attachment (att) sites. The positions of the attP sites have been localized on the restriction maps of RP2 and RP3 to restriction fragments of 800 and 300 bp, respectively. The prophages did not affect the level of oxytetracycline production or the genetic instability of this trait.
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Optimization of Streptomyces aureofaciens transformation and disruption of the hrdA gene encoding a homologue of the principal σ factor
More LessSUMMARY: Conditions for the preparation of protoplasts from Streptomyces aureofaciens and transformation with several replicative and integrative plasmids were optimized. Of all the replicative plasmids used, carrying various Streptomyces replication origins, only plasmid pGM9 produced transformants, with a transformation efficiency of 2 × 103 (μg DNA)-1. When plasmid DNA isolated from S. aureofaciens was used, the transformation efficiency increased to 105 transformants (μg DNA)-1. Transformation of protoplasts with integrative plasmids containing part of the hrdA gene gave rise to transformants with an efficiency of approximately 30 transformants (μg DNA)-1. Integration frequency was strongly reduced when plasmid DNA was modified by dam or dcm methylase. By integrative transformation, via a double crossover, a stable null mutant of the hrdA gene was prepared. This mutation appeared to have no obvious effect on growth, morphology, differentiation and production of chlortetracycline.
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- Pathogenicity And Medical Microbiology
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Albumin-binding proteins on the surface of the Streptococcus milleri group and characterization of the albumin receptor of Streptococcus intermedius C5
More LessSUMMARY: Members of the Streptococcus milleri group (SMG) that react with Lancefield group C antisera were shown to bind large amounts of albumin although there was no direct relation between these two properties as polyclonal antisera to Lancefield group C antigen did not prevent the binding of albumin. There was a specificity for albumin binding, with albumin from man, monkeys, cat, dog and mouse being bound to a greater degree than albumin from cow, horse, goat or rabbit. Gold-labelled albumin was shown to be located close to the surface of strains by transmission electron microscopy. A cell-surface protein of Mr 24000, which was liberated by Iysozyme treatment of cells, was shown to be the cell-surface receptor on Streptococcus intermedius C5. The receptor was physically dissimilar from protein G, an albumin- and IgG-binding protein of ‘large-colony’ Lancefield group C and G streptococci.
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Transformation of Helicobacter pylori by chromosomal metronidazole resistance and by a plasmid with a selectable chloramphenicol resistance marker
More LessSUMMARY: Most strains of Helicobacter pylori are naturally competent for uptake of chromosomal DNA. Transformation frequencies for streptomycin resistance or rifampicin resistance markers ranged from 1 10-4 to 1 10-3 per viable cell using a plate transformation procedure. Transformation of a metronidazole resistance marker (MtrR) was demonstrated when either a laboratory-derived mutant or a MtrR clinical isolate were used as the source of donor DNA. MtrR was transformed at a frequency of 3 10-5 per viable cell. All H. pylori strains tested produce large amounts of DNAase, which may reduce DNA available for transformation. Four H. pylori plasmids were isolated. DNA fragments from H. pylori plasmids were deleted or rearranged when cloned in pUC19 and propagated in Escherichia coli DH5a. An H. pylori plasmid, pUOA26 which contained a chloramphenicol resistance determinant from Campylobacter coli, was constructed in H. pylori. This plasmid could be successfully introduced by natural transformation only into H. pylori recipients which contained a homologous resident plasmid. Transformation of pUOA26 into plasmid-free cells of H. pylori was achieved by electroporation. Transformation frequencies were 1 10-4 transformants per viable cell when plasmid DNA was isolated from the same strain; however, introduction of pUOA26 DNA derived from H. pylori 8091 into a different H. pylori strain, NCTC 11639, resulted in transformation at much lower frequencies (? 1 10-7 per viable cell). Changes in plasmid restriction patterns were also noted when pUOA26 was isolated from H. pylori NCTC 11639, suggesting the presence of at least two different DNA restriction and modification systems in H. pylori which may interfere with uptake of plasmid DNA.
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- Physiology And Growth
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Proteolysis and orientation in Dictyostelium slugs
More LessSUMMARY: It has been long known that the migrating slugs of the cellular slime moulds are highly sensitive to their environment and orient towards light and in temperature and chemical gradients. There is considerable evidence from past work that these orientations are governed by NH3 which affects the rate of movement of cells within the slug with such precision that orientation to the external stimuli is achieved. In order to test this hypothesis further, various ways to alter the internal NH3 concentration were devised. Substances that either increased or decreased proteolysis were applied to one side of the tip of a slug, thereby affecting its orientation. Some of the treatments strongly support the role of internally produced NH3 in orientation, and all the treatments produce results that are consistent with the hypothesis.
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Protection by sterols against the cytotoxicity of syringomycin in the yeast Saccharomyces cereuisiae
More LessSUMMARY: A brief exposure (ca 20 min) of the yeast Saccharomyces cerevisiae to the phytotoxin syringomycin was sufficient to kill the cell. The protective effect of sterols against this cytotoxicity of syringomycin was investigated. Syringomycin was much more toxic to growing cells than to stationary-phase cells. The cytotoxicity of syringomycin was reduced in an environment containing sterols. Cytotoxicity of syringomycin at 3 fig ml-1 (ca 2.5 μm) was completely abolished by the simultaneous presence of 10 μM-cholesterol in the medium. Cholesterol acetate had no protective effect. Ergosterol, sitosterol and stigmasterol also protected against syringomycin, but they were less effective than cholesterol. The protective effect of sterols against the action of syringomycin is consistent with our hypothesis that membrane ergosterol is a critical component for syringomycin-binding as suggested by recent genetic studies.
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Growth-phase-dependent expression of the lipolytic system of Acinetobacter calcoaceticus BD413: cloning of a gene encoding one of the esterases
More LessSUMMARY: Acinetobacter calcoaceticus BD413, when grown in batch culture in nutrient broth, produces both extracellular lipase activity and cell-bound esterase activity during and after the transition between exponential growth and the stationary phase. From a library of A. calcoaceticus DNA in Escherichia coli, plasmids were isolated that enabled E. coli to grow on media with tributyrin as the sole carbon source. Assays with model substrates classified the product of the cloned gene as an esterase. Via deletion analysis, the esterase gene was mapped on a 1.8 kbp chromosomal DNA fragment. This fragment was sequenced and found to contain one open reading frame, termed estA, which encodes a protein of 40.0 kDa. The amino acid sequence of this protein shows homology to a number of lipolytic enzymes, most notably to esterases. Deletion of estA only partially abolished cell-bound esterase activity in A. calcoaceticus, indicating that BD413 forms at least two esterases. Both esterases show the same temporal regulation of expression. β-Galactosidase activity was measured in strains in which a promoterless lacZ gene was inserted into estA. Induction of lacZ expression in these strains also occurred at the end of exponential growth in batch cultures, indicating that production of the esterase is regulated at the genetic level.
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Formation of melanin pigment by a mutant of Bacillus thuringiensis H-14
More LessSUMMARY: A mutant of Bacillus thuringiensis H-14 produced a dark brown pigment during sporulation. Production of the pigment depended on the nutritional properties of the growth medium. The pigment was identified as melanin, based on chemical tests and its infra-red spectrum. Incorporation of L-tyrosine in the culture medium enhanced the level of melanin production, and L-3,4-dihydroxyphenylalanine (L-DOPA) was detected in the culture broth during the late-exponential phase of growth. This indicates that the pathway of melanin synthesis is from L-tyrosine, via L-DOPA, to melanin.
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A halophilic thermophilic bacterium isolated from a coastal hot spring in Lutao, Taiwan
More LessSUMMARY: A heterotrophic, moderately thermophilic bacterial strain, designated strain T501, was isolated from a hot spring on the coast of Lutao, Taiwan. This bacterial strain was a strictly aerobic, Gram-negative rod, normally 0.6-0.7 m in diameter and 4-10 m in length, and motility was achieved by one to several lateral flagella. Its minimum and maximum growth temperatures were approximately 30 and 60 C, respectively, with an optimal temperature at about 50 C. The G + C ratio of its DNA was 38.1 mol%. Strain T501 tolerated a pH range from 6 to 9. It was halophilic, growing optimally in about 2% (w/v) salt, and Na+ was indispensable unless Mg2+ or Ca2+ were available. The addition of either cation to Na--sufficient media significantly enhanced growth. Halophilic heterotrophic bacteria capable of aerobic growth over a temperature range of 40-55 C have not been reported previously.
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- Systematics
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Helicobacter canis sp. nov., a new species from dogs: an integrated study of phenotype and genotype
SUMMARY: A group of Campylobacter-like organisms (CLOs) were isolated from the faeces of diarrhoeic or healthy dogs, constituting 4% of all CLOs from this source. Since they formed a unique DNA homology group within the genus Helicobacter, and exhibited distinctive phenotypic properties, they were collectively termed the HC group. A polyphasic taxonomic analysis was made of this group. The phenotype of four dog isolates and a single human isolate was unique and could be distinguished bacteriologically from other helicobacters. Electron microscopic ultrastructure revealed defining characteristics of Helicobacter. The 16S rRNA gene of the nominated type strain NCTC 12739T was sequenced, and its analysis delineated the group as a new species of Helicobacter. This conclusion was supported by relative DNA homology and whole-cell protein electrophoretic patterns. We therefore propose the name Helicobacter canis sp. nov. for this group. The species most closely related to H. canis sp. nov. were H. cinaedi, ‘Flexispira rappini’ and H. fennelliae. A species-specific recombinant DNA probe was cloned from NCTC 12739T for use in routine laboratory identification and epidemiological studies. The faecal source, bile tolerance and lack of urease activity of H. canis sp. nov. suggest that this new Helicobacter species colonizes the lower bowel rather than the stomach.
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- Genome Analysis
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“Analysis of the Anaplasma mavginale genome by pulsed-field electrophoresis”
More LessSUMMARY: Anaplasma marginale is a rickettsial parasite of bovine erythrocytes causing world-wide economic losses in livestock production. Despite its importance, little is known about this rickettsia at a molecular level because it has not been cultured in vitro, and there is no small-animal model. Although several genes have been cloned and sequenced, the gross genome structure of the organism has not yet been well characterized. We separated intact bovine erythrocytes from leucocytes, and determined the genome size of A. marginale by use of restriction endonuclease cleavage and pulsed-field gel electrophoresis (PFGE). A value of 56 mol% G + C was obtained for this genome by spectral analysis. Undigested A. marginale DNA failed to migrate under several different electrophoretic conditions, indicating a circular genome. Digestions of intact A. marginale DNA were performed using restriction endonucleases NotI, SfiI and PacI. Complete digestion with SfiI resulted in 12 distinct bands ranging in size from 14 to 170 kbp. Total size determined by addition of SfiI-digested fragments was approximately 1200 kbp. PacI cleaved the A. marginale genome from three different isolates into just three fragments, of 598, 557 and 97 kbp. Incomplete digestion produced a band measuring 1250 kbp. These results indicate that A. marginale has a circular genome between 1200 and 1260 kbp, with a G + C content of 56 mol%.
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Variation in the size of the ospA-containing linear plasmid, but not the linear chromosome, among the three Borrelia species associated with Lyme disease
More LessSUMMARY: The aetiological agents of Lyme disease form a phylogenetically heterogeneous group, composed of three species, Borrelia burgdorferi, Borrelia garinii, and group VS461. We have compared the sizes of the linear plasmid that carries the genes encoding the major outer-surface proteins OspA and OspB as well as the size and structure of the chromosome among the Lyme disease spirochaetes. We have found differences in the sizes of the ospA-containing plasmids, but not the linear chromosomes among the three species. The ospA-containing plasmid size of 50 kb in B. burgdorferi isolates is significantly smaller than the size of 55 kb in B. garinii isolates and 56 kb in group VS461 isolates. The chromosome was found to be linear in all three Borrelia species, but not significantly different in size.
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Volumes and issues
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Volume 138 (1992)
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Volume 137 (1991)
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Volume 136 (1990)
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Volume 135 (1989)
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Volume 134 (1988)
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Volume 133 (1987)
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Volume 132 (1986)
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Volume 131 (1985)
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Volume 130 (1984)
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Volume 129 (1983)
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Volume 128 (1982)
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Volume 127 (1981)
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Volume 126 (1981)
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Volume 125 (1981)
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Volume 124 (1981)
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Volume 123 (1981)
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Volume 122 (1981)
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Volume 121 (1980)
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Volume 120 (1980)
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Volume 119 (1980)
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Volume 118 (1980)
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Volume 117 (1980)
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Volume 116 (1980)
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Volume 115 (1979)
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Volume 114 (1979)
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Volume 113 (1979)
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Volume 112 (1979)
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Volume 111 (1979)
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Volume 110 (1979)
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Volume 109 (1978)
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Volume 108 (1978)
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Volume 107 (1978)
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Volume 106 (1978)
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Volume 105 (1978)
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Volume 104 (1978)
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Volume 103 (1977)
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Volume 102 (1977)
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Volume 101 (1977)
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Volume 100 (1977)
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Volume 99 (1977)
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Volume 98 (1977)
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Volume 97 (1976)
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Volume 96 (1976)
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Volume 95 (1976)
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Volume 94 (1976)
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Volume 93 (1976)
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Volume 92 (1976)
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Volume 91 (1975)
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Volume 90 (1975)
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Volume 89 (1975)
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Volume 88 (1975)
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Volume 87 (1975)
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Volume 86 (1975)
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Volume 85 (1974)
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Volume 84 (1974)
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Volume 83 (1974)
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Volume 82 (1974)
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Volume 81 (1974)
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Volume 80 (1974)
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Volume 79 (1973)
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Volume 78 (1973)
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Volume 77 (1973)
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Volume 76 (1973)
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Volume 75 (1973)
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Volume 74 (1973)
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Volume 73 (1972)
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Volume 72 (1972)
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Volume 71 (1972)
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Volume 70 (1972)
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Volume 69 (1971)
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Volume 68 (1971)
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Volume 67 (1971)
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Volume 66 (1971)
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Volume 65 (1971)
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Volume 64 (1970)
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Volume 63 (1970)
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Volume 62 (1970)
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Volume 61 (1970)
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Volume 60 (1970)
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Volume 59 (1969)
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Volume 58 (1969)
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Volume 57 (1969)
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Volume 56 (1969)
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Volume 55 (1969)
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Volume 54 (1968)
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Volume 53 (1968)
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Volume 52 (1968)
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Volume 51 (1968)
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Volume 50 (1968)
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Volume 49 (1967)
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Volume 48 (1967)
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Volume 47 (1967)
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Volume 46 (1967)
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Volume 45 (1966)
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Volume 44 (1966)
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Volume 43 (1966)
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Volume 42 (1966)
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Volume 41 (1965)
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Volume 40 (1965)
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Volume 39 (1965)
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Volume 38 (1965)
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Volume 37 (1964)
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Volume 36 (1964)
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Volume 35 (1964)
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Volume 34 (1964)
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Volume 33 (1963)
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Volume 32 (1963)
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Volume 31 (1963)
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Volume 30 (1963)
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Volume 29 (1962)
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Volume 28 (1962)
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Volume 27 (1962)
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Volume 26 (1961)
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Volume 25 (1961)
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Volume 24 (1961)
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Volume 23 (1960)
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Volume 22 (1960)
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Volume 21 (1959)
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Volume 20 (1959)
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Volume 19 (1958)
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Volume 18 (1958)
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Volume 17 (1957)
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Volume 16 (1957)
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Volume 15 (1956)
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Volume 14 (1956)
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Volume 13 (1955)
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Volume 12 (1955)
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Volume 11 (1954)
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Volume 10 (1954)
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Volume 9 (1953)
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Volume 8 (1953)
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Volume 7 (1952)
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Volume 6 (1952)
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Volume 5 (1951)
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Volume 4 (1950)
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Volume 3 (1949)
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Volume 2 (1948)
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Volume 1 (1947)