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  • 1
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Examination of the properties of ColE1 derivatives containing either deletions or insertions of transposable genetic elements, has enabled a functional map of plasmid ColE1 to be constructed.
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  • 2
    ISSN: 1617-4623
    Keywords: P22 transduction ; Salmonella ; Cosmids ; aro gene ; Virulence
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A cosmid gene bank of the virulent Salmonella typhimurium C5 was constructed in Escherichia coli K12. The bank was repackaged into bacteriophage heads and transduced into the semi-rough S. typhimurium strain AS68 which expresses the LamBλ receptor protein. Approximately 6000 ampicillin-resistant transductants were pooled and used as host for the propagation of bacteriophage P22. The P22 lysate was able to transduce cosmid recombinants to smooth strains of S. typhimurium and individual transductants were selected which complemented various S. typhimurium auxotrophic mutations. A stable mutation was introduced into the aroD gene of S. typhimurium C5. The resulting aroD - mutant, named CU038, was highly attenuated compared with the wild-type parent strain and BALB/c mice immunised orally with CU038 were well protected against challenge with the virulent C5 parental strain. Using the cosmid bank repackaged into bacteriophage P22 heads it was possible to isolate cosmid recombinants that could complement the aroD mutation of CU038 either by in vitro selection using minimal medium or in vivo selection for restoration of virulence in BALB/c mice. Repackaged P22 cosmid banks could provide a simple system for selecting in vivo for Salmonella virulence determinants. A Salmonella typhi strain harbouring mutations in aroA and aroD was constructed for potential use as a live oral typhoid vaccine in humans.
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  • 3
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Insertion of the transposable genetic element Tn1 into different sites of plasmid ColE1 results in a number of mutant phenotypes. Whereas all plasmids examined were present in normal amount, all showed reduced immunity to killing by colicin E1. Of six insertions isolated after conjugation, five fail to produce colicin, are conjugally proficient (transmissible), and map within a 500 nucleotide region of the genome. The other is conjugally deficient, produces colicin normally and maps close to two others with a similar phenotype isolated after transformation. Of four others isolated after transformation, two have similar properties to the original five transmissible plasmids. The other two are nontransmissible and produce colicin. Non-transmissibility is correlated with reduced relaxation complex. Patterns of protein synthesis in minicels by ColE1 and ColE1:: Tn1 plasmids have been examined: all ColE1 plasmids containing Tn1 show an altered pattern of ColE1 protein synthesis in addition to three presumptive Tn1-specified proteins, one of which is shown to be β-lactamase. ColE1:: Tn1 plasmids can be inserted into the conjugative plasmid R64drd11 to form a cointegrate in which ColE1 and Tn1 function can be expressed.
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  • 4
    ISSN: 1617-4623
    Keywords: Salmonella ; Typhoid ; Vaccine ; Aromatic
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Derivatives of the Salmonella typhi strain Ty2 carrying stable mutations in the aroA gene were isolated. The mutations were generated by transducing an aroA::Tn10 marker into Ty2 and selecting for derivatives which were tetracyline sensitive and dependent on aromatic compounds for growth. Isolates that did not revert to aromatic compound independence at a detectable frequency were obtained. An S. typhimurium derived aroA specific DNA probe was used to demonstrate the presence of DNA rearrangements in the aroA region of the chromosome of some of the S. typhi aroA mutants. Most of these isolates still expressed Vi antigen. Aromatic compound dependent mutants of S. typhi were less virulent in mice than S. typhi Ty2 following intraperitoneal challenge with bacteria suspended in mucin. Mice immunised with one of these mutants, named WBL85-1, were protected against a potentially lethal challenge of S. typhi Ty2.
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  • 5
    ISSN: 1617-4623
    Keywords: aro mutants ; Dehydroquinate synthase ; 3-Dehydroquinase ; Mycobacterium tuberculosis ; Quinic acid utilisation ; Shikimate pathway
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The Mycobacterium tuberculosis shikimate pathway genes designated aroB and aroQ encoding 3-dehydroquinate synthase and 3-dehydroquinase, respectively were isolated by molecular cloning and their nucleotide sequences determined. The deduced dehydroquinate synthase amino acid sequence from M. tuberculosis showed high similarity to those of equivalent enzymes from prokaryotes and filamentous fungi. Surprisingly, the deduced M. tuberculosis 3-dehydroquinase amino acid sequence showed no similarity to other characterised prokaryotic biosynthetic 3-dehydroquinases (bDHQases). A high degree of similarity was observed, however, to the fungal catabolic 3-dehydroquinases (cDHQases) which are active in the quinic acid utilisation pathway and are isozymes of the fungal bDHQases. This finding indicates a common ancestral origin for genes encoding the catabolic dehydroquinases of fungi and the biosynthetic dehydroquinases present in some prokaryotes. Deletion of genes encoding shikimate pathway enzymes represents a possible approach to generation of rationally attenuated strains of M. tuberculosis for use as live vaccines.
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  • 6
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract Cell envelope preparations of Treponema hyodysenteriae (strain CN 8368) were examined using biochemical and immunochemical methods. Several major polypeptides were detected with molecular weight between 24-kDa and 45-kDa. The majority of these polypeptides were recognised by serum from a pig vaccinated with an experimental whole-cell T. hyodysenteriae vaccine and hyperimmune anti-T. hyodysenteriae rabbit sera. Immune electron microscopy confirmed that the major antigens detected were associated with the cell envelope. Triton X-100, in the presence of EDTA, completely solubilised a polypeptide with an approximate molecular weight of 36-kDa. Antibodies to this polypeptide were not absorbed by whole T. innocens cells.
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  • 7
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract Plasmid pKT274 encoding a determinant for the Escherichia coli K1 polysaccharide was introduced into the Salmonella typhimurium aroA vaccine strain SL3261 and cells harbouring the plasmid were shown to express K1 polysaccharide at their cell surface. SL3261 (pKT274) could be detected in the livers and spleens of BALB/c mice infected by the intravenous route and viable organisms persisted for several weeks. SL3261 (pKT274) was cleared from the livers more rapidly and from the spleens more slowly than SL3261. Unlike mice infected with SL3261 those infected with SL3261 (pKT274) did not exhibit gross splenomegaly during the first three weeks after infection. Mice vaccinated with viable SL3261 (pKT274) were protected against challenge with virulent S. typhimurium but failed to produce detectable levels of humoral anti-K1 polysaccharide antibodies.
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  • 8
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract Attenuated Salmonella strains are currently being evaluated as live vectors for the delivery of heterologous antigens to the mammalian mucosal and systemic immune systems. An approach to improving the stability of heterologous antigen expression during vaccination is to drive expression of the foreign protein from promoters e.g. nirB, that become activated when Salmonella enter the host. Salmonella strains were constructed that harboured similar multicopy plasmids encoding the lacZ gene. In each strain, lacZ expression was driven from either the nirB, htrA or groE promoters. Expression of LacZ increased in all vaccine strains as they were shifted from conditions of low to high temperature. In addition, expression of lacZ driven from the htrA and nirB promoters significantly increased when the Salmonella entered eukaryotic cells, including macrophages. Expression of lacZ from the groE promoter was significantly elevated in macrophages but not in cells derived from epithelia. These promoters may be useful for optimising heterologous antigen expression within immune cells of the host.
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  • 9
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Enteropathogenic Escherichia coli (EPEC) encode a type III secretion system located on a pathogenicity island known as the locus for enterocyte effacement. Four proteins are known to be exported by this type III secretion system – EspA, EspB and EspD required for subversion of host cell signal transduction pathways and a translocated intimin receptor protein (Tir) required for intimin-mediated intimate attachment and attaching and effacing lesion formation. The espA gene is located within the locus for enterocyte effacement and the EspA polypeptide from the prototype EPEC strain E2348/69 (O127:H6) has recently been shown to be a component of a filamentous structure involved in bacteria-host cell interaction and locus for enterocyte effacement-encoded protein translocation involved in attaching and effacing lesion formation. In this study we have extended our investigation of EspA to strains belonging to other classical EPEC serotypes. DNA sequencing demonstrated that the espA gene from the different EPEC strains share at least 65% DNA identity. In addition, we detected morphologically and antigenically similar EspA filaments in all but one of the bacterial strains examined including recombinant, non-pathogenic E. coli expressing espA from a cloned locus for enterocyte effacement region (HB101(pCVD462)).
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  • 10
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 143 (1996), S. 0 
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract As part of a study of genes whose transcription is maintained in stationary phase, cloned segments of DNA were selected from a Lambda ZAP II library of Campylobacter jejuni NCTC 11168. One such clone was found to encode a homologue of the Escherichia coli cell division gene ftsA. Examination of mRNA by transcription mapping revealed that the Campylobacter gene has one major and three minor transcription start sites. There were several significant differences in the structure and organisation of the C. jejuni ftsA promoter compared to that of E. coli.
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