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  • 1990-1994  (107,280)
  • 1970-1974  (45,346)
  • 1993  (107,280)
  • 1972  (45,346)
Collection
Years
Year
  • 1
    Electronic Resource
    Electronic Resource
    Amsterdam : Elsevier
    Physics Letters B 294 (1992), S. 466-478 
    ISSN: 0370-2693
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Physics
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Amsterdam : Elsevier
    Physics Letters B 317 (1993), S. 474-484 
    ISSN: 0370-2693
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Physics
    Type of Medium: Electronic Resource
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  • 3
    facet.materialart.
    Unknown
    Totowa, NJ : Humana Press
    Keywords: Molecular Biology / methods
    Notes: This is a series title, single volumes see link below.
    ISSN: 1940-6029
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  • 4
    Call number: 01-Beschaffungsw:70 ; M230:3/1 ; M230:3/2
    Pages: loose-leaf
    ISBN: 3-8073-0843-1
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    01-Beschaffungsw:70 departmental collection or stack – please contact the library
    M230:3/1 departmental collection or stack – please contact the library
    M230:3/2 departmental collection or stack – please contact the library
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  • 5
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    Unknown
    New York, NY : Elsevier
    Keywords: Biochemistry ; Enzymes
    Notes: This is a series title, single volumes see link below.
    ISSN: 1557-7988
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  • 6
    Keywords: Leukemia / therapy ; Prognosis
    Notes: Last volumes with varying subtitle and editor.
    ISSN: 0949-7021
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  • 7
    Keywords: Hazardous Substances / toxicity
    Notes: Ceased with vol. 15(1999).
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  • 8
    facet.materialart.
    Unknown
    Königswinter : Petersberg Verlag
    Keywords: Arbeitsgemeinschaft der Großforschungseinrichtungen (Germany) ; Research institutes / Germany ; Research ; Germany
    Notes: Ceased with ed. 1995.
    ISSN: 0935-2236
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  • 9
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Computational intelligence 9 (1993), S. 0 
    ISSN: 1467-8640
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Computer Science
    Type of Medium: Electronic Resource
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  • 10
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Computational intelligence 9 (1993), S. 0 
    ISSN: 1467-8640
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Computer Science
    Type of Medium: Electronic Resource
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  • 11
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Computational intelligence 9 (1993), S. 0 
    ISSN: 1467-8640
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Computer Science
    Type of Medium: Electronic Resource
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  • 12
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Computational intelligence 9 (1993), S. 0 
    ISSN: 1467-8640
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Computer Science
    Type of Medium: Electronic Resource
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  • 13
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Computational intelligence 9 (1993), S. 0 
    ISSN: 1467-8640
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Computer Science
    Type of Medium: Electronic Resource
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  • 14
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Computational intelligence 9 (1993), S. 0 
    ISSN: 1467-8640
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Computer Science
    Notes: We define an argument system to be a pair consisting of a set of inference rules and a set of completeness conditions. Inference rules are used to build arguments. Completeness conditions are used to define argument structures, which are sets of arguments supporting belief sets. We reformulate Reiter's default logic as special argument systems. This enables us, among other things, to apply the negation-as-failure rule to general default theories. We also speculate on some other potential uses of our argument systems.
    Type of Medium: Electronic Resource
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  • 15
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Computational intelligence 9 (1993), S. 0 
    ISSN: 1467-8640
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Computer Science
    Notes: This paper describes an original approach to semantics representation based on the use of a non-strict functional programming language with polymorphic typing. This approach provides a unified formalism needing no preprocessing or postprocessing to the functional language itself: parsing and semantics are declared naturally using function definition and evaluation is done by lambda application along the lines of Montague. We show that by changing only the model we can, after parsing, compute either the truth value of a sentence or its parse tree.
    Type of Medium: Electronic Resource
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  • 16
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Computational intelligence 9 (1993), S. 0 
    ISSN: 1467-8640
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Computer Science
    Type of Medium: Electronic Resource
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  • 17
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Computational intelligence 9 (1993), S. 0 
    ISSN: 1467-8640
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Computer Science
    Notes: Search is fundamental to artificial intelligence (AI) and numerous sophisticated search methods have been developed. We present a general, simple model of search processes and use it to analytically determine some typical behavior when applied to large problems. In particular, this identifies abrupt changes in overall search cost as small improvements are made in the underlying method. We also examine the robustness of this model's predictions in a range of more realistic cases. More generally, we introduce a criterion for determining when average case results reflect typical behavior which allows the method developed here to be used for investigating other large-scale behaviors of complex AI systems.
    Type of Medium: Electronic Resource
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  • 18
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Computational intelligence 9 (1993), S. 0 
    ISSN: 1467-8640
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Computer Science
    Notes: Glasgow's revival of the “imagery debate” in computational terms provides a renewed opportunity to review the role of logical reasoning in general problem solving. Of special interest is the long-standing distinction between analogical or depictive problem representations, and the more abstract linguistic forms typified by traditional formal logic syntax.In our brief statement, we recall that logical reasoning rests on semantics not syntax, and that the concepts of soundness, completeness, and consistency are manifest in both depictive and linguistic representations. We emerge with two conclusions: (1) enduring confusion regarding computational aspects of the “imagery debate” arise from long-standing confusion regarding key logical concepts, and related notions such as epistemological versus heuristic adequacy, logical versus probabilistic independence, and direct versus indirect representations; (2) the desire for depictive reasoning methods is ultimately motivated by human needs, not computational needs.
    Type of Medium: Electronic Resource
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  • 19
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Computational intelligence 9 (1993), S. 0 
    ISSN: 1467-8640
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Computer Science
    Notes: It might be said that there are five basic tree search algorithms for the constraint satisfaction problem (csp), namely, naive backtracking (BT), backjumping (BJ), conflict-directed backjumping (CBJ), backmarking (BM), and forward checking (FC). In broad terms, BT, BJ, and CBJ describe different styles of backward move (backtracking), whereas BT, BM, and FC describe different styles of forward move (labeling of variables). This paper presents an approach that allows base algorithms to be combined, giving us new hybrids. The base algorithms are described explicitly, in terms of a forward move and a backward move. It is then shown that the forward move of one algorithm may be combined with the backward move of another, giving a new hybrid. In total, four hybrids are presented: backmarking with backjumping (BMJ), backmarking with conflict-directed backjumping (BM-CBJ), forward checking with backjumping (FC-BJ), and forward checking with conflict-directed backjumping (FC-CBJ). The performances of the nine algorithms (BT, BJ, CBJ, BM, BMJ, BM-CBJ, FC, FC-BJ, FC-CBJ) are compared empirically, using 450 instances of the ZEBRA problem, and it is shown that FC-CBJ is by far the best of the algorithms examined.
    Type of Medium: Electronic Resource
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  • 20
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Computational intelligence 9 (1993), S. 0 
    ISSN: 1467-8640
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Computer Science
    Notes: We investigate the problem of learning two-layer neural nets of nonoverlapping perceptrons where each input unit is connected to one and only one hidden unit. We first show that this restricted problem with no overlap at all between the receptive fields of the hidden units is as hard as the general problem (with total overlap) if the learner uses examples only. However, if membership queries are allowed, the restricted problem is indeed easier to solve. We give a learning algorithm that uses examples and membership queries to PAC learn the intersection of K-nonoverlapping perceptrons, regardless of whether the instance space in Boolean, discrete, or continuous. An extension of this algorithm is proven to PAC learn two-layer nets with K-nonoverlapping perceptrons. The simulations performed indicate that both algorithms are fast and efficient.
    Type of Medium: Electronic Resource
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  • 21
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Computational intelligence 9 (1993), S. 0 
    ISSN: 1467-8640
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Computer Science
    Notes: A method is presented of establishing bounds on the number of classification rules in such applications as credit worthiness assessment, investment decisions, premium determination, consumer choices, employee selection, and editorial preferences, to name just a few. A function that relates the maximum number of classification rules to the problem space size of such application domains is established. It is shown that in this important class of ordinal classification problems, the maximum possible number of rules is significantly lower than the relative problem space sizes. The approach grants the ability to a priori estimate worst case response time and memory requirements, and to better predict the effectiveness of knowledge acquisition efforts.
    Type of Medium: Electronic Resource
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  • 22
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Teaching statistics 15 (1993), S. 0 
    ISSN: 1467-9639
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Mathematics
    Type of Medium: Electronic Resource
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  • 23
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Teaching statistics 15 (1993), S. 0 
    ISSN: 1467-9639
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Mathematics
    Type of Medium: Electronic Resource
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  • 24
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Teaching statistics 15 (1993), S. 0 
    ISSN: 1467-9639
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Mathematics
    Type of Medium: Electronic Resource
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  • 25
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Teaching statistics 15 (1993), S. 0 
    ISSN: 1467-9639
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Mathematics
    Type of Medium: Electronic Resource
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  • 26
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Teaching statistics 15 (1993), S. 0 
    ISSN: 1467-9639
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Mathematics
    Type of Medium: Electronic Resource
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  • 27
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Teaching statistics 15 (1993), S. 0 
    ISSN: 1467-9639
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Mathematics
    Type of Medium: Electronic Resource
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  • 28
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Teaching statistics 15 (1993), S. 0 
    ISSN: 1467-9639
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Mathematics
    Notes: Books Reviewed in this Article:Teaching Large Classes in Higher Education.Third World Guide 91/92.PM649 Supporting Primary Mathematics, The Open University.Exploring Statistics with Minitab.Ability, Partial information, Guessing: Statistical Modelling applied to Multiple-choice Tests.Mathematical Statistics.The Crest of the Peacock.
    Type of Medium: Electronic Resource
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  • 29
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Teaching statistics 15 (1993), S. 0 
    ISSN: 1467-9639
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Mathematics
    Type of Medium: Electronic Resource
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  • 30
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Teaching statistics 15 (1993), S. 0 
    ISSN: 1467-9639
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Mathematics
    Notes: A classical experiment on the tasting of tea is used to show that many standard methods of analysis of the resulting data are unsatisfactory. A similar experiment with wine is used to show how a more sensible method may be developed.
    Type of Medium: Electronic Resource
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  • 31
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Teaching statistics 15 (1993), S. 0 
    ISSN: 1467-9639
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Mathematics
    Type of Medium: Electronic Resource
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  • 32
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Teaching statistics 15 (1993), S. 0 
    ISSN: 1467-9639
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Mathematics
    Type of Medium: Electronic Resource
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  • 33
    Electronic Resource
    Electronic Resource
    Amsterdam : Elsevier
    Nuclear Physics, Section B 403 (1993), S. 3-24 
    ISSN: 0550-3213
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Physics
    Type of Medium: Electronic Resource
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  • 34
    Electronic Resource
    Electronic Resource
    Amsterdam : Elsevier
    Physics Letters B 318 (1993), S. 249-262 
    ISSN: 0370-2693
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Physics
    Type of Medium: Electronic Resource
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  • 35
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The process of SOS mutagenesis in Escherichia coli requires (i) the replisome enzymes, (ii) RecA protein, and (iii) the formation of the UmuD'C protein complex which appears to help the replisome to resume DNA synthesis across a lesion. We found that the UmuD'C complex is an antagonist of RecA-mediated recombination. Homologous recombination in an Hfr x F- cross decreased as a function of the UmuD'C cell concentration; this effect was challenged by increasing RecA concentration. Recombination of a u.v.-damaged F-lac with the lac gene of an F- recipient was reduced by increasing the UmuD'C concentration while lac mutagenesis increased, showing an inverse relationship between recombination and SOS mutagenesis. We explain our data with the following model. The kinetics of appearance of the UmuD'C complex after DNA damage is slow, reaching a maximum after an hour. Within that period, excision and recombinational repair have had time to occur. When the UmuD'C concentration relative to the number of residual RecA filaments, not resolved by recombinational repair, becomes high enough, UmuD'C proteins provide a processive factor for the replisome to help replication bypass and repel the standing RecA filament. Thus, at a high enough concentration, the UmuD'C complex will switch repair from recombination to SOS mutagenesis.
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  • 36
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 10 (1993), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: A novel ompC mutation was isolated that not only lowered the amount of its own product, OmpC27, but also reduced the level of OmpF present in the outer membrane. ompC27 codes for a mutant OmpC protein that contains two non-native cysteine residues. The ompC27 allele confers phage resistance by lowering the level of OmpC present in the outer membrane. This effect on OmpC27 was manifested at the level of assembly as a result of disulphide bond formation between the two cysteine residues. This disulphide bonding in OmpC27 also produced a novel phenotype by specifically influencing OmpF levels. The effect of OmpC27 on OmpF was partly a result of a lowering of ompF transcription, and partly a result of an effect at the post-transcription level. The transcriptional effect is likely to be brought about by a defective membrane as a result of the insertion of the disulphide bond containing OmpC27. The post-transcriptional effect of OmpC27 on OmpF could be due to interference at the assembly level. In a dsbA::kan1 background where the in vivo disulphide bonding ability was dramatically reduced, the OmpC27-mediated effects were also curtailed.
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  • 37
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: In Escherichia coli the OmpR and EnvZ proteins regulate the expression of the outer membrane porin proteins OmpC and OmpF. EnvZ and OmpR belong to a family of sensor/effector protein pairs that control adaptation to a variety of environmental conditions. EnvZ acts as the sensor protein that phosphorylates OmpR, which in turn regulates porin gene expression. The level of phosphorylated OmpR appears to be a determining factor for ompC and ompF regulation. Phosphorylation of OmpR is considered to occur at one or more aspartic acid residues (Asp-11, Asp-12 and/or Asp-55) that are highly conserved among the effector proteins. In this report we biochemically characterized the aspartic acid residue(s) in OmpR that were phosphorylated by EnvZ. Reduction of aspartyl phosphate residues in the amino-terminal domain of OmpR with [3H]-NaBH4 indicated that Asp-55 was a primary site of modification. We further studied the role of the highly conserved aspartate residues by creating OmpR mutants having aspartate to alanine substitutions at positions 11 (D11A), 12 (D12A) and 55 (D55A). Studies of ompF and ompC expression as well as in vivo and in vitro phosphorylation experiments also demonstrated that while Asp-55 is the primary phosphate acceptor site in OmpR, Asp-11 may also serve as a phosphorylation site, particularly in the absence of Asp-55.
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  • 38
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Adherence of capsulate Neisseria meningitidis to endothelial and epithelial cells is facilitated in variants that express pili. Whereas piliated variants of N. meningitidis strain C311 adhered to endothelial cells in large numbers (〈150 bacteria/cell), derivatives containing specific mutations that disrupt pilE encoding the pilin subunit were both non-piliated and failed to adhere to endothelial cells (〈1 bacterium/ cell). In addition, meningococcal pili recognized human endothelial and epithelial cells but not cells originating from other animals. Variants of strain C311 were obtained that expressed pilins of reduced apparent Mr and exhibited a marked increase in adherence to epithelial cells. Structural analysis of pilins from two hyper-adherent variants and the parent strain were carried out by DNA sequencing of their pilE genes. Deduced molecular weights of pilins were considerably tower compared with their apparent Mr values on SDS-PAGE. Hyper-adherent pilins shared unique changes in sequence including substitution of Asn-113 for Asp-113 and changes from Asn-Asp-Thr-Asp to Thr-Asp-Ala-Lys at residues 127-130 in mature pilin. Asn residues 113 and 127 of‘parental’pilin both form part of the typical eukaryotic N-glycosylation motif Asn-X-Ser/Thr and could potentially be glycosylated post-translationally. The presence of carbohydrate on pilin was demonstrated and when pilins were deglycosylated, their migration on SDS-PAGE increased, supporting the notion that variable glycosylation accounts for discrepancies in apparent and deduced molecular weights. Functionally distinct pilins produced by two fully piliated variants of a second strain (MC58) differed only in that the putative glycosylation motif Asn-60-Asn-61-Thr-62 in an adherent variant was replaced with Asp-60-Asn-61-Ser-62 in a non-adherent variant. Fully adherent backswitchers obtained from the non-adherent variant always regained Asn-60 but retained Ser-62. We propose, therefore, that functional variations in N. meningitidis pili may be modulated in large part by primary amino acid sequence changes that ablate or create N-linked glycosylation sites on the pilin subunit.
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  • 39
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The binding of Streptococcus pyogenes to fibronectin (FN) enables the adherence of this pathogen to target epithelial cells, which is the first necessary step for initiation of infection. Binding is mediated by a bacterial surface protein termed protein F. Here we provide the complete structure of protein F and identify two domains responsible for binding to fibronectin. The first domain is located towards the C-terminal end of the molecule and is composed of five repeats of 37 amino acids that are completely repeated four times and a fifth time partially. The second domain is adjacent to the first domain and is located on the /V-terminal side of it. It is composed of a single stretch of 43 amino acids. Protein F expressed in Escherichia coli completely blocked the binding of fibronectin to S. pyogenes. However, mutant proteins that contained only one or the other of the two domains were only capable of partial blockage of binding. Complete blockage of binding of fibronectin could be achieved when a protein extract containing the N-terminal domain was mixed in a binding reaction with a protein extract containing the C-terminal domain. Similarly, a purified recombinant protein containing the two domains only, blocked the binding completely. In contrast, a purified recombinant protein containing just the C-terminal domain, blocked the binding partially. A clone exclusively expressing the C-terminal domain, completely blocked the binding of the 30 kDa N-terminal fragment of fibronectin to S. pyogenes, whereas a clone expressing the N-terminal domain failed to block the binding of this FN fragment. Thus, the two FN-binding domains of protein F are necessary for maximal bacterial binding and act in concert to enhance the binding to fibronectin. The possibility that the two domains bind to two different regions on the fibronectin molecule is discussed.
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  • 40
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Mycobacterium tuberculosis complex strains contain a unique chromosomal region, which consists of multiple 36bp direct repeats (DRs), which are interspersed by unique spacers 35 to 41 bp in length. In this study we investigated the nature of the DNA polymorphism of this DR cluster by sequencing part of this region in a large number of M. tuberculosis complex strains. Two types of genetic rearrangements were observed. One type consists of the variation in one or a few discrete, contiguous DRs plus spacer sequences. This variation is probably driven by homologous recombination between adjacent or distant DRs. The other type of polymorphism is probably driven by transpositional events of the insertion sequence, IS6110, which is almost invariably present in the DR cluster of M. tuberculosis complex strains. Based on the nature of the DNA polymorphism in the DR cluster, we developed a novel method of strain differentiation, direct variable repeat polymer chain reaction (DVR-PCR), which enables typing of individual M. tuberculosis strains in a single PCR. The method allows an excellent differentiation of epidemiologically unrelated isolates and, in principle, the DVR-PCR allows the detection of M. tuberculosis and strain differentiation at the same time.
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  • 41
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The Pseudomonas aeruginosa genes pilB-D and pilQ are necessary for the assembly of type 4 fimbriae. Homologues of these genes and of the subunit (pilin) gene have been described in various different bacterial species, but not always in association with type 4 fimbrial biosynthesis and function. Pil-like proteins are also involved in protein secretion, DNA transfer by conjugation and transformation, and morphogenesis of filamentous bacteriophages. It seems likely that the Pil homologues function in the processing and export of proteins resembling type 4 fimbrial sub-units, and in their organization into fimbrial-like structures. These may either be true type 4 fimbriae, or components of protein complexes which act in the transport of macromolecules (DNA or protein) into or out of the cell. Some PilB-like and PilQ-like proteins are apparently also involved in the assembly of non-type 4 polymeric structures (filamentous phage virions and conjugative pili). The diverse studies summarized in this review are providing insight into an extensive infrastructural system which appears to be utilized in the formation of a variety of cell surface-associated complexes.
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  • 42
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 10 (1993), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Galactose transport and metabolism in Escherichia coli involves a multicomponent amphibolic pathway. Galactose transport is accomplished by two different galactose-specific transport systems. At least four of the genes and operons involved in galactose transport and metabolism have promoters containing similar regulatory sequences. These sequences are recognized by at least three regulators, Gal repressor (GalR), Gal isorepressor (GalS) and cAMP receptor protein (CRP), which modulate transcription from these promoters. The negative regulators, GalR and GalS, discriminate between utilization of the high-affinity (regulated by GalS) and low-affinity (regulated by GalR) transport systems, and modulate the expression of genes for galactose metabolism in an overlapping fashion. GalS is itself autogenously regulated and CRP dependent, while the gene for GalR is constitutive. The gal operon encoding the enzymes for galactose metabolism has two promoters regulated by CRP in opposite ways; one (P1) is stimulated and the other (P2) inhibited by CRP. Both promoters are strongly repressed by GalR but weakly by GalS. All but one of the constituent promoters of the gal regulon have two operators. The gal regulon has the potential to coordinate galactose metabolism and transport in a highly efficient manner, under a wide variety of conditions of galactose availability.
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  • 43
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The Bacillus subtilis ccpA gene has previously been shown to be involved in repression of amyE expression when cells are grown in excess glucose. The region of the B. subtilis chromosome downstream from ccpA was characterized to determine if additional genes involved in carbohydrate metabolism were present. Two open reading frames that exhibited sequence similarity to the Escherichia coli and B. subtilis motA and motB motility genes were found immediately downstream from ccpA; disruption of this region had no effect on growth, sporulation or motility. Two divergent transcriptional units containing the acsA and acuABC genes were also found in this region. The acsA gene encodes acetyl-CoA synthetase, and inactivation of this gene resulted in loss of the ability to utilize acetate as a carbon source for growth or sporulation. Disruption of the acuABC genes resulted in poor growth or sporulation on acetoin or butanediol. The acsA and acuABC promoter sequences were identified by primer extension, and are in close proximity. Two sequences resembling the amyO regulatory target site necessary for glucose repression of amyE were identified in the acsA-acuABC promoter regions.
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  • 44
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The oprF gene, expressing Pseudomonas aeruginosa major outer membrane protein OprF, was subjected to semi-random linker mutagenesis by insertion of a 1.3 kb Hincll kanamycin-resistance fragment from plasmid pUC4KAPA into multiple blunt-ended restriction sites in the oprF gene. The kanamycin-resistance gene was then removed by Pstl digestion, which left a 12 nucleotide pair linker residue. Nine unique clones were identified that contained such linkers at different locations within the oprF gene and were permissive for the production of full-length OprF variants. In addition, one permissive site-directed insertion, one non-permissive insertion and one carboxy-terminal insertion leading to proteolytic truncation were also identified. These mutants were characterized by DNA sequencing and reactivity of the OprF variants with a bank of 10 OprF-specific monoclonal antibodies. Permissive clones produced OprF variants that were shown to be reactive with the majority of these monoclonal antibodies, except where the insertion was suspected of interrupting the epitope for the specific monoclonal antibody. In addition, these variants were shown to be 2-mercaptoethanol modifiable, to be resistant to trypsin cleavage in intact cells and partly cleaved to a high-molecular-weight core fragment in outer membranes and, where studied, to be accessible to indirect immunofluorescenee labelling in intact cells by monoclonal antibodies specific for surface epitopes. Based on these data, a revised structural model for OprF is proposed.
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  • 45
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: In this paper we show that the Escherichia coli protein Fis has a regulatory function in Mu transposition in the presence of Mu repressor. Fis can lower the transposition frequency of a mini-Mu 3–80-fold, but only if the Mu repressor is expressed simultaneously. In this novel type of regulation of transposition by the concerted action of Fis and repressor, the IAS, the internal activating sequence, is also involved as deletion of this site leads to the loss of the Fis effect. As the IAS contains strong repressor binding sites these are probably the target for the repressor in the observed negative regulation by Fis and repressor. However, the role of Fis and repressor is not only to inactivate the IAS, since a 4bp insertion in the IAS, which changes the spacing of the repressor-binding site, abolishes the enhancing function of the IAS but leaves the repressor-Fis effect intact. A likely target for Fis in this regulation is a strong Fis-binding site, which is located adjacent to the L2 transposase-binding site. However, when this Fis-binding sequence was substituted by a random sequence and Fis no longer showed specific binding to this site, the Fis effect was still observed. Although it is still possible that Fis can function by binding to this non-specific site in a particular complex, it seems more likely that Fis is directly or indirectly involved in determining the level of the repressor.
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  • 46
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Expression of a promoterless cat gene fused to a DNA fragment of approximately 400 bp, beginning at –313 of Escherichia coli hns, was significantly repressed in E. coli and Salmonella typhimurium strains with wild-type hns but not in mutants carrying hns alleles. CAT expression from fusions containing a shorter (110 bp) segment of hns was essentially unaffected in the same genetic backgrounds. The stage of growth was found to influence the extent of repression which was maximum (approximately 75%) in mid-log cultures and negligible in cells entering the stationary phase. The level of repression in early-log phase was lower than in mid-log phase cultures, probably because of the presence of high levels of Fis protein, which counteracts the H-NS inhibition by stimulating hns transcription. The effects observed in vivo were mirrored by similar results obtained in vitro upon addition of purified H-NS and Fis protein to transcriptional systems programmed with the same hns caf fusions. Electrophoretic gel shift assays, DNase I footprinting and cyclic permutation get analyses revealed that H-NS binds preferentially to the upstream region of its own gene recognizing two rather extended segments of DNA on both sides of a bend centred around –150. When these sites are filled by H-NS, an additional site between approximately –20 and –65, which partly overlaps the promoter, is also occupied. Binding of H-NS to this site is probably the ultimate cause of transcriptional auto-repression.
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  • 47
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The interaction between the DNA replication terminator, IRI, of Bacillus subtilis and its cognate replication terminator protein (RTP) has been examined by the technique of missing nucleoside interference (MNI). IRI contains two adjacent binding sites (A and B) for RTP dimers. The B site is proximal to the replication fork arrest site. The present results have shown that nucleoside contacts with RTP in the two sites are very different. There are more extensive contacts of nucleosides in both strands of the B site with RTP compared with the A site. The data also strongly suggest that filling by RTP of the B site occurs first and is needed for subsequent co-operative filling of an overlapping A site. The A site alone binds RTP poorly. The findings are consistent with interaction occurring between RTP dimers bound to adjacent sites of IRI, which would explain why RTP bound to the B site alone cannot cause replication fork arrest.
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  • 48
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Global gene expression is dramatically altered by genomic rearrangements in Streptomyces ambofaciens RP181110. Partial genome mapping of two derivatives of strain RP181110 (strains NSA205 and NSA228) revealed rearrangements located in the unstable region of the genome (deletion in strain NSA228; deletion and amplification in strain NSA205). Computerized comparisons of pulse-labelled proteins separated by two-dimensional electrophoresis have revealed numerous differences in gene expression among the three strains during both exponential and stationary phases of growth: 31 proteins were absent in both mutant strains, 16 were absent only in strain NSA228, 17 were absent only in strain NSA205 and 9 were found to be present or over expressed in strain NSA205. Thus, in spite of the scarcity of genetic markers in the unstable region and its dispensability for growth under laboratory conditions, these results suggest that it includes genes which are actively expressed. Spontaneous gene amplifications, which occur frequently in this region of the chromosome, can further activate their expression.
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  • 49
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 10 (1993), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Microorganisms have numerous strategies for coping with environmental changes. In many systems, a single cell has the capacity to generate a seemingly infinite array of phenotypic variants in just a few generations of growth. The resulting heterogeneous population is well equipped for sudden environmental change; even if only a few cells in the population possess a phenotype needed for survival, these cells have the capacity to regenerate a similarly diverse population. Phenotypic switching in these systems usually results from high-frequency DNA rearrangements which are the subject of this review.
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  • 50
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Six putative ATP-binding motifs of SecA protein were altered by oligonucleotide-directed mutagenesis to try to define the ATP-binding regions of this multifunctional protein. The effects of the mutations were analysed by genetic and biochemical assays. The results show that SecA contains two essential ATP-binding domains. One domain is responsible for high-affinity ATP binding and contains motifs AO and BO, located at amino acid residues 102-109 and 198-210, respectively. A second domain is responsible for low-affinity ATP binding and contains motifs A3 and a predicted B motif located at amino acid residues 503-511 and 631-653, respectively. The ATP-binding properties of both domains were essential for SecA-dependent translocation ATPase and in vitro protein translocation activities. The significance of these findings for the mechanism of SecA-dependent protein translocation is discussed.
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  • 51
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Neisseria meningitidis (Nm) isolates from disease or during carriage express, on their outer membranes, one or more of a family of closely related proteins designated Opa proteins. In this study, we have examined the potential rotes of Nm Opa proteins in bacterial attachment and invasion of endothelial as well as epithelial cells and compared the influence of Opa proteins with that of Ope protein, which has been previously shown to increase bacterial interactions with eukaryotic cells. Several variants expressing different Opa proteins (A, B, D) or Opc were selected from a culture of capsule-deficient non-piliated bacteria of strain C751. Although the Opa proteins increased bacterial attachment and invasion of endothelial cells, Opc was the most effective protein in increasing bacterial interactions with these cells. In contrast, attachment to several human epithelial cells was facilitated at least as much by OpaB as Opc protein. OpaA was largely without effect whereas OpaD conferred intermediate attachment. OpaB also increased invasion of epithelial cells; more bacteria were internalized by Chang conjunctival cells compared with Hep-2 larynx carcinoma or A549 lung carcinoma cells. Monoclonal antibody reacting with OpaB inhibited bacterial interactions with the host cells. Opa-mediated interactions were also eliminated or significantly reduced in variants expressing capsule or those with sialylated lipopolysaccharide. These data are consistent with the notion that environmental factors controlling capsule and lipopolysaccharide phenotype may modulate bacterial interactions mediated by these OM proteins. In permissive microenvironments, some Opa proteins may be important in bacterial colonization and translocation in addition to Opc. The data also support the notion that Nm Opa may confer tissue tropism.
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  • 52
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The recent emergence of indolent and rapidly fatal drug-resistant strains of Mycobacterium tuberculosis has renewed interest in defining the molecular mechanisms of drug resistance in the tubercle bacilli. In this report, we have examined the mechanism of resistance to streptomycin (Sm) in M. tuberculosis through the cloning and nucleotide sequence analysis of the gene encoding the ribosomal SR protein (rpsL gene) from streptomycin-resistant strains and their streptomycin-sensitive parental strains. We have demonstrated that five singly SmRM. tuberculosis strains and an SmR isolate that has reduced sensitivity to multiple antibiotics have identical point mutations at codon 43 of the rpsL gene. Mutations at this same site confer SmR in Escherichia coli. In contrast, two other multiple drug-resistant M. tuberculosis strains that are resistant to Sm have rpsL genes that have the same nucleotide sequence as their drug-sensitive parent strains, suggesting that different resistance mechanisms are involved in these strains.
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  • 53
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The pheromone N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) regulates expression of bioluminescence in the marine bacterium Vibrio fischeri, the production of carbapenem antibiotic in Erwinia carotovora and exoenzymes in both E. carotovora and Pseudomonas aeruginosa. A characteristic feature of this regulatory mechanism in V. fischeri is that it is cell density-dependent, reflecting the need to accumulate sufficient pheromone to trigger the induction of gene expression. Using a lux plasmid-based bioluminescent sensor for OHHL, pheromone production by E. carotovora, Enterobacter agglomerans, Hafnia alvei, Rahnella aquatilis and Serratia marcescens has been demonstrated and shown also to be cell density-dependent. Production of OHHL implies the presence in these bacteria of a gene equivalent to luxl. Chromosomal banks from all five enteric bacteria have yielded clones capable of eliciting OHHL production when expressed in Escherichia coli. The luxl homologue from both E. carotovora (carl) and E. agglomerans (eagl) were characterized at the DNA sequence level and the deduced protein sequences have only 25% identity with the V. fischeri Luxl. Despite this, carl, eagl and luxl are shown to be biologically equivalent. An insertion mutant of eagl demonstrates that this gene is essential for OHHL production in E. agglomerans.
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  • 54
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Pseudomonas aeruginosa strain K437 is defective in the production of a 90 kDa ferripyoverdine receptor and is unable to grow in an iron-deficient medium in the presence of the non-metabolizable iron chelator 2,2′-dípyrídyl (0.25mM). An attempt to clone the ferripyoverdine receptor gene was made by complementing this growth defect. A number of clones restoring growth of K437 on dipyridyl-containing medium were obtained and several of these restored moderate expression of the 90 kDa receptor. A 5.5 kb Xhol-HindIII fragment derived from one of these clones was similarly capable of complementing the dipyridyl growth defect although it failed to restore expression of the 90 kDa ferripyoverdine receptor. Nucleotide sequencing of the 5.5 kb fragment revealed two large open reading frames (ORFs), designated ORFA and ORFB, which appeared to form an operon and were capable of encoding products of 41 kDa and 112 kDa, respectively. Using a phage T7-based expression system, products of 42 kDa and c. 108 kDa were produced from the cloned DNA, confirming that the ORFs were, indeed, expressed. The cloned ORFAB operon was inducible under conditions of iron limitation in both P. aeruginosa and Escherichia coli. In addition, mutants expressing ORFAB constitutively were constitutive for pyoverdine and ferripyoverdine receptor production suggesting that components of the pyoverdine-mediated iron-transport system are co-regulated with ORFAB. The predicted products of ORFA and ORFB showed significant homology to the Escherichia coli EnvC and EnvD polypeptides which are reportedly involved in septum formation. In addition, the ORFB product showed moderate homology to the CzcA polypeptide identified as a component of a membrane-associated plasmid-encoded cation efflux system in Alcaligenes eutrophus. Using in vitro mutagenesis and gene replacement, ORFA- and ORFB-deficient mutants of K372, the parent strain of K437, were constructed. These mutants were unable to grow on iron-deficient minimal medium containing 0.25 mM dipyridyl although they expressed the ferripyoverdine receptor and were proficient in pyoverdine-mediated iron uptake. Despite the homology of the ORFA and ORFB products to EnvC and EnvD, respectively, the ORFA-ORFB-deficient mutants were not defective in septum formation. Introduction of an ORFB mutation into the pyoverdine-producing strain ML5087 resulted in poor growth of the mutant in an iron-limited medium. The same mutation in a pyoverdine-deficient derivative of ML5087 did not adversely affect growth. These data are discussed in the context of a possible role in pyoverdine secretion for the ORFAB products.
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  • 55
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The genes encoding urease were cloned from Bordetella bronchiseptica and the 5.2 kb of DNA essential for expression analysed in a T7 RNA polymerase transcription-translation system. At least four poly-peptides with predicted molecular weights of 69 000, 26 000, 12 200 and 11 000 were found. Partial DNA sequence of the gene encoding the 69 000 Da polypeptide revealed high amino acid identity to the α-subunit of Proteus mirabilis urease, UreC and jack bean urease. A stable, unmarked deletion was constructed in this gene to create a urease-negative mutant of B. bronchiseptica. To assess colonization in a guinea-pig model, the urease-negative strain was inoculated with the urease-positive parental strain in a mixed infection. The urease-negative strain out competed the urease-positive strain in the trachea, lungs and caecum. We demonstrate that urease is not essential for B. bronchiseptica colonization of the guinea-pig respiratory and digestive tracts.
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  • 56
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 10 (1993), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The periplasm of Neisseria gonorrhoeae should be similar to other Gram-negative bacteria, but no published reports confirm this assumption. We used a periplasmic isolation procedure developed in Escherichia coli to release the periplasmic contents of N. gonorrhoeae. The resultant periplasmic extract lacked lipopolysaccharide, protein markers of inner or outer membranes, surface-radiolabelled protein components, or ribosomal proteins. The periplasmic extract contained a single haem protein believed to be a c-type cytochrome known to exist in the periplasm of other Gram-negative species, and retained significant alkaline phosphatase activity. The dominant protein species released in the periplasmic extract was the gonococcal homologue of elongation factor Tu, a major component released in similar periplasmic extracts of E. coli. These data showed that the extraction procedure selectively released periplasmic components and that the gonococcal periplasm was comparable to that of E. coli. Further analysis of the gonococcal periplasm may provide important insights into the physiology of this pathogen of humans.
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  • 57
    Electronic Resource
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    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 10 (1993), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The AmpR transcriptional activator for the chromosomal ampCβ-lactamase gene of Citrobacter freundii was found to interact with an operator sequence located in the 5′ half of the 38 bp region protected by AmpR in DNase I footprinting experiments. AmpR binding was associated with significant DNA bending of target DNA. A glycine to glutamic acid alteration at position 102 in AmpR converts AmpR into a transcriptional activator even in the absence of β-lactam inducer. AmpRG102E interacted with the operator binding sequence and induced DNA bending. A glycine to lysine alteration at residue 102 completely abolished the ability of AmpR to transcriptionally affect the ampC promoter, i.e. to repress in the absence of β-lactam inducer and induce in the presence of β-lactam. Nevertheless, AmpRG102K could repress the oppositely orientated ampR promoter. AmpRG102K could also specifically interact with the operator but the resulting complex migrated faster in gel retardation experiments and no significant DNA bending was observed.
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  • 58
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: A gene (VRP1) encoding a novel proline-rich protein (verprolin) has been isolated from the yeast Saccharomyces cerevisiae as a result of its hybridization to a chick vinculin cDNA probe. The deduced protein sequence contains 24% proline residues present as proline-rich motifs throughout the verprolin sequence. Several of these motifs resemble recently identified sequences shown to bind Src homology 3 (SH3) domains in vitro. Replacement of the wild-type VRP1 allele with a mutant allele results in strains that grow slower than wild-type strains and are temperature sensitive. The vrp1 mutants are impaired in both cell shape and size and display aberrant chitin and actin localization. We propose that verprolin is involved in the maintenance of the yeast actin cytoskeleton, through interactions with other proteins, possibly containing SH3 domains.
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  • 59
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Transcriptional levels of the Escherichia coli mioC and gidA genes, which flank the chromosomal origin of replication (oriC) and the dnaA gene, were correlated with the time of initiation of chromosome replication. The transcripts were measured either in dnaC2(ts) mutants that had been aligned for initiation of chromosome replication by a temperature shift or in synchronous cultures of cells obtained using the baby machine technique. In both types of experiments, mioC transcription was inhibited prior to initiation of chromosome replication and resumed several minutes after initiation. Conversely, gidA and dnaA transcription were both inhibited after initiation of replication, coincident with the period of hemimethylation of oriC DNA. It is proposed that mioC transcription prevents initiation of chromosome replication, and must terminate before replication can begin. It is further proposed that the eclipse period between rounds of replication, i.e. the minimum interval between successive initiations, encompasses the time required to methylate GATC sequences in newly replicated oriC plus the time required to terminate mioC transcription. Conversely, the active transcription of gidA and dnaA prior to initiation is consistent with their positive effects on initiation, and their shutdown after initiation could serve to limit premature reinitiation.
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  • 60
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Protective antigen (PA) of anthrax toxin forms ion-conductive channels in planar lipid bilayers and liposomes under acidic pH conditions. We show here that PA has a similar permeabilizing action on the plasma membranes of CHO-K1 and three other mammalian cell lines (J774A.1, RAW264.7 and Vero). Changes in membrane permeability were evaluated by measuring the efflux of the K+ analogue, 86Rb+, from prelabelled cells, and the influx of 22Na+. The permeabilizing activity of PA was limited to a proteolytically activated form (PAN) and was dependent on acidic pH for membrane insertion (optimal at pH 5.0), but not for sustained ion flux. The flux was reduced in the presence of several known channel blockers: tetrabutyl-, tetrapentyl-, and tetrahexylammonium bromides. PAN facilitated the membrane translocation of anthrax edema factor under the same conditions that induced changes in membrane permeability to ions. These results indicate that PAN permeabilizes cellular membranes under conditions that are believed to prevail in the endosomal compartment of toxin-sensitive cells; and they provide a basis for more detailed studies of the relationship between channel formation and translocation of toxin effector moieties in vivo.
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  • 61
    Electronic Resource
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    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 10 (1993), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Deletions or substitutions of amino acids at the carboxyl-terminus of the heat-labile enterotoxin B sub-unit (EtxB) affect its assembly into pentamers in a temperature-dependent manner. At 42°C, the mutations prevent the B subunits from achieving their final pentameric structure resulting in membrane association of the monomers. However, mutant B subunits produced at 30°C assemble, in the periplasm, into pentamers that remain stable when transferred to 42°C, indicating that the mutant pentamers are stable under conditions where their formation is inhibited. The mutant pentamers are, similarly to wild-type pentamers, SDS-resistant and stable, in vitro, at temperatures up to 65°C. This suggests that although the C-terminal amino acids are part of the subunit interface, they appear not to contribute significantly to the stability of the final pentameric complex, but are instead essential for the formation or stabilization of an assembly intermediate in the pentamerization process. Single second site mutations suppress the assembly defect of mutant EtxB191.5, which carries substitutions at its C-terminus. The Thr→IIe replacement at position 75 in the α2-helix probably restores the van der Waals contact between residues 75 and 101, which had been greatly reduced by the Met→Leu substitution at position 101 in the β6-strand of EtxB191.5. Interaction between the α2-helix and β6-strand which contains the c-terminus probably stabilizes a conformation essential for assembly and is therefore required for the formation of pentamers.
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  • 62
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: We have established the genomic cleavage map of Salmonella enteritidis strain SSU7998 using pulsed-field gel electrophoresis. The chromosome of 4600kb was analysed by XbaI (16 fragments), I-CeuI (7 fragments) and BlnI (12 fragments); the genome also contains a plasmid of 60 kb. Cleavage sites of I-CeuI, in the large subunit ribosomal RNA gene, are conserved from Salmonella typhimurium and Escherichia coli K-12, and the XbaI and BinI sites in glt-tRNA are also conserved, but other sites are less conserved. Transposon Tn10, located at 60 different positions in the chromosome of S. typhimurium, was transduced by bacteriophage P22 into S. enteritidis and the insertion mapped using the XbaI and BlnI sites on Tn10. Gene order in S. enteritidis is identical to S. typhimurium LT2 and similar to E. coli K-12 except for an inversion of 815 kb, which covers the terminus region including T1 and T2. Endpoints are in the NDZs, or non-divisible zones, in which inversion endpoints were not detected in experiments in E. coli K-12 and S. typhimurium LT2. This inversion resembles the inversion between S. typhimurium and E. coli, but is longer at both ends.
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  • 63
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 10 (1993), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The Escherichia coli chromosome is compacted into 40-50 negatively supercoiled domains. It has been proposed that these domains differ in superhelical density. Here, we present evidence that this is probably not the case. A modified Tn10 transposable element was inserted at a number of locations around the E. coli chromosome. This element, mTn10-plac-lacZ+, contains the lac operon promoter, plac, whose activity increases with increasing superhelical density, fused to a lacZ+ reporter gene. Although mTn10-plac-lacZ+ fusion expression varies as much as approximately threefold at different insertion sites, the relative levels of expression from these elements are unaffected by replacing plac with the gyrA promoter, pgyrA, which has a reciprocal response to changes in superhelical density. Importantly, topoisomerase mutations and coumermycin, which inhibits DNA gyrase activity, alter mTn10-plac-lacZ+ and mTn10-pgyrA-lacZ+ fusion expression in expected ways, showing that the elements remain responsive to supercoiling and that topoisomerase activity is required for maintaining superhelical density. Fusion expression is not affected by anaerobic growth or osmotic shock, two physiological conditions thought to alter supercoiling. The approximately threefold difference in mTn10-plac-lacZ- and mTn10-pgyrA-lacZ+ fusion expression observed at different sites may be explained by regional differences in chromosomal copy number that arise from bidirectional replication. Together, these results strongly suggest that the E. coli chromosomal domains do not differ in functional superhelical density.
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  • 64
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: It has been proposed that the four type IV pilin-like proteins that are required for extracellular protein secretion by the general secretory pathway (GSP) might assemble into a trans-periplasm complex resembling a type IV pilus. To test this idea, we examined the subcellular distribution and oligomeric state of PulG, one of the type IV pilin-like proteins required for pullulanase secretion in Klebsiella oxytoca. Fractionation of Escherichia coli cells carrying a single copy of each pul gene showed that PulG protein was located in two distinct envelope fractions corresponding to the outer and cytoplasmic membranes. The protein was partially released by treating the membranes with Triton X-100 + EDTA or at high pH, but not by Triton X-100 atone or by 8M urea, 6M guanidine hydrochloride or 1 M NaCl. Like type IV pilins, non-sedimentable PuiG that had been released from the membranes at high pH could be sedimented by centrifugation when the pH was lowered. Treatment of whole cells, sphaeroplasts or isolated membranes with a cleavable cross-linking agent produced mainly PulG homodimers. Previous studies showed that both PulO, which cleaves and N-methylates the PulG precursor, and PulE, a putative ATP-binding protein, share extensive sequence identity with proteins known to be required for type IV pilus processing and assembly. However, mutations which disrupted either pulE or pulO, or indeed the complete absence of all other components of the pullulanase secretion apparatus, had little or no effect on any of the properties of PulG protein described above. We conclude that there is no evidence that PulG protein assembles into a stable multiprotein complex or that processing of the PulG precursor causes a detectable change in its subcellular distribution.
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  • 65
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    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 10 (1993), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Lysyl-tRNA synthetases are synthesized from two distinct genes in Escherichia coli, lysS (constitutively) and lysU (inducibly); however, the physiological significance and the differential control mechanism of these two genes have been a long-standing puzzle. Recent studies have successfully uncovered a significant control mechanism of lysU expression, which involves the leucine-responsive regulatory protein (Lrp) and a translational enhancer element called‘downstream box'. Moreover, it is likely that there is a mechanism underlying co-ordinate expression of lysU with other genes outside the leucine-Lrp regulon under harsh conditions such as low pH and anaerobiosis. A possible mechanism of lysyl-tRNA synthetase expression and function is reviewed.
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  • 66
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Phosphorylation of the α subunit of eukaryotic initiation factor 2 (elF-2α) is one of the best-characterized mechanisms for down-regulating total protein synthesis in mammalian cells in response to various stress conditions. Recent work indicates that regulation of the GCN4 gene of Saccharomyces cerevisiae by amino acid availability represents a gene-specific case of translational control by phosphorylation of elF-2α, Four short open reading frames in the leader of GCN4 mRNA (uORFs) restrict the flow of scanning ribosomes from the cap site to the GCN4 initiation codon. When amino acids are abundant, ribosomes translate the first uORF and reinitiate at one of the remaining uORFs in the leader, after which they dissociate from the mRNA. Under conditions of amino acid starvation, many ribosomes which have translated uORFI fail to reinitiate at uORFs 2-4 and utilize the GCN4 start codon instead. Failure to reinitiate at uORFs 2-4 in starved cells results from a reduction in the GTP-bound form of elF-2 that delivers charged initiator tRNAiMet to the ribosome. When the levels of elF-2·GTP·Met-tRNAiMet ternary complexes are low, many ribosomes will not rebind this critical initiation factor following translation of uORF1 until after scanning past uORF4, but before reaching GCN4. Phosphorylation of elF-2 by the protein kinase GCN2 decreases the concentration of elF-2·GTP·Met-tRNAiMet complexes by inhibiting the guanine nucleotide exchange factor for elF-2, which is the same mechanism utilized in mammalian cells to inhibit total protein synthesis by phosphorylation of elF-2.
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  • 67
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Bschehchia coli Shiga-like toxin I is a type II ribosome-inactivating protein composed of an A subunit with RNA-specific N-glycosidase activity, non-covalently associated with a pentamer of B subunits possessing affinity for galabiose-containing glycolipids. The A subunit contains a single intrachain disulphide bond encompassing a hydrophilic sequence containing two trypsin-sensitive arginine residues. By analogy with other bacterial toxins it has been proposed that proteolytic nicking, deemed essential for a cytotoxic effect, occurs within this disulphide-bonded loop to generate the A1 and A2 fragments. Reduced A1 is then believed to translocate an internal membrane to inactivate protein synthesis in the cytosol. In this report, the disulphide-loop arginines of the SLT I A subunit were mutated to block the specific proteolysis presumed to occur. However, the mutant generated remained an effective toxin having similar catalytic activity to wild-type toxin and only a marginally reduced cytotoxicity towards cultured cells. We conclude that the disulphide-loop arginine residues are not the unique and essential processing sites previously assumed, but that processing may occur at alterNatlve accessible sites to compensate for loss of target sites within the loop.
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  • 68
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 10 (1993), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Haem A, a prosthetic group of many respiratory oxidases, is probably synthesized from haem B (protohaem IX) in a pathway in which haem O is an intermediate. Possible roles of the Bacillus subtilis ctaA and CtaB gene products in haem O and haem A synthesis were studied. Escherichia coli does not contain haem A. The CtaA gene on plasmids in E. coli resulted in haem A accumulation in membranes. The presence of CtaB together with ctaA increased the amount of haem A found in E. coli. Haem O was not detected in wild-type B. subtilis strains. A previously isolated B. subtilis CtaA deletion mutant was found to contain haem B and haem O, but not haem A. B. subtilis ctaB deletion mutants were constructed and found to tack both haem A and haem O. The results with E. coli and B. subtilis strongly suggest that the B. subtilis CtaA protein functions in haem A synthesis. It is tentatively suggested that it functions in the oxygeNatlon/oxidation of the methyl side group of carbon 8 of haem O. B. subtilis CtaB, which is homologous to Saccharomyces cerevisiae COX10 and E. coli CyoE, also has a role in haem A synthesis and seems to be required for both cytochrome a and cytochrome o synthesis.
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  • 69
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: During phosphate-limited growth the marine phycoerythrin-containing picoplanktonic cyanobacterium Synechococcus sp. WH7803 synthesizes novel polypeptides, including two abundant species of 100 kDa and 32kDa. The 32kDa polypeptide was localized to the cell wall, although in a related strain, Synechococcus sp. WH8103, it could be detected in both the cell wall fraction and the periplasm. The gene (designated pstS) encoding this polypeptide was cloned and shown to be present in a single copy. The deduced amino acid sequence indicated a polypeptide consisting of 326 amino acids with a calculated Mr of 33763. Comparison of this sequence with that obtained by microsequencing the N-terminus of the 32kDa polypeptide showed that the mature protein was synthesized as a precursor, the first 24 amino acid residues being cleaved between two alanine residues at positions 24 and 25. The amino acid sequence of the mature polypeptide showed 35% identity and 52% similarity to the periplasmic phosphate-binding protein (PstS) from Escherichia coli, including three regions of much stronger homology which, by comparison with E. coli PstS, are directly involved in phosphate binding. Northern blot analysis revealed a pstS transcript of 1.2 kb in RNA extracted from cells grown in Pi-replete conditions and one of 1.4 kb in considerably increased abundance under Pi-depleted conditions. Homologues of the pstS gene were detected in other marine phycoerythrin-containing Synechococcus strains, but not in freshwater or halotolerant species.
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  • 70
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: We have analysed a new gene, CEM1, from Saccharomyces cerevisiae. Inactivation of this gene leads to a respiratory-deficient phenotype. The deduced protein sequence shows strong similarities with β-keto-acyl synthases or condensing enzymes. Typically, enzymes of this class are involved in the synthesis of fatty acids or similar molecules. An analysis of the mitochondrial lipids and fatty acids shows no major difference between the wild type and deleted strains. Implying that the CBM1 gene product is not involved in the synthesis of the bulk fatty acids. Thus it Is possible that the CEM1 protein is involved in the synthesis of a specialized molecule, probably related to a fatty acid, which is essential for mitochondrial respiration.
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  • 71
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Two genomic sequences from the halophilic archaeon Haloferax mediterranei, where we had found PstI restriction-pattern modifications depending on the salinity of the growth medium, have been studied. A markedly salt-dependent differential expression has been detected in the nearby regions. Two of the open reading frames characterized correspond to two of the differentially expressed transcripts. In both cases the PstI sites were included in purine–pyrimidine alternancies suggestive of Z-DNA structures and located in non–coding regions with frequent repetitive motifs. A long alternating adenine-thymine tract also appears in the upstream regions of one of these open reading frames. A possible role of local DNA configuration in osmoregulation in this organism is discussed.
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  • 72
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: We report the purification of a minor Bordetella pertussis fimbrial subunit, designated FimD, and the identification of its gene (fimD.) FimD could be purified from the bulk of major fimbrial subunits by exploiting the fact that major subunit-subunit interactions are more stable in the presence of SDS than minor-major subunit interactions. To locate the gene for FimD, internal peptides of FimD were generated, purified and sequenced. Subsequently, an oligonucleotide probe, based on the primary sequence of one peptide, was used to clone fimD. The primary structure of FimD, derived from the DNA sequence of its gene, showed homology with a number of fimbrial adhesins. Most pronounced homology was observed with MrkD, a fimbrial adhesin derived from Klebsieila pneumoniae. These observations suggest that FimD may represent a B. pertussis fimbrial adhesin. With a fimD-specific probe we detected the presence of a fimD homologue in Bordetella parapertussis and Bordetella bron-chiseptica but not in Bordetella avium. Cloning and sequencing revealed that the B. parapertussis and B. bronchiseptica fimD product differed from the B. pertussis fimD product in 20 and 1 amino acid residues, respectively. Since B. bronchiseptica is normally not a human pathogen, but causes respiratory disease in a wide range of non-human mammalian species, this may suggest that FimD recognizes a receptor that is well conserved in mammalian species. An in-frame deletion in fimD completely abolished FimD expression and also affected the expression of the major subunits Fim2 and Fim3 suggesting that, in contrast to other adhesins that are minor components of fimbriae, FimD is required for formation of the fimbrial structure.
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