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  • 1
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    International journal of immunogenetics 16 (1989), S. 0 
    ISSN: 1744-313X
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: MHC class II molecules and self antigens, such as Mls, influence T-cell selection by clonal deletion of potentially self-reactive T cells. In order to examine the role of various class II molecules in the T-cell receptor-self antigen interaction, class II transgenic and recombinant mice were analysed for TCR expression. Our studies indicate that the Aα and Eα chains can present Mls gene products for the clonal deletion of Vβ6-bearing T cells, and that the Aαq chain is defective in this process. We have also shown that Eα Aβ heterodimer in transgenic and recombinant mice is expressed and functions to delete I-E reactive Vβ11 T cells, demonstrating again the role of the Eα molecule.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 2
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    International journal of immunogenetics 16 (1989), S. 0 
    ISSN: 1744-313X
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The localization of TNF genes on the short arm of chromosome 6 between HLA B and the complement genes focused attention to that genetic region which harbours many immunologically relevant genes and is also thought to hold susceptibility genes for a variety of autoimmune diseases that are linked to specific alleles of particular loci in the HLA D region. Since the recently established HLA-DR-DQ variation accounts only for part of the genetic susceptibility to insulin-dependent diabetes mellitus (IDDM) we searched for genomic variation of the tumour necrosis factor (TNF) alpha. We have identified a TNF-alpha restriction fragment length polymorphism (RFLP) with N coI and analysed diabetic patients including their families, controls and homozygous typing cell lines (HTC) defined by the 10th International Histocompatibility Workshop. Segregation analysis in families and HTC results show a strong linkage of the TNF-alpha 5.5 kb allele with DR types in particular with AIB8DR3. This tight linkage of TNF-alpha alleles with extended haplotypes and the significant increase of heterozygotes in patients could lead to some explanation of the DR3 association with a variety of autoimmune diseases particularly IDDM.
    Materialart: Digitale Medien
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  • 3
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    International journal of immunogenetics 16 (1989), S. 0 
    ISSN: 1744-313X
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The role of the MHC class II antigens in the activation of resting human B lymphocytes (B-Go) was examined with respect to both early and late events in the activation process.The (Ca2+)i induced by anti-IgM was enhanced in the presence of, or following pre-incubation with, an anti-MHC class II DR antibody (D1.12). Pre-incubation with a sepharose conjugated antibody (Seph.-D1.12) augmented the proliferation of B-Go in response to a sub-optimal concentration of anti-IgM.The 2D PAGE profile of B-Go differed from that of in vivo activated B lymphocytes. The 2D PAGE profile of B-Go activated by Seph.-D1.12 was not identical to the profile of B-Go activated by either anti-IgM or PMA.These data suggest that the activation of B-Go via the class II antigens shares part of the pathway of anti-IgM induced activation but does not follow an identical pathway.
    Materialart: Digitale Medien
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  • 4
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    International journal of immunogenetics 16 (1989), S. 0 
    ISSN: 1744-313X
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: We established the organization of the AKR Qa region and determined the sequence of the 44 and Q5 genes. Restriction mapping and genomic Southern blot analysis revealed that the AKR strain codes for only three H-2K homologous genes in this region. The AKR Q5 gene is not homologous to the Q5 gene of the C57BL strain, but is presumably allelic to the Q5 gene isolated from Balb/c. The organization and structure of the AKR Qa family is virtually identical to the Qa genes of the C3H mouse. The AKR Q5 gene, in contrast to other H-2K homologous Qa region genes, codes for a typical transmembrane region, and upon transfection into BHK cells, a 1.6 kb Q5 transcript is detected.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 5
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    International journal of immunogenetics 16 (1989), S. 0 
    ISSN: 1744-313X
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The metastatic B16 mouse melanoma shows a low cell surface expression of H-2Kb and H-2Db class I antigens on cells of both the high-metastatic line B16-F10 and the low-metastatic line B16-F1. Similarly, newly generated clones of these lines, having different metastatic properties, all express low levels of major histo-compatibility antigens. One of these clones, the high-metastatic F10.9, was transfected with H-2Kb genes to generate H-2Kb-expressing transfectants. The resulting clones showed reduced tumourigenicity and a low metastatic phenotype. Unlike the parental cells, H-2Kb-positive transfectants are potent inducers and sensitive targets of H-2Kb-restricted syngeneic cytotoxic T cells. Immunization of mice with H-2Kb-positive transfectants conferred protection against a subsequent challenge with Kb-positive transfectants but had only a small effect on growth and metastatic spread of parental cells.
    Materialart: Digitale Medien
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  • 6
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    International journal of immunogenetics 16 (1989), S. 0 
    ISSN: 1744-313X
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: In this report we demonstrate that lowered expression of the H-2 antigens on RadLV-induced tumour cells is a result of depressed levels of stable mRNA in these cells. Whether this observation is a result of lowered transcription or of mRNA instability is under investigation. In an effort to determine which viral sequences are essential for mediating both the H-2 regulatory function and the transforming function of RadLV, we have begun to assemble newly integrated proviral genomes from tumours. The restriction enzyme cleavage sites of four isolates are presented; these isolates differ substantially from RadLV genomes previously presented. One of these molecular clones is shown to encode a non-defective B-tropic, ecotropic virus which when reinjected into resistant mouse strains can mediate the up-regulation of H-2Dd antigen expression. Finally, possible mechanisms of H-2 regulation are discussed.
    Materialart: Digitale Medien
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  • 7
    ISSN: 1744-313X
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: Expression of beta human chorionic gonadotropin (βhCG) by bladder tumours has been shown to be associated with increased metastases and resistance to treatment with radiotherapy and chemotherapy. Preliminary results from typing frozen tumours using monoclonal antibodies against HLA determinants show reduced or lost expression of one or more antigens in two thirds of patients studied with a trend for more malignant behaviour and inability to generate tumour infiltrating lymphocyte expression using Interleukin-2 in those patients whose tumours demonstrate loss. In this series βhCG expression was only seen in a subgroup of those demonstrating loss of HLA antigen expression. Studies of βhCG secreting bladder cancer cell lines showed that it was possible to induce class II HLA antigen expression with gamma Interferon, and that this treatment but not alpha Interferon reduced βhCG production by the cell line.
    Materialart: Digitale Medien
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  • 8
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    International journal of immunogenetics 16 (1989), S. 0 
    ISSN: 1744-313X
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 9
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    International journal of immunogenetics 16 (1989), S. 0 
    ISSN: 1744-313X
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: mAb KUL/05, a novel murine monoclonal antibody, reacts with molecules displaying the typical tissue distribution and molecular profile of class II MHC antigens. An extensive scrutiny employing serological and immunochemical assays on DR homozygous and DRα- mutant cell lines has shown that this reagent displays some additional, interesting features, namely mAb KUL/05 (a) binds in a broadly monomorphic fashion to cells of DR1 through seven specifities, (b) recognizes a determinant shared by a large proportion of DR, DQ and DPβ chains from most haplotypes, in both their monomeric and α chain-associated forms, and (c) reacts with frozen, acetone-fixed, as well as conventional, formalin-fixed, paraffin embedded tissues. Thus, mAb KUL/05 is likely to represent a useful adjunct for the study of the expression of class II MHC products in normal and pathological tissue specimens.
    Materialart: Digitale Medien
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  • 10
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    International journal of immunogenetics 16 (1989), S. 0 
    ISSN: 1744-313X
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: Substrains of NZB mice have been compared by Southern blot analysis using several probes. The restriction fragment length polymorphism of probes derived from the Igh-V, Igk-V, Tcrα-C loci and of the long terminal repeat of the mouse mammary tumour virus revealed that NZB/BlLwPtIbm were grossly different from NZB/BlNJ and NZB/BlOla. Comparison with mouse strains of the Igk-V haplotypes a and d suggested that NZB/BiLwPtIbm contain genetic material of the C58 mouse strain.
    Materialart: Digitale Medien
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  • 11
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    International journal of immunogenetics 16 (1989), S. 0 
    ISSN: 1744-313X
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: Kaposi's sarcoma is associated with an increased frequency of HLA-DR5. The hypothesized model of a susceptibility gene in linkage disequilibrium with DR5 may be tested by haplotype analysis in familial Kaposi's sarcoma. Our finding of no common haplotype among afflicted members of a family provides evidence against the hypothesized linkage.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 12
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    International journal of immunogenetics 16 (1989), S. 0 
    ISSN: 1744-313X
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 13
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    International journal of immunogenetics 16 (1989), S. 0 
    ISSN: 1744-313X
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: Magnetic filtration of labelled cells as a way of separating leucocyte subpopulations was tested with a very simple and easy filtration device, using colloidal magnetite as the labelling reagent. In order to quantitate cell enrichment, a double label (both fluorescent and magnetic) was used, under conditions which labelled less than 10% of the cells in the initial sample. Up to 20 million cells were simply passed through a small magnetic filter with a hand-held syringe. Depletion of labelled cells in the suspension that passed through was threefold, and enrichment of labelled cells in the wash of the filter after its removal from the magnet was approximately fivefold. Factors which limited the quality of separation are discussed. Other, more preliminary, experiments found enrichments of 15–to 30-fold with the same colloidal magnetite and hand-held apparatus when the cell labelling system was more selective.
    Materialart: Digitale Medien
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  • 14
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    International journal of immunogenetics 16 (1989), S. 0 
    ISSN: 1744-313X
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: HLA class I phenotyping was performed using T-lymphocyte populations isolated by immunomagnetic beads (IMBs) coated with monoclonal antibodies with specificity for CD2, CD4 or CD8. The results were compared to those obtained using density gradient-separated lymphocytes (PBL). The typing trays were read by the automated simultaneous double-fluorescence (SDF) technique previously established in our laboratory using an Astroscan 2100 system. The aims of the present study were to establish whether the advantages of IMB lymphocyte separation and automated plate reading by SDF were complementary and whether the results obtained by IMB-SDF and PBL-SDF were concordant.Similarity coefficients for paired results obtained by IMB-SDF and PBL-SDF varied between 0.825 using anti-CD8-coated IMBs and 0.914 using anti-CD4-coated IMBs with a consistent excess of stronger results observed with the PBL-SDF technique. The variations observed did not result in incorrect phenotype assignment but would significantly influence a cross-matching test.These results illustrate the feasibility of using IMB-separated lymphocytes for HLA phenotyping by SDF.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 15
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    International journal of immunogenetics 16 (1989), S. 0 
    ISSN: 1744-313X
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: Langerhans cells (LC) play an important role in the skin immune system. They are bone marrow-derived and function as the only accessory and antigen-presenting cells in the skin. Several techniques for enriching these cells have been devised, and four, including density gradient centrifugation, use of cell sorter, panning and immunomagnetic separation, are discussed. It is concluded that the most satisfactory method for isolation of LC is based on density gradient centrifugation and the most satisfactory for depletion of epidermal cell preparations for LC is based on the immunomagnetic principle.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 16
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    International journal of immunogenetics 16 (1989), S. 0 
    ISSN: 1744-313X
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The bifunctional cross-linking reagent dithiobis (succinimidyl propionate) (DSP) was used to cross-link 125I surface-labelled glycoproteins from viable thymocytes. The cells were solubilized, and the cross-linked material immunoprecipitated and analysed by SDS-PAGE. When DSP cross-linked thymocyte material was immunoprecipitated with either anti-ThB or anti-Ly 5 monoclonal antibodies, and then cleaved, molecules with masses identical to Ly 5 (Mr180 kD) and ThB (Mr 16–18 kD) were obtained. However, if the cross-linker was not cleaved, the intact product had a molecular mass of 〉 200 kD. The identity of these co-precipitated, cross-linked moieties was formally proved by limited proteolysis peptide map analysis. The data indicated that the ThB and Ly 5 antigens were associated on the thymocyte cell surface but no such association could be found on peripheral lymphocytes. The ThB-Ly 5 interaction may indicate an association relevant to the differentiation of thymocytes.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 17
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    International journal of immunogenetics 16 (1989), S. 0 
    ISSN: 1744-313X
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: ABH and related antigens appeared a long time ago in the evolution of vertebrates on tissues in contact with the external environment, which suggests that the polymorphism given by these antigens might play a role in the relationships of the species with pathogens. However, they are also oncodevelopmental markers and some recent experimental data suggest that they might play a role in cell-cell recognition at some stages of development. This type of function is difficult to reconcile with the polymorphic nature of these markers unless one considers that the glycosyltransferases necessary for the synthesis of the active structures are encoded by various members of multigene families. Some non-polymorphic members of the families would have their expression limited in time and space during development, leading to the same antigenic patterns in every individual, and these could reappear in some tumours, while the expression of other polymorphic members (A/B/O, H/h, Se/se, Le/le), leading to a variety of antigenic phenotypes, would be expressed at later stages and remain so during the whole life of the individual. The corresponding antigens could disappear from some cancer cells. It is argued that the ABH and related antigens would have primarily been involved in cell-cell recognition phenomena. The polymorphism would have evolved later from gene duplication under environmental pressure, the expression on erythrocytes which occurred very late in evolutionary time probably being of very little biological significance.
    Materialart: Digitale Medien
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  • 18
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    International journal of immunogenetics 16 (1989), S. 0 
    ISSN: 1744-313X
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: CBA/J (H-2k) females, mated with DBA/2 J (H-2d) or DBA/1 J (H-2q) males, exhibit a high rate of fetal resorption. In contrast, when H-2 identical CBA substrains (i.e. CBA/Ca and CBA/N) are used, this phenomenon is not observed. On the other hand, before mating with DBA/2 J males, pre-immunization of CBA/J females with spleen cells coming from BALB/c J or (DBA/2 x BALB/c J) F1 males (and not from other H-2d identical males whatever their Mls alleles) has significantly decreased the fetal resorption rate. Thus, immunization against determinants other than classical H-2d (K, I, D, L) antigens (transmitted as a dominant character and different from Mls determinants) can elicit anti-abortive effects. Furthermore, it was observed that the spleen cell endowed with the anti-abortive effects was neither a T nor a B lymphocyte. In contrast, peritoneal cells were able to reproduce the phenomenon, indicating that it may be mediated by a cell of the macrophage-monocyte lineage. Finally, a first gestation was substituted for allo-immunization of CBA/J females. The anti-abortive effects of a first pregnancy by BALB/c J males (and not by other H-2k syngeneic or H-2d allogeneic males) was observed in the course of a second pregnancy sired by DBA/2 J males. These data can be interpreted in terms of maternal recognition of an antigen present on both macrophages and trophoblast cells and necessary for a successful gestation, which is coded for by genes outside the K, I, D, L regions.
    Materialart: Digitale Medien
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  • 19
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    International journal of immunogenetics 16 (1989), S. 0 
    ISSN: 1744-313X
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The myelo-ablative effects of high-dose therapy for refractory cancer can be overcome by the transplantation of bone marrow from an HLA-matched normal donor. Suitable donors are available for only one patient in three, and even minor disparities at HLA loci can produce graft-versus-host disease (GvHD) in transplant recipients. Depletion of T lymphocytes from the marrow in vitro can reduce the incidence and severity of GvHD.In this paper we review the use of immunomagnetic cell separation for the depletion of mature T cells from bone marrow. This procedure uses monoclonal antibodies to identify the target cells. These are then rosetted with anti-immunoglobulin-coated paramagnetic microspheres and collected by exposure of the marrow to a magnetic field. Factors impacting the efficiency of the separation, including choice of anti-immunoglobulin and monoclonal antibodies, incubation conditions and methods for residual cell detection, are outlined.The relative limitations and advantages of the method are discussed in relation to other techniques. It is concluded that the flexibility of the immunomagnetic procedure, in its ability to be used for both positive and negative selection of T-cell subsets, or for pan-T-cell depletion, could make it the method of choice in this application.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 20
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: We have generated a series of 3’deletions of a cloned copy of the bacteriophage Mu transposase (A) gene. The corresponding truncated proteins, expressed under the control of the λ Pl promoter, were analysed in vivo for their capacity to complement a superinfecting Mu4am phage, both for lytic growth and lysogeny, and for their effect on growth of wild-type Mu following infection or induction of a lysogen. Using crude cell extracts, we have also examined binding properties of these proteins to the ends of Mu. The results allow us to further define regions of the protein important in replicative transposition, establishment of lysogeny and DNA binding.
    Materialart: Digitale Medien
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  • 21
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The 7kb virulence Region-2 of the large (virulence) plasmid in Shigella flexneri 2a encodes several proteins required for invasion of intestinal epithelial cells. Insertion and deletion mutagenesis, DNA subcloning and SDS-polyacrylamide gel electro-phoresis of proteins synthesized in minicells demonstrated five genes in this region. They encode 24, 18, 62 (lpaB), 41 (lpaC) and 37 (lpaD)-kiloDalton (kD) proteins. Complementation of Tn5-induced mutations in Region-2 with the above plasmid constructs indicated that Region-2 consists of two operons and that the three lpa proteins are essential for the virulence phenotype. The transcriptional organization determined by Northern blotting, S1 nuclease protection and the effect of Tn5 insertions on expression of the lpa proteins revealed that Region-2 has three promoters that transcribe RNAs of 4.0, 4.5 and 7.5kb. The 4.0 kb RNA was the transcript for the operon encoding the 24, 18 kD, lpaB and C proteins and the 4.5 kb RNA for the ipsD gene. In addition, the full-length RNA of 7.5 kb which covers Region-2 supplemented full expression of the lpa proteins. The 7663 nucleotides of Region-2 were determined to confirm the five open reading frames encoding 23655, 17755, 62168, 41077 and 36660 Dalton proteins, respectively, and their regulatory sequences.
    Materialart: Digitale Medien
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  • 22
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: We have determined the complete nucleotide sequence of a 4.8 kilobase fragment encompassing the virA locus of the nopaline-type plasmid, pTiC58, of Agrobacterium tumefaciens. virA is composed of a single open reading frame of 2499 nucleotides, capable of encoding a protein of 91.3 kiloDaltons. A trpE::virA gene fusion was used to confirm the reading frame of virA. High nucleotide and amino acid sequence homologies were observed between pTiC58 virA and the virA sequences of three octopine-type plasmids. Strong homologies in amino acid sequence were observed between pTiC58 VirA and seven bacterial proteins which control various regulons. Two hydrophobic domains within VirA are also consistent with a model in which VirA acts as a membrane-bound sensor of plant signal molecules.
    Materialart: Digitale Medien
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  • 23
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The β-lactamase fusion vector, pJBS633, has been used to analyse the organization of penicillin-binding protein 3 (PBP3) in the cytoplasmic membrane of Escherichia coli. The fusion junctions in 84 in-frame fusions of the coding region of mature TEM β-lactamase to random positions within the PBP3 gene were determined. Fusions of β-lactamase to 61 different positions in PBP3 were obtained. Fusions to positions within the first 31 residues of PBP3 resulted in enzymatically active fusion proteins which could not protect single cells of E. coli from killing by ampicillin, indicating that the β-lactamase moieties of these fusion proteins were not translocated to the peri-plasm. However, all fusions that contained: ≥36 residues of PBP3 provided single ceils of E coli with substantial levels of resistance to ampicillin, indicating that the β-lactamase moieties of these fusion proteins were translocated to the periplasm. PBP3 therefore appeared to have a simple membrane topology with residues 36 to the carboxy-terminus exposed on the periplasmic side of the cytoplasmic membrane. This topology was confirmed by showing that PBP3 was protected from proteolytic digestion at the cytoplasmic side of the inner membrane but was completely digested by proteolytic attack from the periplasmic side. PBP3 was only inserted in the cytoplasmic membrane at its amino terminus since replacement of its putative lipoprotein signal peptide with a normal signal peptide resulted in a water-soluble, periplasmic form of the enzyme. The periplasmic form of PBP3 retained its penicillin-binding activity and appeared to be truly water-soluble since it fractionated, in the absence of detergents, with the expected molecular weight on Sephadex G-100 and was not retarded by hydrophobic interaction chromatography on Phenyl-Superose.
    Materialart: Digitale Medien
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  • 24
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: In order to develop a procedure for transformation of the industrial yeast Torulaspora delbrueckii, we have constructed a set of recombinant plasmids carrying Saccharomyces cerevisiae ARS and 2μm origin of replication and kanamycin-G418 resistance gene of Tn903(601) as a selective marker. In this paper we show that S. cerevisiae ARS vectors can replicate autonomously and that vectors bearing the whole S. cerevisiae 2 μ sequence yield stable transform ants. We also present evidence to show that 2μm vectors undergo an FLP-mediated inter- and intramolecular recombination, which suggests that T. delbrueckii can support the amplification and partition mechanisms of these plasmids.
    Materialart: Digitale Medien
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  • 25
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The process of spore formation in the Gram-positive bacterium Bacillus subtilis is a simple developmental system controlled by 50 or more genes. The complex pattern of regulatory interactions between these genes is beginning to be elucidated. spoVJ is a poorly characterized locus in which mutations affect spore development at a relatively late stage (Stage V). We have now cloned and physically characterized the SpoVJ locus, and analysed its expression by lacZ fusion. Expression of spoVJ is temporally delayed until about two hours after the initiation of sporulation. Its expression is also spatially restricted to the mother cell compartment; as such, it represents the earliest known mother-cell-specific event. Control of spoVJ transcription is complex: expression is dependent upon the products of all of the spo0 genes and on some of the spoII genes butitis independent of all later genes except spoIIID. As spoIIID mutations do not affect prespore development, this gene must be an important early determinant of mother-cell-specific gene expression.
    Materialart: Digitale Medien
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  • 26
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: Outer membranes from Haemophilus pleuropneumoniae grown under iron-replete and iron-restricted conditions in vitro were analysed by means of SDS-PAGE and immunoblotting. Iron restriction resulted in the appearance of two or more novel polypeptides in the molecular size range of 96–102 kD and an increased amount of a 79 kD polypeptide. These polypeptides were recognized by porcine immune sera indicating their production by H. pleuropnuemoniae during‘growth in vivo. Although soluble siderophore production could not be detected, growth of the organisms on an iron-restricted medium was enhanced by the presence of porcine transferrin but not by bovine or human transferrin. The results suggest that H. pleuropneumoniae possesses a specific transferrin receptor, perhaps in the form of an iron-regulated outer membrane protein.
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  • 27
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: In clinical isolates of Enterobacter cloacae, resistance to the newer β-lactam antibiotics often results from overproduction of a cephalosporinase encoded by the β-lactam-inducible ampC gene. Regulation of ampC is controlled by the divergently expressed activator gene, ampR, and a second unlinked locus. In this presentation we show that although Escherichia coli has IQst its ampR gene it has retained the second regulatory locus and that this comprises the bicistronic ampDE operon. Genetic and biochemical studies define the ampD gene as encoding a repressor for amp C transcription whereas the ampE gene product is a cytoplasmic membrane protein. Inactivation of the AmpD protein by mutation causes massive overproduction of cephalosporinase which, in E. cloacae, can terminate in therapeutic failure. In contrast, toss of AmpE results in a total block in induction, despite the presence of the activator, AmpR.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 28
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The gene encoding cytotoxin (ctx) was cloned from Pseudomonas aeruginosa 158 and the nucleotide sequence was determined. The structural gene of ctx encodes the procytotoxin of 286 amino acid residues with a molecular mass of 31681 Daltons. Procytotoxin was activated by removal of 20 amino acid residues from the C terminus with trypsin. The cloned ctx gene was not expressed in either an Escherichia coli strain or a cytotoxin non-producing strain of P. aeruginosa. An expression system for the ctx gene was constructed by placing the structural gene of ctx downstream of tac promoter on a broad host-range vector plasmid.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 29
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: Two nodulation regions from the symbiotic plasmid (pSym) of Rhizobium phaseoil CE-3 were identified. The two regions were contained in overlapping cosmids pSM927 and pSM991. These cosmids, in a R phaseoli pSym-cured strain background, induced ineffective nodules on Phaseolus vulgaris roots.Transconjugants of Rhizobium meliloti harbouring pSM991 induced nodule-like structures on bean roots, suggesting that this cosmid contains host-range determinants.Analysis of deletions and insertional mutations in the sequences of pSM991 indicated that the genes responsible for the induction and development of nodules in P. vulgaris are organized in two regions 20kb apart. One region, located in a 6.8kb Eco RI fragment, includes the common nodABC genes. The other region, located in a 3.5kb EcoRI fragment, contains information required for host-range determination.
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  • 30
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The expression of Escherichia coli type 1 fimbriae is phase-variable i.e. the bacterial cell is either fimbriated or non-fimbriated. The transition from one state to the other is caused by the change in configuration of an invertible DNA segment harbouring the promoter of the fimA gene. The position of this phase switch is controlled by two proteins, FimB and FimE, which mediate an ‘on’ or ‘off’ configuration of the switch, respectively. In this study, we have investigated how these proteins control the switch by means of fim-lac fusions on low-copy-number plasmids. It was found, by in trans and cis complementation, that the ratio of fimB to fimE and the total concentration of the gene products determine the configuration of the switch as well as the frequency of phase switching. It was also shown that transcription occurs from the promoter located at the phase switch when this is in the ‘off’ configuration. This suggests a regulatory mechanism, since the resulting transcript would be anti-sense to the fimE transcript.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 31
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The activation of the Agrobacterium virulence system is known to be induced by certain phenolic compounds. We have tested the vir-inducing ability of fifty compounds, by using a virB-lacZ gene fusion, and analysed the relationship between structure and activity of these compounds. In this way we have identified several new vir-inducers: coniferylalcohol, 3,5-dimethoxy-4-hydroxybenzene, homovanillic acid, ferulic acid, 3-ethoxy-4-hydroxybenzaldehyde and guaiacol, all of which are compounds with strong or moderate activity and four compounds with weak vir-inducing activity. In view of the specificity of vir-inducers, our data extended observations of others and enabled us to define the specific structural features of a vir-inducer molecule. In addition we show here that induction of the octopine Ti vir-genes is (i) optimal at 29° C and totally abolished at 37° C., and (ii) strongly inhibited at low concentrations of sodium chloride. The implications for plant transformation are discussed.
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  • 32
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: In Aspergillus nidulans, the gene prnB encoding the major proline transport system is one of a cluster of four genes necessary and sufficient for the utilization of proline as sole nitrogen and/or carbon source. The prn cluster has been cloned and the sequence and transcript map of the prnB gene are presented in this paper. The predicted translated sequence consists of 570 amino acids, resulting in a molecular weight of 63028 Daltons. Its hydropathy profile shows 10 hydrophobic segments typical of integral membrane proteins. No N-terminal hydrophobic signal peptide is present, the N-terminal and C-terminal ends of the protein being hydrophilic. Similar results were previously found for the arginine and histidine transporters of Saccharomyces cerevisiae, with which the prnB transporter shares regions of highly conserved amino acid sequences. Using S1 mapping and Northern blot analyses, we confirm the presence of a unique inducible prnB transcript of 1.9 kb.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 33
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: A 22 kb region of the 90kb virulence-associated plasmid, plP1350, of Typhimurium strain C52 was cloned into the mobilizable vector pSUP202, yielding plasmid plP1352. This recombinant plasmid restored full virulence to plasmidless strain C53 in a mouse model. Transposon Tn5 insertion mutagenesis demonstrated the existence of two DNA sequences in plP1352 designated VirA and VirB, both of which are essential for the expression of virulence. A recombinant plasmid containing only the VirA and VirB regions markedly increased the virulence of the plasmidless strain C53, but did not confer full virulence. These results suggested that a third virulence-associated region might be present on plP1352. Eleven proteins encoded by the 22 kb insert sequence of pl P1352 were identified in Escherichia coli SE5000 maxicells. The VirA region encoded at least two proteins with apparent molecular weights of 71000 and 28000 and the VirB region encoded two proteins of 43000 and 38000.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 34
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: A collection of transposon-induced mutants of Rhizobium meliloti 1021 defective in siderophore-mediated iron assimilation were obtained and classified as biosynthetic, transport or regulatory. Several of the mutations were cloned and the adjacent sequences were used to acquire complementing DNA from the wild type. A single genomic region of about 35kb complemented all of the mutants deficient in production of the siderophore.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 35
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The complete nucleotide sequence of the xynA gene coding for a xylanase (XYLA) expressed by Pseudomonas fluorescens subspecies cellulosa, has been determined. The structural gene consists of an open reading frame of 1833 bp followed by a TAA stop codon. Confirmation of the nucleotide sequence was obtained by comparing the predicted amino acid sequence with that derived by N-terminal analysis of purified forms of the xylanase. The signal peptide present at the N terminus of mature XYLA closely resembles signal peptides of other secreted proteins. Truncated forms of the xylanase gene, in which the sequence encoding the N-terminal signal peptide had been deleted, still expressed an enzyme which was secreted in Escherichia coli. XYLA contains domains which are homologous to an endoglucanase expressed by the same organism. These structures include serine-rich sequences. Bal31 deletions of xynA revealed the extent to which these conserved sequences, in XYLA, were essential for xylanase activity. Downstream of the TAA stop codon is a G + C-rich region of dyad symmetry (δG = 24kcal) characteristic of E. coli Rho-independent transcription terminators.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 36
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The rodC1 mutation of Bacillus subtilis is a temperature-sensitive marker which affects the levels of teichoic acid synthesis and the cellular morphology. We have determined the nucleotide sequence of the bicistronic operon which contains the rodC gene and the nucleotide sequence of the rodC1 mutant allele. The temperature-sensitive phenotype of the rodC mutant is the result of a single base-pair change. A cytosine to thymine transition in the non-coding strand results in the replacement of a serine residue in the wild-type protein with a phenylalanine residue in the mutant protein. The other gene in the operon, the rodD gene, appears to be equivalent to the gtaA gene which encodes uridine diphosphate-glucose poly-(glycerol phosphate) α-glucosyl transferase, an enzyme involved in techoic acid synthesis. This is the first nucleotide sequence analysis of both the wild-type and mutant alleles of a morphogene in B. subtilis.
    Materialart: Digitale Medien
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  • 37
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: Iron limitation, a condition encountered within mammalian hosts, induces the synthesis of a number of proteins in pathogenic Shigella species. These include several outer membrane proteins, Shiga toxin, and proteins involved in the biosynthesis and transport of high-affinity iron-binding compounds or siderophores. Although siderophores have been shown to play a major role in the virulence of some bacterial pathogens, these compounds do not appear to be essential for the virulence of Shigella species. Unlike those pathogens which are restricted to the extracellular compartments of the host, the Shigella species invade and multiply within host cells. Alternative iron-acquisition systems, such as the ability to utilize haem-iron, permit growth of the intracellular bacteria. Virulent shigellae also possess a cell-surface haem-binding protein, and synthesis of this protein correlates with infectivity and virulence. This protein does not appear to be involved in iron acquisition. Rather, it may allow the bacteria to coat themselves with haem compounds, thus enhancing their ability to interact with target host cells.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 38
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: We have constructed a novel promoter probe plasmid pSB40, containing a unique lac-α-tetracycline marker gene tandem, which allows for both positive and negative selection of active promoters. Promoters cloned in pSB40 can be readily mobilized as EcoR1 cassettes. Using this vector we have performed a non-invasive analysis of the E. coli chromosome for promoters regulated by osmotic upshift. Only one such promoter, subsequently identified as part of the proU operon, was isolated. A sequence of 253bp, sufficient to mediate osmotic regulation of the proU promoter, was defined. This E coli promoter was normally regulated in Salmonella typhimurium, Klebsiella and Citrobacter but not in Shigella. A proU-luxAB fusion plasmid was constructed and used to monitor in vivo real-time kinetics of proU induction following osmotic upshock.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 39
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The transcription initiation site of the meloperon from Streptomyces glaucescens, determined by S1 mapping and primer elongation experiments, lies 32 to 34bp upstream of the translation initiation codon of the first open reading frame. A total of 172 to 219bp upstream of the transcription start point are necessary for a fully active and regulated mel promoter. Deletion analysis, gel retardation assays and DNAse I footprint experiments facilitated division of the promoter into three functional domains, which include the RNA polymerase recognition site up to nucleotides -33 to -42, the binding region of a protein of assumed regulatory function between nucleotides -65 and -93, and an upstream activator site, located between positions -158 and -219.
    Materialart: Digitale Medien
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  • 40
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The cloned Citrobacter freundii ampC β-lactamase is inducible in the presence of its regulatory gene ampR in Escherichia coli (Lindberg et al., 1985). The basal level of expression and inducibility are affected by two E. coli proteins encoded by the closely linked ampD and ampE genes. Deletion of both genes led to constitutive ampR-dependent overproduction of β-lactamase, whereas an out-of-frame deletion in AmpD caused the basal expression to increase twofold. This ampD1 mutant was inducible at lower β-lactam concentrations than the wild type. An IS1 insertion in ampD was polar on ampE expression and increased basal β-lactamase expression 30-fold while mediating a semi-constitutive phenotype. AmpE expressed from a recombinant plasmid in an ampD ampE deletion mutant reduced basal β-lactamase expression to wild-type levels but did not markedly reduce β-lactam resistance since the cells became hyperinducible. in the absence of AmpD, increasing levels of AmpE therefore decrease the basal expression of AmpC β-lactamase in an AmpR-dependent manner. AmpD modulated the response exerted on β-lactamase expression by AmpE. The ampD gene encodes a 20.5kD cytoplasmic protein while the 32.1kD ampE gene product is an integral membrane protein with a likely ATP-binding site between the second and third putative transmembrane region. Since neither AmpD nor AmpE are needed for β-lactam induction and since these proteins could not be covalently labelled by benzylpenicillin, they are not thought to act as β-lactam-binding sensory tranducers. Instead it is suggested that AmpD and AmpE sense the effect of β-lactam action on peptidoglycan biosynthesis and relay this signal to AmpR.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 41
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: Reverse transcriptase, discovered in 1970 in retroviruses, has until recently been found only in eukaryotic organisms. Recently it was shown to occur in two groups of bacteria: myxobacteria and Escherichia coli. The gene for reverse transcriptase is part of a chromosomal genetic element that codes for the production of a branched DNA-RNA compound. In this compound a single-stranded DNA is connected to RNA at a specific G residue by a 2′-5’phosphodiester linkage. The precursor for the DNA-RNA compound is a folded messenger RNA, in which the specific G residue is the initiation point for reverse transcription. In the final DNA-RNA compound, the portion of the RNA transcribed by reverse transcriptase is eliminated by RNase H. The DNA-RNA compound is present in several hundred copies per cell. Its biological function is unknown at present.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 42
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The translational initiation rates directed by the translational initiation regions (TIRs) of the atpB, atpH, atpA and atpG genes of Escherichia coli were investigated using lacZ fusions present on plasmids as well as integrated into the chromosome. This was the first investigation of the translational efficiency of the atpB gene, whose unfused product (subunit a) can be toxic to the cell. The specific mRNA levels, rates of in vivo protein synthesis and β-galactosidase activities encoded by the atp:: lacZ fusions were compared in order to obtain valid estimates of relative translation rates. The results indicate that in the E. coli atp operon, translation directed by the atpB, atpH and atpG TIRs is less efficient than that directed by the atpA TIR, and are thus consistent with earlier measurements of direct atp gene expression. Initiation is, however, to differing extents, controlled by coupling to the translation of upstream neighbours. There is particularly tight coupling between atpH and atpA. Increasing the distance between these two genes whilst maintaining the original atpA TIR structure decreased the degree of coupling. The influence of manipulations of the atpG TIR structure upon translational efficiency was quantitatively more pronounced when the atpG fusions were present as a single copy per chromosome. This is likely to be related to the mRNA binding characteristics of 30S ribosomal subunits and/or to the influence of other (trans-acting) factors. The control of independent and coupled initiation at the atp TIRs is discussed in relation to mRNA structure and possible CiS and trans regulatory phenomena.
    Materialart: Digitale Medien
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  • 43
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: We describe the construction of an RFLP-based genetic map of the white rot fungus Phanerochaete chrysosporium ME446. The map is deduced from the allele distributions of 38 RFLP markers in a test set of 53 meiotically derived haploid recombinants of this strain. The map includes a cellulase gene, a mating-type locus and a family of lignin peroxidase (and related) genes that are arranged in two unlinked clusters.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 44
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The nifA gene has been identified between the fixX and nifB genes in the clover microsymbiont Rhizobium leguminosarum biovar trifolii (R.I. bv. trifolii) strain ANU843. Expression of the nifA gene is induced in the symbiotic state and site-directed mutagenesis experiments indicate that nifA expression is essential for symbiotic nitrogen fixation. Interestingly, the predicted R.I. bv. trifolii NifA protein lacks an N-terminal domain that is present in the homologous proteins from R.I. bv. viciae, Rhizobium meliloti, Brady rhizobium japonicum, Klebsiella pneumoniae and all other documented NifA proteins. This indicates that this N-terminal domain is not essential for NifA function in R.I. bv. trifolii.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 45
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: We have determined the DNA sequences of eight different insertions of IS91 in a specifically engineered recipient plasmid of known DNA sequence (pSU300). The sequences at the termini of IS91 are 5′ -CGAG-TAGG… CCTATCGAT. IS91 inserts specifically 5′ to either one of the tetranucleotides 5′-GAAC or 5′-CAAG, and always in the same relative orientation with respect to the sequence of the target. Except in one special case, no duplications of the recipient DNA were produced at the site of insertion.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 46
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: Hybrid 5′ regulatory regions were constructed in which the upstream activator sequence (UAS) and promoter of various nif genes were exchanged with the upstream regulatory sequence (URS) of the fdhF gene from Escherichia coli. They were analysed for their regulatory response under different growth conditions with the aid of fdhF′-′lacZ or nif′-′lacZ fusions. Placement of the UAS from the Bradyrhizobium japonicum nifH gene in front of the spacer (DNA region between URS and promoter) plus promoter from fdhF renders fdhF expression activatable by the Klebsiella pneumoniae NIFA protein, both under aerobic and anaerobic conditions. This excludes the possibility that the spacer of the fdhF 5’flanking region contains a site recognized by a putative oxygen- or nitrate-responsive repressor. There was also considerable activation by NIFA of fdhF expression in a construct lacking the nifH UAS but containing the fdhF spacer plus promoter. Further experimental evidence suggests that this reflects a direct interaction between NIFA and RNA polymerase at the nfrA-dependent promoter. A second set of hybrid constructs in which the URS from fdhF (E. coli) was placed In front of the nifD spacer plus promoter from B. Japonicum or in front of the K. pneumoniae nifH, nifU, nifB spacers and promoters, delivered inactive constructs in the case of the nifD, nifU and nifB genes. However, a nifH′-′lacZ fusion preceded by its own spacer and promoter plus the foreign fdhF URS displayed all the regulatory characteristics of fdhF expression, i.e. anaerobic induction with formate and repression by oxygen and nitrate. Although it is not known why only one out of the four nif promoters could be activated by the fdhF URS, this result nevertheless demonstrates that the various regulatory stimuli affecting expression of fdhF in E. coli have their target at the upstream regulatory sequence.
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  • 47
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The Rhizobium meliloti nod region lib is involved in host-range determination: (i)the presence of region lib is necessary for transfer of alfalfa root hair curling ability to Rhizobium legumlnosarum biovar trifolii; (ii) a mutation in region llb extends the R. meliloti infection host range to Vicia sativa nigra; (iii) dominance of R. meliloti nod genes over R. leguminosarum biovar viciae nod genes is abolished by mutations in region llb. The nucleotide sequence of this region has been determined. Genes corresponding to the two open reading frames identified are designated nodP and nodQ. The predicted amino acid sequence of the NodQ protein shows homology with translation initiation and elongation factors. The consensus sequence involved in the GTP-binding domain is conserved.
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  • 48
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The nucleotide sequence of the Escherichia coli enterobactin biosynthesis gene entD has been determined. entD specifies a predicted 23579 Dalton protein containing several helical regions, a trans-membrane segment and one positively charged domain. The EntD polypeptide was overexpressed and identified in electrophoretic gels as a membrane protein. Although results of conventional membrane fractionation techniques were inconclusive, protease accessibility studies provided evidence that EntD domains are exposed on the inner leaflet of the cytoplasmic membrane. The presence of repetitive extragenic palindromic (REP) sequences within the fepA-entD intercistronic region was confirmed. Lack of a canonical promoter and an iron control region 5’to entD, along with RNA hybridization data, suggest that an iron-regulated transcript contains both fepA and entD.
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  • 49
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: Expression and phase variation of Neisseria gonorr-hoeae P.ll genes in Escherichia coli viere studied using TnphoA fusions. Fusions were created in the P.llc gene of N. gonorrhoeae JS3 using λTnphoA-1 and were characterized by restriction digestion and dideoxy sequencing. Three fusions were chosen for further study; Tnp7 (fusion junction at mature amino acid 7), Tnp57 (amino acid 57), Tnp66 (amino acid 66). All fusions were in frame with the P.llc coding sequence but were out of frame with the purported initiation codon. All fusion constructions were shown to phase vary in E. coli in an analogous fashion to that seen in N. gonorrhoeae, i.e. phase changes (in a recA background) at a frequency of c. 10−3 accompanied by an alteration at the DNA level of the number of coding repeats (CRs). In vitro mutagenesis of the fusion constructions indicated that expression of out of frame P.ll genes in E. coli was probably the result of ribosomal frameshifting within the run of ‘A’ residues immediately preceding the CR region and not due to low-level false initiation at codons other than the ATG initiation codon (as had previously been suggested). The mechanism for P.llc::phoA phase variation appears to be related to the ‘slipped-strand mispairing’ mechanism responsible for frameshift mutations in a number of other bacterial genes containing short, direct, tandem repeats.
    Materialart: Digitale Medien
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  • 50
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: srnB is an F-plasmid encoded gene, otherwise silent, whose expression is induced by added rifampicin, leading to the release of cellular Mg2+ and degradation of stable RNA. In the absence of rifampicin, transcripts from the srnB gene were relatively short. S1 nuclease mapping revealed that the short mRNA species terminated within the leader, at the 3’end of a potential stem-and-loop structure. A deletion in the stem-loop resulted in constitutive synthesis of the mRNA that extended beyond the termination site into the structural gene. Even with the wild-type gene, transcription continued beyond the terminator sequence in the presence of added rifampicin. Most of the transcripts synthesized in the presence of rifampicin were long enough to encode the srnB protein. We hypothesize from these results that RNA polymerase associated with rifampicin can read through the terminator to induce srnB expression.
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    Standort Signatur Erwartet Verfügbarkeit
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  • 51
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The effect of temperature on coagulase and fibrinolysin expression (Pla) by Yersinia pestis has been implicated in the transmission of plague by fleas. In an attempt to improve our understanding of this process, we have cloned, sequenced and characterized the gene encoding the Pla phenotypes in Y. pestis, and examined its temperature-dependent regulation. The coding region for this gene overlaps a 900bp Y. pestis-specific DNA fragment that we have previously shown to be capable of detecting plague bacilli in fleas. The pla gene contains a single open reading frame encoding 312 amino acids with a predicted molecular weight of 34.7 kD and a putative signal sequence of 20 amino acids. This coding region appears to be sufficient for both coagulase and fibrinolytic activities. In Y. pestis, modulation between coagulase and fibrinolytic activities is temperature-dependent: coagulase activity is most evident at temperatures below 30°C but fibrinolytic activity increases with higher temperatures (〉30°C), regardless of the temperature at which the bacteria are grown. Our results lead us to believe that this regulation occurs post-translationally. It is possible that the alternative forms of the Pla protein are essential to ‘flea blockage’ and subsequent transmission of the plague bacillus to animals.
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  • 52
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: Plasmid pLAX3, carrying the colicin E3 gene, was used to direct the in vitro synthesis of a colicin E3*molecule totally devoid of its immunity protein. We established that this molecule is able to kill sensitive Escherichia coli cells in the total absence of immunity protein. Therefore, all of the information required for colicin E3 action is located on the colicin potypeptide itself. Furthermore, our studies indicated that immunity protein protects the C-terminal enzymatic part of native colicin E3 protein against proteolytic degradation before or during its translocation across the cell envelope. These results are discussed in relation to the mode of entry of colicin E3 into bacterial cells.
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  • 53
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The DNA encoding the zinc metalloprotease of Legionella pneumophila Philadelphia 1 has been isolated and expressed in Escherichia coli. This protein, which is 38000 Daltons in size, possesses immunological and biochemical properties identical to those previously described for the purified L. pneumophila protease. Periplasmic extracts of E. coli clones expressing the recombinant protease are also capable of causing the haemolysis of canine erythrocytes and the cytotoxic destruction of CHO cells. Using trans-poson mutagenesis, it was determined that a maximum of 1.2 kb of DNA encoded all three biological activities. Inactrvation of proteolytic activity by trans-poson insertion occurred concomitantly with losses of the haemolytic and cytotoxic phenotypes. A putative regulatory sequence approximately 200-500bp upstream of the gene's coding region was identified. A 4.0 kb fragment encoding these activities hybridized to the chromosomal DNA of the parent strain of L. pneumophila Philadelphia 1 as well as clinical isolates of L. pneumophila.
    Materialart: Digitale Medien
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  • 54
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: A 5.1 kbp DNA fragment was isolated which complemented C4-dicarboxylate transport mutants (dct) of Rhizobium meliloti. Characterization of this fragment by subcloning, transposon mutagenesis, and complementation analysis revealed three loci, designated dctA, dctB, and dctD. TnphoA-generated alkaline phosphatase fusions to dctA suggested that this gene encodes the structural transport protein and allowed the determination of its direction of transcription. Analysis of the fusions in various mutant backgrounds demonstrated that dctB, dctD, and ntrA products are required for dctA expression. The dctA fusion was constitutively expressed in a dctA mutant background, but was not expressed in dctA dctB or dctA dctD double mutants. This suggests that the constitutive expression in a dctA mutant background is mediated through dctB and dctD. Three independent second-site Dct+ revertant mutations in ntrA mutant strains mapped to the dct locus. Succinate transport in these revertant strains was constitutive, whereas in the wild type, succinate transport was inducible. These results are consistent with the direct requirement of the ntrA gene product for dctA expression. Alfalfa plants inoculated with the dctB and dctD mutants showed reduced nitrogen-fixing activity. Nodules induced by dctA mutants failed to fix nitrogen. These symbiotic phenotypes are consistent with previous suggestions that dctA expression in bacteroids can occur independently of dctB and dctD.
    Materialart: Digitale Medien
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  • 55
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: A computer search for repeated sequences led us to identify five REP (repetitive extragenic palindromic) sequences in the 3′ -terminal region of the Escherichia coli ribonucleoside diphosphate reductase gene (nrdA). These REP sequences are located within a putative duplicated DNA region, the first of them being part of the carboxy-terminal coding region of the nrdA gene. This is the first report of a REP sequence within a structural gene and also the first example of a REP sequence apparently generated by DNA duplication.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 56
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The nucleotrde sequence of the Azorhizobium caulinodans ORS571 nlfA locus was determined and the deduced NifA amino acid sequence compared with that of NifA from other nitrogen-fixing species. Highly conserved domains, including helix-turn-helix and ATP-binding motifs, and specific conserved residues, such as a cluster of cysteines, were identified. The nifA 5’upstream region was found to contain DNA sequence motifs highly homologous to promoter elements involved in nifAlntr-mediated control and a consensus element found in the 5’upstream region of the Bradyrhizobium japonicum 5-aminolevulinic acid synthase (hemA) gene and of Escherichia coli genes activated during anaerobiosis via the fnr (fumarate nitrate reduction) control system. A nifA-lac fusion was constructed using miniMu-lac and its activity measured in different genetic backgrounds and under various physiological conditions (in culture and in planta.) NifA expression was found to be negatively autoregulated, repressed by rich nitrogen sources and high oxygen concentrations, and controlled (partially) by the ntrC gene, both in culture and in planta. DNA supercoiling was also implicated in nifA regulation, since DNA gyrase inhibitors severely repressed nifA-lac expression.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 57
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: In this paper we describe the chromosomal location of various loci in Erwinia chrysanthemi strain 3937. Auxotrophic markers were obtained by chemical mutagenesis, antibiotic resistances were isolated spontaneously and mutations in sugar utilization were obtained by means of Mu insertions. These markers were located on the genetic linkage map of strain 3937 by using a conjugative system mediated by RP4::mini-Mu plasmids which permitted transfer of genetic material from any point of origin. The location of these markers was compared to that of previously located mutations. Many genes involved in pectinolysis were also located on the E. chrysanthemi 3937 map. These results permitted us to present a new genetic map containing 61 markers distributed over 34 widely scattered loci on the chromosome. Some pairs of markers giving high cotransfer frequencies were tested for cotransduction mediated by the generalized transducing phage phi-EC2; nine cotransducing pairs were found. It appears that the chromosomal locations of many of these loci are quite different to those of the well-known enterobacterium Escherichia coli but seem similar to those described for other E. chrysanthemi strains.
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  • 58
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The pathogenic Neisseria have multiple genes encoding proteins that bind monoclonal antibody (MAb) H.8. We previously reported the cloning and sequencing of a meningococcal gene (laz) encoding an H.8 Mab-binding protein with a consensus lipoprotein processing site, an N-terminal domain containing the epitope for H.8 MAb binding, and a C-terminal domain with extensive similarity to the sequences of azurins from other organisms. In the current study, we showed that the product of the cloned gene could be labelled with palmitic acid, that it was subject to globomycin-sensitive processing, and that it was immunologically cross-reactive with azurin from Pseudomonas aeruginosa. All neisserial species tested, both pathogens and commensals, produced a protein recognized by anti-azurin serum. Southern blots with oligonucleotide probes specific for the azurin domain of the gene showed that it was present in a single copy in the chromosome; it was highly conserved in gonococci and meningococci, and less conserved in commensal Neisseria species.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 59
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: FNR, the transcriptional regulator of gene expression of anaerobic respiration in Escherichia coli, contains a cluster of cysteine residues at the amino terminus which resembles the metal-binding domains of metal-binding proteins. It is possible, therefore, (i) that FNR binds metals with the cysteines as ligands and (ii) that this property is related to the regulatory function of FNR. These questions were investigated, with the following results.Approximately 2.4 of the 4 cysteine residues of FNR can be alkylated with iodoacetate in permeabilized aerobic or anaerobic bacteria without the addition of reducing agents. The time required for half-maximal labelling of the cysteines was 50 min in anaerobic bacteria and 6 min in aerobic bacteria. The difference in the reactivity was specific for the cysteines of FNR. These cysteine residues were also highly reactive in anaerobically grown bacteria, when the growth medium contained chelating agents such as 1,10-phe-nanthroline (15 μM). The effect of the chelating agents was reversed by an excess of divalent metal ions such as Fe(11) or Cu(11) in the medium. The presence of 1,10-phenanthroline (10 μM) also inhibits the expression of fumarate reductase, an FNR-dependent enzyme. These results suggest that FNR exists in two different forms which differ in terms of the reactivity of their cysteine residues to iodoacetate. The interconversion of both forms appears to be regulated by the availability of O2and by the binding of metal ions. The two forms of FNR may be involved in the regulation of O2-dependent gene expression.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 60
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The induction of several amino acid decarboxylases under anaerobic conditions at low pH has been known for many years, but the mechanism associated with this type of regulation has not been elucidated. To study the regulation of the biodegradative arginine and lysine decarboxylases of Escherichia coli K12, Mudlac fusions to these genes were isolated. Mudlac fusion strains deficient for lysine decarboxylase or arginine decarboxylase were identified using decarboxylase indicator media and analysed for their regulation of β-galactosidase expression. The position of the Mud-lac fusion in lysine decarboxylase-deficient strains has been mapped to the cadA gene at 93.7 minutes, while the Mudlac fusions exhibiting a deficiency in the inducible arginine decarboxylase have been mapped to 93.4 minutes.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 61
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The ndh gene of Escherichia coli which encodes an NADH dehydrogenase contains a putative FNR-binding site in its upstream non-coding region, and its expression has been investigated using an ndh-lacZ fusion. Expression of the fusion was found to be reduced during anaerobic growth, and experiments with hosts containing an fnr mutation and/or a multicopy fnr+ plasmid indicated that the anaerobic repression of the ndh gene is mediated by the FNR protein. Thus FNR can function as an anaerobic repressor as well as an anaerobic transcriptional activator. The results are consistent with the FNR-binding function attributed to the proposed consensus sequence. Using frdA-and ndh-lacZ fusions exhibiting positive and negative regulation by FNR, it was further shown that the depletion of metal ions in growth media with chelating agents mimics oxygen with respect to the activity of FNR. Possible roles for metal ions in the oxygen-sensing pathway associated with FNR function are discussed.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 62
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The expression of plasmid-encoded, invasion-related antigens lpa b, c and d of Shigella flexneri was found to be positively regulated at transcriptional level by a 33kD protein produced by the previously defined, virulence-associated Region 1 on the SalI fragment B of the 230 kb invasion plasmid. The gene (designated virB) was identified and its nucleotide sequence determined. No Ipa b or c was produced in the absence of an intact virB gene although lower levels of d were produced. The previously reported regulatory activity of the virF gene some 30 kb distance away was shown to act exclusively through virB. In contrast, the activation of the virG gene necessary for intercellular spread occurred directly by virF without the requirement for virB. This study thus ascribes a critical function to a previously recognized, but functionally undefined, virulence locus on the large invasion plasmid of S. flexneri. The virF gene appears to have a central role in activation of the 230kb plasmid-encoded virulence genes.
    Materialart: Digitale Medien
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  • 63
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The gene encoding monophosphatidylinositol inositol phosphohydrolase (PI-specific phospholipase C, PI-PLC) of Bacillus thuringiensis was cloned in Staphylococcus carnosus TM300. The complete coding region comprises 987 base pairs corresponding to a precursor protein of 329 amino acids (molecular weight, 38095). The NH2-terminal sequence of the purified enzyme from Escherichia coli indicated that the mature PI-PLC consists of 299 amino acid residues with a molecular weight of 34586. Polyacrylamide gel electrophoresis revealed the same molecular weight for the purified enzyme isolated from the DNA-donor strain of B. thuringiensis and from the E. coli clone. By computer analysis, the secondary structure was predicted. The enzyme from the E. coli recombinant shows no activity on other phospholipids and sphingo-myelin. The cleaving specifity of PI-PLC was examined by thin layer chromatography.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 64
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: Four transformant strains of Neisseria gonorrhoeae were generated, two of which (WS3 and WS5) had protein I subclass A (P.IA) and two which (WS2 and WS4) had protein I subclass B (P.IB). Analysis of the strains demonstrated that the two P.IA-bearing strains differed in lipooligosaccharide (LOS) and H.8 antigen, as assessed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immuno-blotting. The WS5 strain had slow-migrating LOS and H.8 antigen, and the WS3 strain had fast-migrating LOS and H.8 antigen. The P.IB-bearing strains also had either slow-migrating LOS and H.8 antigen (WS4) or fast-migrating LOS and H.8 antigen (WS2). Structural and exposure analysis revealed that although the P.IAs were identical in the WS3 and WS5 strains, there was a slight alteration of the exposure of the proteins which correlated with altered LOS and/or H.8 antigen. The P.IBs were also shown to be structurally identical, but the LOS and/or H.8 antigen variation in these strains correlated with a more pronounced alteration in the exposure of the P.IB molecules. The differences in protein I (P.I) exposure were generally found in highly negatively charged regions of the molecule, suggesting that the immunogenicity and/or antigenicity of the P.I molecules may vary as a result of LOS and/or H.8 antigen alterations.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 65
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: An apo form of cytochrome cd1, (nitrite reductase) of Paracoccus denitrificans has been detected immunologically in the periplasm of a mutant that lacks all c-type cytochromes. A method for the preparation of apo-nitrite reductase (lacking both c- and d-type haem) from the holoenzyme of wild-type cells has been developed. The apoprotein synthesized by the mutant is indistinguishable from the chemically prepared apoprotein in respect of: (i) subunit molecular weight; (ii) formation of a homodimer; (iii) properties on anion exchange chromatography. The holoenzyme has similar properties in respect of (i) and (ii) but behaves differently during anion exchange. A suggested mode of assembly of cytochrome cd1, is trans-location into the periplasm of a precursor polypeptide, maturation by a signal peptidase to give an apoprotein identical to that prepared chemically from the holoenzyme, followed by insertion of c-type and d-type haem in an as yet unknown order.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 66
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: Aprotein II (P.II) gene from Neisseria gonorrhoeae was cloned in Escherichia coli and characterized by DNA sequence analysis. As with other reported P.II sequences, this gene contains an ATG initiation codon which is out of frame with respect to the remainder of the P.II amino acid sequence. A translational fusion was constructed in E. coli which linked the P.II sequence to the signal peptide of β-lactamase. This P.II fusion differs from the gonococcal protein only in the first seven residues at the N terminus. In E. coli, the P.II fusion product exhibits properties analogous to those of P.II in N. gonorrhoeae. The P.II fusion product is a major component of the E. coli outer membrane and it is exposed on the cell surface. The P.II fusion protein also exhibits the heat-modifiable phenotype of gonococcal P.II.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 67
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: A tribrid gene containing ompF, faeC, and lacZ sequences was constructed by subcloning a large central segment of the K88ab gene encoding the fibrillar subunit-like protein FaeC into the open reading frame expression vector pORF2. The resulting tribrid protein was isolated and used to raise antibodies against the FaeC protein. These antibodies were then used for the detection and subcellular localization of the FaeC protein in Escherichia coli harbouring the K88ab-encoding plasmid pFM205 or mutant derivatives. Immunoblotting of subcellular fractions and of purified fibrillae, and agglutination experiments using whole cells revealed that the FaeC protein is present in the periplasm and as a minor component in the K88ab fibrillae. FaeC was also detected in purified K88ac and K88ad fibrillae. Immunoelectron microscopy confirmed the presence of FaeC in K88ab fibrillae, particularly at the tips of the longer fibrillae.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 68
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The complete nucleotide sequence of the gene for a cell-surface protein antigen (PAc) of Streptococcus mutans MT8148 (serotype c) was determined. The pac gene consisted of 4695 bp and coded for a 170 773 D protein. The pac gene product contained a putative 38 amino acid signal peptide, resulting in a 166817D mature protein. A potential promoter sequence and a putative Shine-Dalgarno sequence preceded the open reading frame. Two internal repeating amino acid sequences were present in the PAc. One repeating region located in the N-terminal region was rich in alanine, and the other located in the central region was rich in proline. Southern blot analysis under the less stringent condition (allowing up to 35% base mismatch) revealed that the probe covering the prolinerich region hybridized to DNA preparations from strains of Streptococcus cricetus, Streptococcus sobrinus and Streptococcus downei as well as Streptococcus mutans.
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  • 69
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The topology of the integral membrane protein Cai (colicin A immunity protein), which is required to protect producing cells from the pore-forming colicin A, was analysed using fusions to alkaline phosphatase. The properties of these fusion proteins support the model for Cai topology previously proposed on theoretical grounds. The protein was found to contain four transmembrane sequences and its N-and C-terminal regions were found to be directed towards the cytoplasm. Oligonucleotide-directed mutagenesis and sequence comparisons between Cai, Cbi (colicin B immunity protein), and Cni (colicin N immunity protein) were carried out to determine the functional regions of Cai. The possible roles of the various regions of Cai in its protective function and in its topological organization are discussed.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 70
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: Seven genes coding for two different enzymes of the penicillin/cephalosporin biosynthetic pathway have been cloned from fungal and bacterial sources. Using amino acid sequences derived from the purified enzymes, oligonucleotide probes were designed to hybridize to their cognate genes in a genomic library. The high degree of similarity (57%) between enzymes of bacterial and fungal origin suggests a horizontal transfer of a primordial beta-lactam pathway, probably from a bacterial cell to a fungal cell. Overproduction of the proteins in Escherichia coli has allowed further study of the mechanism of action of these important enzymes.
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  • 71
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: We show, using gel retardation, that crude Escherichia coli cell extracts contain a protein which binds specifically to DNA fragments carrying either end of the phage Mu genome. We have identified this protein as Fis, a factor involved in several site-specific recombinational switches. Furthermore, we show that induction of a Mucte62 prophage in a fis lysogen occurs at a lower temperature than that of a wild-type strain, and that spontaneous induction of Mucfs62 is increased in the fis mutant. DNasel footprinting using either crude extracts or purified Fis indicate that binding on the left end of Mu occurs at a site which overlaps a weak transposase binding site. Thus, Fis may modulate Mu growth by influencing the binding of transposase, or other proteins, to the transposase binding site(s), in a way similar to its influence on Xis binding in phage λ.
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  • 72
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The nucleotide sequence has been determined for the Streptococcus mutans wall-associated protein A (wapA) gene from serotype c strains Ingbritt and GS5. The nucleotide sequence for each wapA gene was virtually identical, although the gene from strain GS5 contained a 24 base pair deletion. A 29 amino acid signal peptide was specified by each wapA gene with a mature protein of 424 amino acids (Mr, 45276) for strain Ingbritt and 416 amino acids (Mr, 44846) for strain GS5. In the C-terminal region of the wall-associated protein A, considerable sequence similarity was found with the membrane anchor region of proteins from other Gram-positive organisms such as the group A streptococcal M protein and the group G streptococcal IgG binding protein. Adjacent to the proposed membrane anchor is a highly hydrophilic region which may span the cell wall; both sequence data and experimental evidence indicate the existence of a region immediately outside the wall at which proteolytic cleavage occurs to release antigen A of Mr 29000 into the culture supernatant. Thus, the wall-associated protein A is a precursor of the 29000 Mr antigen A.
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  • 73
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The ospA and ospB genes encode the major outer membrane proteins of the Lyme disease spirochaete Borrelia burgdorferi. The deduced translation products from the ospA and ospB genes were: (OspA) 273 amino acids long with a molecular weight of 29334, and (OspB) 296 amino acids long with a molecular weight of 31739. The two Osp proteins showed a great degree of sequence similarity indicating a recent evolutionary event. Molecular analysis and sequence comparison of OspA and OspB with other proteins revealed a sequence similarity to the signal peptides of prokaryotic lipoproteins. These are the first sequences from Borrelia and provide interesting data on the evolutionary relationship between spirochaetes and other species as well as providing potential for spirochaete diagnostics and vaccines.
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  • 74
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: Klebsiella pneumoniae strain PAP996 was previously shown to secrete fatty acylated, aggregated (micellar) pullulanase only after the end of exponential growth. Here we show that the closely related strain K21 secretes large amounts of unacylated, non-aggregated (monomeric) pullulanase during exponential growth. Only a small amount (〈10%) of the secreted pullulanase was initially retained by the exponentially growing cells to be subsequently secreted in a fatty acylated, aggregated form. Despite the absence of fatty acids in secreted monomeric pullulanase, the effects of the antibiotic globomycin on pullulanase maturation indicated that all of the enzyme synthesized by strain K21 is processed by lipoprotein signal peptidase.
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  • 75
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: Cells of the thermoacidophilic bacterium Bacillus acidocaldarius express a high-affinity K+-uptake system when grown at low external K+. A vanadate-sensitive, K+ - and Mg2+ -stimulated ATPase was partially purified from membranes of these cells by solubilization with a non-ionic detergent followed by ion-exchange chromatography of the extract. Combinations of non-denaturing and denaturing electrophoretic separation methods revealed that the ATPase complex consisted of three subunits with molecular weights almost identical to those of the KdpA, B and C proteins, which together form the Kdp high-affinity, K+ -translocating ATPase complex of Escherichia coli. The affinity of the partially purified ATPase from B. acidocaldarius for its substrates K+ (Km 2–3 μM) and ATP (Km 80 μM), its stimulation by various divalent cations, and its inhibition by vanadate (Ki 1–2 μM), bafilomycin A1 (Ki, 20 μM), DCCD (Ki 200 μM) or Ca2+ were also similar to those of the E coli enzyme, indicating that the two K+ -translocating ATPases have almost identical properties.
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  • 76
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The DNA sequence of a 3.6kb region downstream of the nagB gene (encoding glucosamJne-6-PO4-deaminase) in Escherichia coli has been determined. Three open reading frames, which are subsequently referred to as nagA, nagC and nagD, were detected in this sequence. Genetic complementation and enzyme assays have shown that the first of these, nagA, encodes N-acetyl glucosamine-6-phosphate deacetylase. Growth on N-acetyl glucosamine induces the synthesis of a 1900 nucleotide long transcript which covers just nagE, encoding EIINag which is transcribed divergently from nagB, and of a 4200 nucleotide long transcript which covers all four ORFs of the nagB, A, C, D operon. More mRNA corresponding to nagB and nagA is detected than that corresponding to the distal genes, nagC and nagD. Considerable amounts of the induced mRNA are truncated molecules having their 3’ends after nagB and after nagA. Multiple 3′ RNA ends have been mapped after nagD and seem to correspond to the ends of transcripts stabilized by mRNA secondary structure (REP sequences) rather than transcription termination sites. A second promoter producing napD-specific transcripts has been mapped just in front of the nagD gene.
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  • 77
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The Yop proteins of Yersinia are important virulence determinants. The Yop1 protein sequences of Yersinia pestis, Yersinia pseudotuberculosis, and two Yersinia enterocolitica serotypes, O:3 and O:8, deduced from the nucleotide sequences of the corresponding yopA genes, were compared. Most differences were found in the hydrophilic domains of the proteins, whereas the hydrophobic domains were conserved. The amino acid sequences revealed a signal sequence 25 amino acids long. No cysteine residues were present, even though Yop1 forms a polymeric structure.The transcription startpoint of yop4 was determined by primer extension. The coding region and transcription startpoint were separated by a leader sequence 270 nucleotides long. The yopA promoter sequence of Y. pestis is identical to the corresponding sequence of Y. pseudotuberculosis and transcription studies revealed that this promoter is active in Y. pestis. Thus, the inability of Y. pestis to express the Yop1 protein is due to a single base pair deletion In the coding region of the yopA gene of Y. pestis (Rosqvist et al., 1988).
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  • 78
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The proline catabolism gene cluster of Aspergillus nidulans was cloned using a ‘brute force’ technique which detects clones hybridizing to restriction fragments overlapping chromosomal rearrangements. A number of deletion mutations and a translocation mutation in the cluster have been physically mapped, and an excellent correlation between the genetic and physical maps was established. Transcripts have been identified and orientated for each of the four genes of the cluster. All are monocistronic by size. All of the transcripts, including that of the regulatory gene prnA, are inducible. Using deletion endpoints and mRNA sizes, approximate gene positions on the physical map have been determined. Finally, the relationship between genetic and physical distance across the cluster has been estimated at 3-4 kilobases per centiMorgan.
    Materialart: Digitale Medien
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  • 79
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The region of the antibiotic resistance plasmid R100 that encodes the plasmid-specific transfer gene traM has two tandemly aligned promoters separated by 145 nucleotides. The principal transcripts are 705 and 562 nucleotides long. Minor transcripts are 1550 and 1700 nucleotides long. The 705-base transcript appears to encode an 11 kD traM protein. The 562-base transcript does not encode a detectable protein. When sub-cloned on short fragments, the promoter for the 562-base transcript initiates efficiently but that for the 705 site does not. The 3′ ends of the 705 and 562 base transcripts end inside the traJ ORF. Thus they provide additional sense RNA to compete with traJ for finP, the antisense translational regulator of traJ. A model is proposed for the participation of these sense and antisense transcripts in the control of expression of the traJ gene.
    Materialart: Digitale Medien
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  • 80
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: Two regions of homology to Anabaena nifH (nitrogenase Fe protein) were detected in the total DNA of the thermophilic nitrogen-fixing archaebacterium Methanococcus thermolithotrophicus. A 2.8 kb Hindlll fragment carrying one of these regions was previously cloned and shown to contain a nifH gene (Souillard et al., 1988) now referred to as 0RFnifH2. A 3.4kb Pstl fragment and an overlapping 3.B kb BglU fragment, containing the second region of homology, were cloned, and a DNA region of 4073bp was sequenced. It contained four complete open reading frames (ORFs) (ORFnifH1, ORF105, ORF128, ORFnifD) and two truncated ORFs (ORFnifK and ORF96). Five ORFs were transcribed in the same direction in the order of ORFnifH1-ORF105-ORF128-ORFnifD-ORFnifK. ORFnifH1, ORFnifD and ORFnifK were assigned from their similarity to eubacterial nifH and nifDK (nitrogenase MoFe protein) genes. Transcription studies showed that ORFnifH1 and ORFnifD were expressed only under nitrogen-fixation conditions, whereas no ORFnifH2 mRNA was detected under the same conditions. A DNA probe containing ORFnifH1 hybridized with a 1.8 kb mRNA, as detected by a Northern blotting experiment. A transcriptional start site was localized 87 and 88 bp upstream from the ATG codon of ORFnifH1, This site is preceded, 21 bp upstream, by the sequence 5′-TTTATATA-3′ already found at the same position in several archaebacterial promoters. ORFnifH1 mRNA was too small to encode ORFnifDK. This was confirmed by the fact that another transcription start site was localized 85bp upstream from the ATG codon of ORFnifD.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 81
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The level of DNA supercoiling is crucial for many cellular processes, Including gene expression, and is determined, primarily, by the opposing actions of two enzymes: topoisomerase I and DNA gyrase. Escherichia coli strains lacking topoisomerase I (topA mutants) normally fail to grow in the absence of compensatory mutations which are presumed to relax DNA. We have found that, in media of low osmolarity, topA mutants are viable in the absence of any compensatory mutation, consistent with the view that decreased extracellular osmolarity causes a relaxation of cellular DNA. At higher osmolarity most compensatory mutations, as expected, are in the gyrA and gyrB genes. The only other locus at which compensatory mutations arise, designated toc, is shown to involve the amplification of a region of chromosomal DNA which includes the tolC gene. However, amplification of tolC alone is insufficient to explain the phenotypes of toc mutants. tolC insertion mutations alter the distribution of plasmid topoisomers in vivo. This effect is probably indirect, possibly a result of altered membrane structure and an alteration in the cell's osmotic barrier. As tolC is a highly pleiotropic locus, affecting the expression of many genes, it is possible that some of the TolC phenotypes are a direct result of this topological change. The possible relationship between toe and tolC mutations, and the means by which tolC mutations might affect DNA supercoiling, are discussed.
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  • 82
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 83
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: Many genes of Escherichia coli have been shown to be sensitive to DNA superhelicity. The superhelicity of the chromosome is itself also supercoiling-dependent. We have developed a general strategy for investigating how a particular gene responds to changes in DNA topology. This approach is used to study the E. coli ligase gene. The thermosensitivity of the E. coli ligts251 mutation can be phenotypically suppressed by mutations which map close to, or in, the gyrB gene and which affect the degree of DNA supercoillng. The level of suppression correlates with the degree of DNA relaxation observed, suggesting that the gene encoding the E. coli DNA ligase is activated by relaxation of the chromosomal DNA.
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  • 84
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The gene encoding plastocyanin (petE1) from Anabaena sp. PCC 7937 was isolated using two sets of mixed oligonucleotide hybridization probes derived from conserved regions in the protein. Plastocyanin is encoded as a preprotein of 139 amino acids. The amino-terminal extension of 34 residues has all the characteristics of a signal peptide and is probably involved in translocation of preplastocyanin over the thylakoid membrane. The level of the petE1 mRNA, a single transcript of about 740 bases, was found to be severely reduced under conditions of Cu2+ deficiency. The petE1 gene was transferred to the genome of Synechococcus sp. PCC 7942, which did not appear to contain a structural gene for plastocyanin itself. The integrated gene becomes expressed at the transcriptional level, regardless of the amount of Cu2+ available.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 85
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The phytopathogenic enterobacterium Erwinia chrysanthemi secretes a number of enzymes involved in plant-tissue degradation, notably the five isoenzymes of pectate lyase. We have cloned a region involved in pectate lyase and cellulase secretion by complementation of non-secretory outJ mutants of E. chrysanthemi strain 3937 using the RP4::miniMu plasmid pULB110. The cloned region maps near the ade-22 marker on the E. chrysanthemi 3937 chromosome. An R-prime containing a chromosomal DNA insert of about 30 kb was first obtained; subcloning into pBR325 permitted the isolation of a 4 kb Cial/Sspl fragment able to complement outJ mutations in E. chrysanthemi. The isolation of phoA fusions in this fragment allowed us to determine the direction of transcription of the encoding region, which extends over about 2.5 kb, and demonstrate that this region encodes exported protein(s). When the TnphoA insertions were transferred back into E. chrysanthemi chromosome, the recombined strains no longer secreted pectate lyases or cellulases. Identification of the products encoded by the Cial/Sspl fragment demonstrated that oufJ encodes an 83 kD polypeptide which is processed to an 81 kD polypeptide by cleavage of a signal sequence. The cloned DNA fragment did not endow Escherichia coli with the ability to secrete pectate lyases.
    Materialart: Digitale Medien
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  • 86
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The gene cluster (rfb region) which determines the synthesis of O101 lipopolysaccharide (LPS) O-antigen was cloned from the Escherichia coli O101:K99:F41 reference strain B41 to give plasmid pPM1301. The smallest subclones represented by pPM1305 and pPM 1330expressed O-antigen in E. coli K-12 similar to (but not identical to) B41, as judged by immunogold electron microscopy and silver staining of LPS separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). At least six proteins were detected by minicell analysis of proteins encoded by pPM1305, which suggests that O-antigen synthesis is genetically complex.Restriction and deletion analysis demonstrated that a minimum of 8.9 kb and a maximum of 11.8 kb are required for O101 O-antigen biosynthesis in E. coli K-12. Examination of LPS banding patterns of other O101 isolates by SDS-PAGE suggested heterogeneity of LPS structure. Southern DNA hybridization analysis using radiolabelled subclones of pPM1305 demonstrated that there was a close relationship among the 0101 ETEC isolates.
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  • 87
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The genes determining the biosynthesis of the colonization factor CS5 have been cloned from Escherichia coli 0115:H40:PCF8775 isolated during an outbreak of diarrhoea among aboriginal children in Central Australia. Electron microscopy has shown purified CS5 to be of semi-rigid fimbrial type. NH2-terminal analysis has shown the CS5 determinant to be distinct from other fimbriae, although there is some conservation of certain residues. Expression in minicells of the cloned fimbrial genes encoded on pPM1312 has shown that proteins of 70 and 46.5 kD which co-purify with the 23kD major fimbrial subunit protein are also co-expressed along with proteins of 45, 31, 17 and 14kD. The major CS5 subunit is synthesized in precursor form (approximately 26 kD). A synthetic oligonucleotide to the NH2-terminal amino acid coding sequence of the purified protein has been used in Southern hybridization analyses to define the region on pPM1312 encoding the structural gene for the major pilin subunit
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  • 88
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The major outer membrane protein (MOMP) is the prime candidate for the development of a chlamydial vaccine. Antibodies to the subspecies-specific epitope neutralize chlamydial infection. Monoclonal antibodies (MAbs) to this epitope were prepared either by immunization with whole chlamydiae or with a 16 amino acid synthetic peptide. The critical binding site on the subspecies epitope for these MAbs was determined to single amino acid resolution using several hundred solid-phase peptides. A frame shift of just one amino acid in critical binding site completely prevented antibody binding to viable chlamydiae. A single MAb to whole organisms was capable of spanning both the surface-exposed, conformation-dependent, subspecies epitope and a buried, conformation-independent species epitope some 10 Å distant. Immunization with peptide generated an MAb with reduced binding constraints which permitted the antibody to bind with broadened species-specificity at the subspecies binding site. The results show for the first time the importance of both critical binding site and conformation at the subspecies epitope. We suggest that the conformational flexibility of short, epitopic peptide vaccines may in some cases be advantageous, giving rise to extended specificity not attained with the natural protein.
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  • 89
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: Protein G (also designated Fc receptor type III) is the IgG-binding protein of group C and G streptococci. Protein G has also been shown to bind human serum albumin but at a site that is structurally separated from the IgG-binding region. From the known gene sequence of protein G, two synthetic oligonucleotides were constructed for use as probes in DNA-hybridization experiments to study the structure and distribution of the albumin- and IgG-binding regions in bacterial strains belonging to different species. Thus, one of the probes corresponded to repeats within the IgG-binding region (I) and the other corresponded to repeats in the albumin-binding encoding region (II). Probe I showed strong hybridization to DNA isolated from 31 human group C and G strains, whereas hybridization to probe II was variable. With the three restriction endonucleases used, three restriction patterns were found in Southern blot experiments. No fundamental difference could be detected in hybridization experiments, either between strains of group C and G streptococci, or between isolates of different clinical origin. No hybridization to DNA from other bacterial species was found.
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  • 90
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: Interaction of the basement-membrane binding O75X adhesin of uropathogenic Escherichia coli with various extracellular matrix proteins was studied. The adhesin showed strong binding to type IV collagen immobilized on microtitre plates, whereas other collagens, laminin and fibronectin, were only weakly recognized. Similarly, specific binding of [125I]-labelled type IV collagen to 075X-positive bacteria was shown. Interaction of the two proteins was also demonstrated by affinity chromatography of the O75X adhesin on immobilized type IV collagen. The adhesin bound strongly to the immobilized N-terminal 7S domain of type IV collagen, and the binding of [125I]-labelled type IV collagen to O75X-positive bacteria was inhibited by the soluble 7S domain. Binding of O75X to type IV collagen and to its 7S domain was specifically inhibited by chloramphenicol but was not affected by periodate or endoglycosidase-H treatment of the glycoproteins. Our results show that the 7S domain of type IV collagen is the basement membrane receptor for the O75X adhesin and suggest an interaction based on protein-protein recognition. Inhibition of the interaction by chloramphenicol favours the supposition that a modified tyrosine is involved in the binding site.
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  • 91
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: A series of expression plasmids containing either the complete insert from plasmid pUCDBKI (Peoples et al., 1987) or sub-fragments thereof were constructed in a tac promoter vector. Analysis of protein lysates of induced cultures of these clones identified the gene encoding NADPH-specific acetoacetyl-CoA reductase in the 2.3 kb of sequence located downstream from the β-ketothiolase gene in plasmid pUCDBK1. The complete nucleotide sequence (2.1 kb) of this region was determined. An open reading frame was located 88 bp downstream from the stop codon of the thiotase gene encoding a potential polypeptide of M, 25000, which is in good agreement with that observed for the overexpressed protein on SDS-PAGE. N-terminal protein sequence data obtained by Edman degradation of the purified Mr= 25000 polypeptide were used to identify the correct start of the NADPH-specific acetoacetyl-CoA reductase gene. Hence in Z. ramigera, the genes encoding β-ketothiolase (phbA) and NADPH-specific acetoacetyl-CoA reductase (phbB) are organized as phbA-phbB. S1-nuclease analysis of Z. ramigera RNA identified a transcription start site 85 bp upstream from the phbA structural gene locating the promoter region.
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  • 92
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: Streptococcus lactis NCDO763 harbours a plasmid designated pLP763. The cells harbouring pLP763 are able to grow to a higher density in milk because of their proteinase-positive phenotype (Prt). The 6.2kb HindIII-Pstl fragment from pLP763 was found to be responsible for the Prt+ phenotype. The DNA fragment contains an incomplete large open reading frame (ORF). Further sequence analysis downstream from the Pstl site revealed that the ORF consists of 5706 bases. It was found that the deduced amino acid sequence consisting of 1902 amino acid residues was extremely similar to that of the Wg2 proteinase, a serine protease from Streptococcus cremoris, suggesting that both genes were derived from a common ancestral gene.
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  • 93
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The chromophoric protein phycocyanin is the major protein in the phycobilisome rod of the cyanobacterium Synechococcus 6301 (formerly designated Ana-cystis nidulans sp. UTEX 625). The gene clusters coding for the β- and α-subunits of phycocyanin are duplicated on the chromosome of Synechococcus 6301 and separated by an intergenic region 2.5 kb long. The structure of the phycocyanin operons of the phycobilisome mutant strains AN112 and AN135 was compared to that of wild-type Synechococcus 6301. Both mutants have an altered phycobilisome structure resulting in the appearance of rods of a shorter overall length. Genetic mapping indicated that the mutant strain AN112 completely lacked the intergenic region and carried only one set of phycocyanin subunit genes. No gross structural difference in the genetic organization of the corresponding region of mutant strain AN135 was detected. Northern blot analysis and primer extension experiments were used to monitor the transcriptional pattern of the phycocyanin rod operon. It was found that AN112 had lost the 3.7 kb minor mRNA, which covers the intergenic region, and only produced two major 1.3 and 1.4 kb mRNA species. These transcripts were identified as fusion products of the 5′ end of the transcriptional unit originating from the promoter region of the left-hand phycocyanin gene cluster and the 3′ end of the transcriptional unit covering the right-hand phycocyanin gene cluster. The mutant strain AN 135 had a transcriptional pattern very similar to that of the wild type. The level of transcription of the major transcripts covering the phycocyanin gene clusters was similar in the wild-type and mutant strains. The results indicate that genes coding for two of the linker proteins, namely the 30 and 33 kD poly-peptides, are located in the intergenic region between the duplicated phycocyanin gene clusters in Synecho-coccus 6301. The results also show that loss of the right-hand phycocyanin promoter does not drastically impair phycocyanin synthesis.
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  • 94
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: A mobilizable plasmid which carries the promoter for the exotoxin A (ETA) structural gene fused to lacZ was integrated into the chromosome of wild-type and mutant strains of Pseudomonas aeruginosa at the toxA locus by homologous recombination. β-galactosidase synthesis in the strains (cointegrates) carrying the tox4-lacZ fusions was regulated like ETA synthesis is in P. aeruginosa.Two multicopy plasmids carrying a positive regulatory gene designated toxR were constructed which are identical except with respect to the orientation of toxR to the lacZ promoter on the plasmid. These plasmids were then introduced into P. aeruginosa cointegrate strains. When toxR was using its own promoter, synthesis of β-galactosidase in the cointegrate strains was increased but the pattern of iron regulation was not altered. In contrast, when the lacZ promoter was directing synthesis of the toxR product in the cointegrate strains, iron regulation of β-galacto-sidase and ETA synthesis were abolished.
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  • 95
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: Virulence genes of the Agrobacterium tumefaciens Ti plasmid are positively regulated by the products of virA and virG. To study the DNA-binding properties of the VirG protein, a translational fusion between virG and the frpEgene of Escherichia coli was constructed, and antiserum was raised against the encoded fusion protein. Using this antiserum, a protein of Mr= 29000, a size similar to that calculated from the virG nucleotide sequence, was detected in an E. coli strain harbouring a virG expression vector. Both the VirG protein and the fusion protein were found, by filter-binding and gel retardation analyses, to bind DNA nonspecifically. These data support an existing model for the two-component regulatory systems of bacteria.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 96
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: Transcription of ribosomal RNAs in Escherichia coli is started from two strong tandem promoters, P1 and P2. tt is known, however, that internal promoter-like structures occur and in a recent report (Mankin et al., 1987) a promoter sequence Pi within the 16S and 23S RNA spacer region showing good homology to the pro-karyotic consensus promoter structure was identified. It was proposed that this putative promoter has a possible function in the transcription of ribosomal RNAs in E. coli. Fusion of various DNA fragments containing the putative promoter sequence and different parts of the 16S/23S spacer region as well as the 23S RNA to the galactokinase gene allowed us to assess the functional activity of the promoter in vivo. To determine any growth rate dependent function of the putative promoter, the measurements were performed under different growth conditions. The promoter activity did not exceed 7% of the lac promoter under in vivo assay conditions. In addition, transcription starting at the promoter Pi did not proceed through the entire 23S RNA gene. We conclude, therefore, that transcription from Pi does not contribute significantly to the synthesis of ribosomal RNAs. Thus Its functional significance, if any, remains elusive.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 97
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The quaternary structure of regulatory proteins undoubtedly plays an important role in the initiation of transcription and DNA replication. To date, the best-characterized regulatory proteins are oligomers in which protomers are bound together by isologous interactions. From the examples presented in this article, it appears that the formation of certain nucleoprotein complexes implicated in transcription initiation might involve heterologous rather than isologous interactions, allowing differentiation between two classes of transcription activators.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 98
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: A 71 kiloDalton antigen from Mycobacterium tuberculosis is recognized by antibodies and by T lymphocytes during infection (Britton et al., 1986a). Partial sequence analysis indicates a relationship between this antigen and the highly conserved family of 70-kiloDalton heat shock proteins (hsp70) (Young et al., 1988). Biochemical and serological characterization of the protein confirms its membership of the hsp70 gene family, and metabolic labelling demonstrates that it is a major component of the mycobacterial response to heat stress. The role of stress proteins as antigens during infection is discussed.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 99
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The significance of Bradyrhizobium japonicum upstream activator sequences (UASs) for differential NifA-mediated fix and nif gene expression was investigated by two means: (i) hybrid fixA- and fixB-lacZ fusions were constructed by transposing a nifH-UAS cartridge in front of their promoters; and (ii) B. japonicum mutants were generated carrying specific chromosomal deletions or UAS cartridge insertions within the fixA, fixB or nifH promoter-upstream regions. Expression of fixA was not affected, and expression of fixB decreased only to 42%, when the respective fixA and fixB promoter-upstream DNAs were deleted. This shows that in B. japonicum the Njf A-dependent activation of at least the fixA promoter does not require the presence of a closely adjacent UAS. Deletion of the UASs in front of the nifH gene not only reduced the expression of nifH down to 2.5% but, surprisingly, also resulted in a reduction of the fixB mRNA level to less than 20%. This suggests that the nifH-UASs may exert a long-range effect on the expression of the 3-kb-distant fixBCX operon in nif cluster I of B. japonicum. Artificial transposition of the nif-UASs in front of the fixA and fixB promoters strongly enhanced fixA and fixB expression.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 100
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    ISSN: 1365-2958
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Biologie , Medizin
    Notizen: The fdhF gene, encoding the selenopolypeptide of formate dehydrogenase (FDHH). has a -12/-24 nif-type consensus promoter. A cis-acting DNA element, which is required for the regulation of the promoter by formate under anaerobic conditions, has been identified. This regulatory sequence of about 25bp in length is located 110 bp upstream of the transcription start site. By analysing a variety of mutant constructs in this region (5’deletions, internal deletions and point mutations) we were able to identify a hexanucleotide sequence -GTCACG-, which is important for the formate regulation of the fdhF promoter. The data also indicate that this element has many of the properties characteristic of eukaryotic enhancers.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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