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  • 1
    Electronic Resource
    Electronic Resource
    Cloning of an insecticidal toxin gene of Bacillus thuringiensis subsp. tenebrionis and its expression in Escherichia coli cells (1987)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 48 (1987), S. 0 
    Details
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract A structural gene of a crystal protein toxic for coleoptera larvae was cloned from plasmid DNA of Bacillus thuringiensis subsp. tenebrionis (BTT). The DNA was partially digested with restriction enzyme BamHI and fragments were inserted into cosmid pHC79. In Western blot analysis extracts from infected Escherichia coli cells revealed expression of the BTT crystal protein in antibiotic-resistant cells. Cell lysates from a selected E. coli clone were toxic for larvae of the Colorado potato beetle (Leptinotarsa decemlineata). The electrophoretic mobility in SDS gels of crystal protein from E. coli cells was 68 kDa and 74 kDa as observed for BTT-toxin in B. thuringiensis extracts. The cosmids obtained were unstable during cellular propagation. The deletion product still carried the δ-endotoxin gene.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1574-6968.1987.tb02615.x
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  • 2
    Electronic Resource
    Electronic Resource
    Enzymatic synthesis of bacteriophage fd viral DNA (1982)
    [s.l.] : Nature Publishing Group
    Nature 296 (1982), S. 828-832 
    Details
    ISSN: 1476-4687
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Notes: [Auszug] An enzyme system with requirements similar to those for replication of phage fd replicative form (RF) DNA in bacteriophage fd-infected cells has been reconstituted with purified fd gene 2 protein, and DNA polymerase III holoenzyme, DNA binding protein I and rep-protein (rep-helicase) of Escherichia ...
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1038/296828a0
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  • 3
    Electronic Resource
    Electronic Resource
    Characterization of Bacillus strains from apple and pear trees in South Africa antagonistic to Erwinia amylovora (2002)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 211 (2002), S. 0 
    Details
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: In order to find reasons for the absence of fire blight in most countries of the Southern hemisphere, bark samples from apple and pear trees in orchards of the Western Cape region in South Africa were extracted for bacteria which could be antagonistic to Erwinia amylovora. Screening was done in the late growth season and mainly Gram-positive bacteria were isolated. Approximately half of them produced growth inhibition zones on a lawn of E. amylovora. Most isolates were classified as Bacillus megaterium by microbiological assays and in API 50 test systems. They were visualized in the light microscope as non-motile large rods. These strains may not be responsible for the absence of fire blight in orchards, but they may indicate unfavourable climatic conditions for Gram-negative bacteria including E. amylovora. They may reduce the ability of E. amylovora to establish fire blight and could also be useful for application in biological disease control.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1574-6968.2002.tb11232.x
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  • 4
    Electronic Resource
    Electronic Resource
    Molecular analysis of the ams operon required for exopolysaccharide synthesis of Erwinia amylovora (1995)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 15 (1995), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: A16 kb transcript of the ams region, which is essential for biosynthesis of amylovoran, the acidic exopolysaccharide of Erwinia amylovora, was detected by Northern hybridization analysis. The positive regulator RcsA enhanced transcription of the targe mRNA from the ams operon. The nucleotide sequence of this area revealed 12 open reading frames (ORFs), which are all transcribed in the same direction. Five ORFs corresponded to the previously mapped genes amsA to amsE, Sequence analysis of the insertion sites of several Tn5 mutations confirmed these data. Tn5 or site-directed mutagenesis of the ORFs 477, 377, 144, and 743 reveated an amytovoran-deficient phenotype, and the newly identified genes were named amsG, amsH, amsI, and amsF, respectivety. The predicted amino acid sequence of AmsG is highly homologous to gatactosyl-1 -phosphate undecaprenyl-phosphate transferases. AmsB and AmsD are similar to other glycosyl transferases, and AmsH may be related to BexD. A significant homology to mammalian phosphatases was observed for Amsl. AmsA shows characteristic motifs for membrane association and ATP binding. AmsF carries a secretory signal sequence in the N-terminus and could be involved in periplasmic processing of the repeating units. Complementation experiments located a promoter region required for gene expression as far as 500 bp upstream of amsG. It is preceded by a typical transcriptional termination sequence. A mutation upstream of the terminator did not affect amylovoran synthesis. Partial nucleotide sequences further upstream of the ams region showed homology to genes mapped at 45min on the Escherichia coli chromosome. A termination sequence was also found downstream of the ams operon at a distance of 16 kb from the promoter. Between amsF and this terminator, three additional ORFs were detected.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1995.tb02361.x
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  • 5
    Electronic Resource
    Electronic Resource
    Heterologous expression of a mutated toxin gene from Bacillus thuringiensis subsp. tenebrionis (1990)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 66 (1990), S. 0 
    Details
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract Using oligonucleotide probes we have isolated a DNA fragment encoding an insecticidal toxin of the coleopteran specific Bacillus thuringiensis subsp. tenebrionis. The gene was altered by site directed mutagenesis at its 5′-end and adapted for general cloning and expression purposes with a linker including a start codon and new restriction sites. The constructs were inserted into several vector plasmids and expressed in Escherichia coli. Expression E. coli was strongly enhanced by the lac-promoter. A fusion protein with phage MS2-polymerase was produced together with a 76 kDa protein also found for normal expression of the toxin gene. Synthesis of the latter protein indicated a second ribosome binding site at the 5′-terminus of the toxin encoding sequence. Toxin-containing proteins were identified by Western blot analysis. The positive cell extracts from E. coli had been insecticidal activity on larvae of the Colorado potato breetle. The cloned gene is not homologous to a gene previously cloned by us whose gene products were also toxic to coleopteran larvae.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1574-6968.1990.tb03978.x
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  • 6
    Electronic Resource
    Electronic Resource
    Synthesis of fructans in tubers of transgenic starch-deficient potato plants does not result in an increased allocation of carbohydrates (1996)
    Springer
    Planta 199 (1996), S. 528-536 
    Details
    ISSN: 1432-2048
    Keywords: Erwinia ; Fructan ; Levan sucrase ; Transgenic potato ; Sink strength ; Solanum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Inhibition of starch biosynthesis in transgenic potato (Solanum tuberosum L. cv. Désirée) plants (by virtue of antisense inhibition of ADP-glucose pyrophosphorylase) has recently been reported to influence tuber formation and drastically reduce dry matter content of tubers, indicating a reduction in sink strength (Müller-Röber et al. 1992, EMBO J 11: 1229–1238). Transgenic tubers produced low levels of starch, but instead accumulated high levels of soluble sugars. We wanted to know whether these changes in tuber development/sink strength could be reversed by the production of a new high-molecular-weight polymer, i.e. fructan, that incorporates sucrose and thereby should reduce the level of osmotically active compounds. To this end the enzyme levan sucrase from the gram-negative bacterium Erwinia amylovora was expressed in tubers of transgenic potato plants inhibited for starch biosynthesis. Levan sucrase was targeted to different subcellular compartments (apoplasm, vacuole and cytosol). Only in the case of apoplastic and vacuolar targeting was significant accumulation of fructan observed, leading to fructan representing between 12% and 19% of the tuber dry weight. Gel filtration and 13C-nuclear magnetic resonance spectroscopy showed that the molecular weight and structure of the fructan produced in transgenic plants is identical to levan isolated from E. amylovora. Whereas apoplastic expression of levansucrase had deleterious effects on tuber development, tubers containing the levansucrase in the vacuole did not differ in phenotype from tubers of the starch-deficient plants used as starting material for transformation with the levansucrase. When tuber yield was analysed, no increase but rather a further decrease relative to ADP-glucose pyrophosphorylase antisense plants was observed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00195183
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  • 7
    Electronic Resource
    Electronic Resource
    The use of luciferase as a reporter for response of plant cells to the fireblight pathogen Erwinia amylovora (1995)
    Springer
    Plant cell reports 14 (1995), S. 497-500 
    Details
    ISSN: 1432-203X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We have introduced the gene encoding luciferase from Photinus pyralis into pear and tobacco cells in order to judge the reaction of plant tissue to damaging conditions such as incubation at high temperature or inoculation with a pathogen. The constitutive expression of the luciferase gene via a strong promoter slowly decreased during propagation of the transformed pear cell line. After various stress treatments the resulting luciferase activity and the ATP content of the plant cells were determined by bioluminescence and found to correspond to each other. Inoculation of transformed pear cells with Erwinia amylovora resulted in a continuous decrease of luciferase activity in contrast to tobacco cells, where the enzyme activity was significantly higher in the first period after inoculation with bacteria compared to the untreated control cells. The pattern of the luciferase activity reflected the slow damage of the host-plant cells by E. amylovora and the elevated metabolism of the non-host cells after inoculation with the pathogen.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00232782
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  • 8
    Electronic Resource
    Electronic Resource
    Transient transfection of mammalian cells with DNA of the plant-pathogenic Ti-plasmid and expression of marker and resident sequences (1989)
    Springer
    Molecular and cellular biochemistry 85 (1989), S. 19-28 
    Details
    ISSN: 1573-4919
    Keywords: Ti-plasmid ; transfection ; mammalian cells ; T-DNA expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Summary The large Ti-plasmid from Agrobacterium tumefaciens strain C58 has been used for transfection experiments with mammalian cells. In DNA from Tupaia baby fibroblasts Ti-plasmid sequences could be identified by filter hybridization as long as four weeks after transfection including two cell passages. The hybridization signals decreased rapidly after addition of the Ti-plasmid DNA-coprecipitate to the cells. The signals were often not detected any more after the first day, but were visible one week after transfection. Nuclei prepared from Ti-plasmid-transfected cells hybridized to pTi-specific RNA. With the chloramphenicol acetyl transferase-gene as marker no discrimination in DNA uptake was found between the Ti-plasmid and much smaller plasmids. According to the number of nuclei with homology to pTi-sequences it is assumed that about 0.2% of the cells carry Ti-plasmid DNA in the nucleus. Analysis of RNA isolated from cells transfected with cloned segments of the Ti-plasmid revealed that the TDNA region of the Ti-plasmid was predominantly transcribed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00223510
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  • 9
    Electronic Resource
    Electronic Resource
    Promoter activity and expression of sequences from Ti-plasmid stably maintained in mammalian cells (1989)
    Springer
    Molecular and cellular biochemistry 90 (1989), S. 9-18 
    Details
    ISSN: 1573-4919
    Keywords: heterologous gene expression ; neomycin-phosphotransferase assay ; nopaline synthase gene ; plant cell promoters ; polymerase-chain reaction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Summary Sequences of the plant-pathogenic Ti-plasmid were found to be constitutively expressed in LTK- and in HeLa-cells. Activity of the nopaline-synthase (nos) promoter in these cells was demonstrated by directing expression of G418 resistance from a connected neomycin-phosphotransferase 11 (NPT 11) gene. Control transfections with the widely used thymidine-kinase (TK) promoter gave comparable transfection rates as found for the nos-promoter with NPT 11. The function of the nos-promoter was also confirmed by assaying neomycin-phosphotransferase synthesized in cells containing a plasmid with the NPT 11-gene under control of this promoter. Several LTK+ clones stably transfected with Ti-plasmid propagated the total Ti-plasmid DNA in a colinear state presumably as an episomal unit. Dot blot analysis and polymerase chain reaction showed predominant transcription of Ti-sequences from the T-DNA area reflecting transcriptional activity of this region not only in plant cells but also in animal cells. These results provide new information about promoter functions in systems unrelated to their natural environment.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00225216
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  • 10
    Electronic Resource
    Electronic Resource
    Transformation of soybean protoplasts from permanent suspension cultures by cocultivation with cells of Agrobacterium tumefaciens (1987)
    Springer
    Plant molecular biology 9 (1987), S. 135-145 
    Details
    ISSN: 1573-5028
    Keywords: azacytidine ; DNA/RNA hybridization ; gene expression ; marker rescue ; nopaline-synthase ; plant tumor cells ; Ti-plasmid
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Cell wall regenerating protoplasts from soybean cells kept in suspension culture were cocultivated with bacteria which were derived from the nopaline strain C58 of Agrobacterium tumefaciens. When the bacteria carried an oncogenic Ti-plasmid, about 5% of the surviving protoplasts were able to form calli on hormone-free agar in contrast to controls, where bacteria without Ti-plasmid were applied, and where no calli were formed. After isolation of DNA from hormone-independently growing cells further evidence for transformation was obtained by hybridization to Ti-plasmid specific RNA and by rescue of a segment with a bacterial resistance gene which had been inserted before into the T-DNA. Transfer of T-DNA harboring a neomycin-resistance gene activated by the nos-promoter resulted in calli growing on kanamycin. Verification of segments located at the left and the right part of the T-DNA indicated the presence of its entire length in transformed soybean cells. Expression of T-DNA genes was measured by the assay of nopaline-synthase. Cells cultured on agar had a much higher level of nopaline-synthase than fast growing cells in suspension culture. Transferring them to agar or treatment with azacytidine strongly increased synthesis of nopaline-synthase indicating a reversible repression presumably via a methylation mechanism.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00015646
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