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  • 1
    Electronic Resource
    Electronic Resource
    Molecular genetic analysis of enoyl-acyl carrier protein reductase inhibition by diazaborine (1999)
    Oxford BSL : Blackwell Science Ltd
    Molecular microbiology 31 (1999), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Diazaborine and isoniazid are, at first sight, unrelated anti-bacterial agents that inhibit the enoyl-ACP reductase (ENR) of Escherichia coli and Mycobacterium tuberculosis respectively. The crystal structures of these enzymes including that of the diazaborine-inhibited E. coli ENR have been obtained at high resolution. Site-directed mutagenesis was used to study the importance of amino acid residues in diazaborine susceptibility and enzyme function. The results show that drug binding and inhibition require the presence of a glycine residue at position 93 of E. coli ENR or at the structurally equivalent position in the plant homologue, which is naturally resistant to the drug. The data confirm the hypothesis that any amino acid side-chain other than hydrogen at this position within the three-dimensional structure of these enzymes will affect diazaborine resistance by encroaching into the drug binding site. Substitutions of Gly-93 by amino acids with small side-chains, such as serine, alanine, cysteine and valine, hardly affected the catalytic parameters and rendered the bacterial host resistant to the drug. Larger amino acid side-chains, such as that of arginine, histidine, lysine and glutamine, completely inactivated the activity of the enzyme.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.1999.01182.x
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  • 2
    Electronic Resource
    Electronic Resource
    Lipid production of revertants of Ufa mutants from the oleaginous yeast Apiotrichum curvatum (1990)
    Springer
    Applied microbiology and biotechnology 33 (1990), S. 176-182 
    Details
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary From six unsaturated fatty acid auxotrophs (Ufa mutants) of the oleaginous yeast Apiotrichum curvatum blocked in the conversion of stearic to oleic acid, were isolated revertants able to grow in the absence of unsaturated fatty acids, in a search for strains that can produce cocoa butter equivalents. A broad range in the percentage of saturated fatty acids (%SFA) was observed in the lipids of individual revertants (varying from 27%–86% SFA), compared with the wild-type (44% SFA). Further analysis of fatty acid composition indicated that: (i) not all six Ufa mutants had the same genetic background and (ii) one specific Ufa mutation could be reverted in more than one way. Revertants that produced lipids with a %SFA〉56%, were examined further. These strains were cultivated for 50 generations and half of them produced lipids with high %SFA after that time and were defined as stable. The viability of revertant strains with extremely high %SFA (〉80%) may be explained by our finding that polar lipids, which are part of yeast membranes, contained much more polyunsaturated fatty acids and a significantly lower %SFA than neutral (storage) lipids. One revertant (R25.75) was selected that was able to produce lipids in whey permeate at a rate comparable with wild-type A. curvatum and with a fatty acid composition and congelation curve comparable with cocoa butter.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00176521
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  • 3
    Electronic Resource
    Electronic Resource
    Isolation and characterization of fatty acid auxotrophs from the oleaginous yeast Apiotrichum curvatum (1989)
    Springer
    Applied microbiology and biotechnology 32 (1989), S. 76-84 
    Details
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary In order to improve the economic value of lipids produced by the oleaginous yeast strain Apiotrichum curvatum ATCC 20509, a search was made for mutants defective in the conversion of stearic acid to oleic acid. Mutants could be selected as unsaturated fatty acid auxotrophs, since unsaturated fatty acids are essential componenets in membrane lipids. After treatment of A. curvatum wild-type with N-methyl-N′-nitro-N⇔-nitrosoguanidine, 58 fatty-acid-requiring mutants were isolated. On the basis of (1) the growth response to saturated and unsaturated fatty acids and (2) the fatty acid composition of lipids produced by these mutants, it was concluded that only 18 of them were real unsaturated fatty acid (Ufa) mutants, while the other 40 were designated as fatty acid synthetase (Fas) mutants. It is further shown that Ufa mutants of A. curvatum are able to produce high amounts of lipids consisting of more than 90% triacylglycerols with a percentage of saturated fatty acids resembling that of cocoa butter, when grown in the presence of relatively small amounts of oleic acid in the growth medium. This may offer an economically favourable alternative in comparison with other methods that have been developed for the production of cocoa butter equivalents by microorganisms.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00164826
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  • 4
    Electronic Resource
    Electronic Resource
    Identification of mutations affecting replication control of plasmid Clo DF13 (1981)
    [s.l.] : Nature Publishing Group
    Nature 290 (1981), S. 264-267 
    Details
    ISSN: 1476-4687
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Notes: [Auszug] Both nonconditional3 and conditional13 replication-control mutants of plasmid Clo DF13 (8.6 kilobases) have been isolated after chemical mutagenesis, and characterized13 25. As reported elsewhere18, the Clo DF13-cop3 mutation (Table 1) was mapped by insertion of transposons and successive deletion ...
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1038/290264a0
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  • 5
    Electronic Resource
    Electronic Resource
    Two potential Petunia hybrida mitochondrial DNA replication origins show structural and in vitro functional homology with the animal mitochondrial DNA heavy and light strand replication origins (1991)
    Springer
    Current genetics 20 (1991), S. 503-513 
    Details
    ISSN: 1432-0983
    Keywords: Mitochondrial DNA ; Petunia hybrida ; Physical map ; Replication origin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Four Petunia hybrida mitochondrial (mt) DNA fragments have been isolated, sequenced, localized on the physical map and analyzed for their ability to initiate specific DNA synthesis. When all four mtDNA fragments were tested as templates in an in vitro DNA synthesizing lysate system, developed from purified P. hybrida mitochondria, specific initiation of DNA synthesis could only be observed starting within two framents, oriA and oriB. When DNA synthesis incubations were performed with DNA templates consisting of both the A and B origins in the same plasmid in complementary strands, DNA synthesis first initiates in the A-origin, proceeds in the direction of the B-origin after which replication is also initiated in the B-origin. Based on these observations, a replication model for the P. hybrida mitochondrial genome is presented.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00334779
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  • 6
    Electronic Resource
    Electronic Resource
    Supported PCR: an efficient procedure to amplify sequences flanking a known DNA segment (1993)
    Springer
    Plant molecular biology 21 (1993), S. 723-728 
    Details
    ISSN: 1573-5028
    Keywords: supported polymerase chain reaction (sPCR) ; target enrichment ; T-DNA tagging ; transposition
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We describe a novel modification of the polymerase chain reaction for efficient in vitro amplification of genomic DNA sequences flanking short stretches of known sequence. The technique utilizes a target enrichment step, based on the selective isolation of biotinylated fragments from the bulk of genomic DNA on streptavidin-containing support. Subsequently, following ligation with a second universal linker primer, the selected fragments can be amplified to amounts suitable for further molecular studies. The procedure has been applied to recover T-DNA flanking sequences in transgenic tomato plants which could subsequently be used to assign the positions of T-DNA to the molecular map of tomato. The method called supported PCR (sPCR) is a simple and efficient alternative to techniques used in the isolation of specific sequences flanking a known DNA segment.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00014557
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  • 7
    Electronic Resource
    Electronic Resource
    Characterization of theAc/Ds behaviour in transgenic tomato plants using plasmid rescue (1992)
    Springer
    Plant molecular biology 20 (1992), S. 61-70 
    Details
    ISSN: 1573-5028
    Keywords: transposition ; Ac/Ds ; transgenic tomato plants ; RFLP mapping ; plasmid rescue
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We describe the use of plasmid rescue to facilitate studies on the behaviour ofDs andAc elements in transgenic tomato plants. The rescue ofDs elements relies on the presence of a plasmid origin of replication and a marker gene selective inEscherichia coli within the element. The position within the genome of modifiedDs elements, rescued both before and after transposition, is assigned to the RFLP map of tomato. Alternatively to the rescue ofDs elements equipped with plasmid sequences,Ac elements are rescued by virtue of plasmid sequences flanking the element. In this way, the consequences of the presence of an (active)Ac element on the DNA structure at the original site can be studied in detail. Analysis of a library ofAc elements, rescued from the genome of a primary transformant, shows thatAc elements are, infrequently, involved in the formation of deletions. In one case the deletion refers to a 174 bp genomic DNA sequence immediately flankingAc. In another case, a 1878 bp internalAc sequence is deleted.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00029149
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  • 8
    Electronic Resource
    Electronic Resource
    Developmental specific expression and organelle targeting of the Escherichia coli fabD gene, encoding malonyl coenzyme A-acyl carrier protein transacylase in transgenic rape and tobacco seeds (1994)
    Springer
    Plant molecular biology 26 (1994), S. 189-202 
    Details
    ISSN: 1573-5028
    Keywords: fatty acid synthesis ; malonyl CoA-ACP transacylase ; seed development ; transgenic rape ; transgenic tobacco
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In both plants and bacteria, de novo fatty acid biosynthesis is catalysed by a type II fatty acid synthetase (FAS) system which consists of a group of eight discrete enzyme components. The introduction of heterologous, i.e. bacterial, FAS genes in plants could provide an alternative way of modifying the plant lipid composition. In this study the Escherichia coli fabD gene, encoding malonyl CoA-ACP transacylase (MCAT), was used as a model gene to investigate the effects of over-producing a bacterial FAS component in the seeds of transgenic plants. Chimeric genes were designed, so as not to interfere with the household activities of fatty acid biosynthesis in the earlier stages of seed development, and introduced into tobacco and rapeseed using the Agrobacterium tumefaciens binary vector system. A napin promoter was used to express the E. coli MCAT in a seed-specific and developmentally specific manner. The rapeseed enoyl-ACP reductase transit peptide was used successfully, as confirmed by immunogold labelling studies, for plastid targeting of the bacterial protein. The activity of the bacterial enzyme reached its maximum (up to 55 times the maximum endogenous MCAT activity) at the end of seed development, and remained stable in mature transgenic seeds. Significant changes in fatty acid profiles of storage lipids and total seed lipid content of the transgenic plants were not found. These results are in support of the notion that MCAT does not catalyse a rate-limiting step in plant fatty acid biosynthesis.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00039531
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  • 9
    Electronic Resource
    Electronic Resource
    Trans-activation of an artificial dTam3 transposable element in transgenic tobacco plants (1991)
    Springer
    Plant molecular biology 16 (1991), S. 39-47 
    Details
    ISSN: 1573-5028
    Keywords: transposition ; transposon tagging ; two-element system ; Tam3 rearrangements ; transgenic plants ; phenotypic assay
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In Antirrhinum majus only autonomous Tam3 transposons have been characterized. We investigated whether an artificial dTam3 element, with a deletion in the presumptive transposase coding region, can be trans-activated in tobacco by an activator Tam3 element, which was immobilized by the deletion of one inverted repeat. A phenotypic assay based on restored hygromycin resistance demonstrates that a dTam3 element harbouring a bacterial plasmid can be trans-activated with a low frequency. Molecular analysis confirms that the dTam3 element has been excised from the HPTII marker gene. Reintegration of the dTam3 element into the tobacco genome is detected only in one out of six hygromycin-resistant plants analysed. PCR analysis of empty donor sites shows that excision of the dTam3 element in tobacco results in rearrangements (deletions and additions), that have been shown to be characteristic of Tam3 excision in the original host Antirrhinum majus. This trans-activation assay allowed us to establish that, in contrast to what has been detected in Antirrhinum majus, a periodical temperature shift down to 15°C does not enhance dTam3 transposition in regenerating tobacco calli.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00017915
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  • 10
    Electronic Resource
    Electronic Resource
    Transposition pattern of a modified Ds element in tomato (1993)
    Springer
    Plant molecular biology 21 (1993), S. 1109-1119 
    Details
    ISSN: 1573-5028
    Keywords: transposition ; Ac/Ds ; transgenic tomato plants ; RFLP mapping ; plasmid rescue
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Several aspects of transposition of an in vitro modified Ds element are described. This Ds element, designated ds-r, is equipped with bacterial plasmid sequences and can, therefore, be rescued from the plant genome. Our results indicate that the Ds-r element has a ‘late’ timing of transposition from T-DNAs. This feature of the element might be advantageous for tagging experiments because it leads to independently transposed germinally transmitted elements. Furthermore, it is shown that Ds-r transposition generates clusters of insertions, indicating that ‘genes to be tagged’ should be located in genomic regions covered by insertions.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00023607
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