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  • 1
    Electronic Resource
    Electronic Resource
    Induction, purification and characterisation of acyl-ACP thioesterase from developing seeds of oil seed rape (Brassica napus) (1992)
    Springer
    Plant molecular biology 20 (1992), S. 763-780 
    Details
    ISSN: 1573-5028
    Keywords: acyl-(acyl carrier protein) thioesterase ; acyl carrier protein ; Brassica napus ; fatty acid synthesis ; rape ; seed development
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The level of two thioesterases, acyl-CoA thioesterase and acyl-ACP thioesterase was determined during seed maturation in oil seed rape. Both thioesterase activities rose markedly prior to the onset of lipid accumulation, but the induction kinetics suggest that the activities reside on distinct polypeptides. Acyl-ACP thioesterase (EC 3.1.2.14) was purified 2000-fold using a combination of ion exchange, ACP-affinity chromatogr aphy, chromatofocusing and gel filtration. Using native gel electrophoresis, and assays for enzymic activity, two polypeptides were identified on SDS-PAGE as associated with the activity. Cleveland mapping of these polypeptides, of 38 kDa component and 33 kDa respectively, demonstrated that they are related. An antibody was prepared against the 38 kDa component, and this also recognises the 33 kDa polypeptide in highly purified preparations. Western blotting of a crude extract identifies one band at 38 kDa consistent with the 33 kDa component being a degradation product generated during purification. The native molecule has a Mr of 70 kDa indicating a dimeric structure. The enzyme has a pH optimum of 9.5 and shows strong preference for oleoyl-ACP as substrate. The intact enzyme has an N-terminus blocked to protein sequencing. We also found that two other polypeptides co-purify with acyl-ACP thioesterase under native conditions. The N-terminal amino-acid sequence of these polypeptides is shown and their possible identity is discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00027148
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  • 2
    Electronic Resource
    Electronic Resource
    A Zea mays GTP-binding protein of the ARF family complements an Escherichia coli mutant with a temperature-sensitive malonyl-coenzyme A:acyl carrier protein transacylase (1995)
    Springer
    Plant molecular biology 27 (1995), S. 629-633 
    Details
    ISSN: 1573-5028
    Keywords: GTP binding ; ADP ribosylation ; Zea mays ; Escherichia coli ; fatty acid biosynthesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In an attempt to isolate a plant malonyl-coenzyme A:acyl carrier protein transacylase cDNA clone, by direct genetic selection in an Escherichia coli fabD mutant (LA2-89) with a maize cDNA expression library, a Zea mays cDNA clone encoding a GTP-binding protein of the ARF family was isolated. Complementation of a mutation affecting bacterial membrane lipid biosynthesis by a plant ARF protein, could indicate the existence of as yet unidentified bacterial equivalents of this ubiquitous eucaryotic GTP-binding protein.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00019329
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    Paper (German National Licenses)
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  • 3
    Electronic Resource
    Electronic Resource
    Cloning of Brassica napus CTP:phosphocholine cytidylyltransferase cDNAs by complementation in a yeast cct mutant (1996)
    Springer
    Plant molecular biology 31 (1996), S. 205-211 
    Details
    ISSN: 1573-5028
    Keywords: Brassica napus ; CDP-choline ; cytidylyltransferase ; freezing stress ; phosphatidylcholine ; yeast mutant complementation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract CTP: phosphocholine cytidylyltransferase is a rate-limiting enzyme in biosynthesis of phosphatidylcholine in plant cells. We have isolated four cDNAs for the cytidylyltransferase from a root cDNA library of Brassica napus by complementation in a yeast cct mutant. The deduced amino-acid sequences of the B. napus enzymes resembled rat and yeast enzymes in the central domain. Although all cytidylyltransferases ever cloned from B. napus and other organisms were predicted to be structurally hydrophilic, the yeast cct mutant transformed with one of the B. napus cDNA clones restored the cytidylyltransferase activity in the microsomal fraction as well as in the soluble fraction. These results are consistent with a recent view that yeast cells contained a machinery for targeting the yeast cytidylyltransferase to membranes, and may indicate that the B. napus enzyme was compatible with the machinery.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00021784
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  • 4
    Electronic Resource
    Electronic Resource
    Isolation and characterization of two Brassica napus embryo acyl-ACP thioesterase cDNA clones (1993)
    Springer
    Plant molecular biology 23 (1993), S. 769-778 
    Details
    ISSN: 1573-5028
    Keywords: acyl ; Brassica napus ; fatty acid synthesis ; plastidial location ; seed ; thioesterase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Acyl-ACP thioesterases are involved in regulating chain termination of fatty acid biosynthesis in plant systems. Previously, acyl-ACP thioesterase purified from Brassica napus seed tissue has been shown to have a high preference for hydrolysing oleoyl-ACP. Here, oligonucleotides derived from B. napus oleoyl-ACP thioesterase protein sequence data have been used to isolate two acyl-ACP thioesterase clones from a B. napus embryo cDNA library. The two clones, pNL2 and pNL3, contain 1642 bp and 1523 bp respectively and differ in the length of their 3′ non-coding regions. Both cDNAs contain open reading frames of 366 amino acids which encode for 42 kDa polypeptides. Mature rape thioesterase has an apparent molecular weight of 38 kDa on SDS-PAGE and these cDNAs therefore encode for precursor forms of the enzyme. This latter finding is consistent with the expected plastidial location of fatty acid synthase enzymes. Northern blot analysis shows thioesterase mRNA size to be ca. 1.6 kb and for the thioesterase genes to be highly expressed in seed tissue coincident with the most active phase of storage lipid synthesis. There is some sequence heterogeneity between the two cDNA clones, but overall they are highly homologous sharing 95.7% identity at the DNA level and 98.4% identity at the amino acid level. Some sequence heterogeneity was also observed between the deduced and directly determined thioesterase protein sequences. Consistent with the observed sequence heterogeneity was Southern blot data showing B. napus thioesterase to be encoded by a small multi-gene family.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00021532
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  • 5
    Electronic Resource
    Electronic Resource
    Towards the genetic engineering of triacylglycerols of defined fatty acid composition: major changes in erucic acid content at the sn-2 position affected by the introduction of a 1-acyl-sn-glycerol-3-phosphate acyltransferase from Limnanthes douglasii into oil seed rape (1996)
    Springer
    Molecular breeding 2 (1996), S. 133-142 
    Details
    ISSN: 1572-9788
    Keywords: 1-acyl-sn-glycerol-3-phosphate acyltransferase ; Brassica napus ; Limnanthes douglasii ; trierucin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract A cDNA encoding a 1-acyl-sn-glycerol-3-phosphate acyltransferase from Limnanthes douglasii was introduced into oil seed rape (Brassica napus) under the control of a napin promoter. Seed triacylglycerols from transgenic plants were analysed by reversed-phase HPLC and trierucin was detected at a level of 0.4% and 2.8% in two transgenic plants but was not found in untransformed rape seed. Total fatty acid composition analysis of seeds from these selected plants revealed that the erucic acid content was no higher than the maximum found in the starting population. Analysis of fatty acids at the sn-2 position showed no erucic acid in untransformed rape but in the selected transgenic plants 9% (mol/mol) and 28.3% (mol/mol) erucic acid was present. These results conclusively demonstrate that the gene from L. douglasii encodes a 1-acyl-sn-glycerol-3-phosphate acyltransferase which can function in rape and incorporate erucic acid at the sn-2 position of triacylglycerols in seed. Additional modifications may further increase levels of trierucin.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00441428
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    Paper (German National Licenses)
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